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National Institute on Drug Abuse
RESEARCH
MONOGRAPH SERIES
Molecular
Approaches to
Drug Abuse
Research
Volume II
1
26
U.S. Department of Health and Human Services • Public Health Service • National Institutes of Health
Molecular Approaches to
Drug Abuse Research
Volume II: Structure,
Function, and Expression
Editor:
Theresa N.H. Lee, Ph.D.
NIDA Research Monograph 126
1992
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service
Alcohol, Drug Abuse, and Mental Health Administration
National Institute on Drug Abuse
5600 Fishers Lane
Rockville, MD 20857
ACKNOWLEDGMENT
This monograph is based on the papers and discussions from a technical
review on “Molecular Approaches to Drug Abuse Research” held on July 30-31,
1991, in Bethesda, MD. The technical review was sponsored by the National
Institute on Drug Abuse (NIDA).
COPYRIGHT STATUS
The National Institute on Drug Abuse has obtained permission from the
copyright holders to reproduce certain previously published material as noted in
the text. Further reproduction of this copyrighted material is permitted only as
part of a reprinting of the entire publication or chapter. For any other use, the
copyright holder’s permission is required. All other material in this volume
except quoted passages from copyrighted sources is in the public domain and
may be used or reproduced without permission from the Institute or the authors.
Citation of the source is appreciated.
Opinions expressed in this volume are those of the authors and do not
necessarily reflect the opinions or official policy of the National Institute on Drug
Abuse or any other part of the U.S. Department of Health and Human Services.
The U.S. Government does not endorse or favor any specific commercial
product or company. Trade, proprietary, or company names appearing in this
publication are used only because they are considered essential in the context of
the studies reported herein.
NIDA Research Monographs are indexed in the “Index Medicus.” They are
selectively included in the coverage of “American Statistics Index,” “Biosciences
Information Service,” “Chemical Abstracts,” “Current Contents,” “Psychological
Abstracts,” and “Psychopharmacology Abstracts.”
DHHS publication number (ADM)92-1945
Printed 1992
ii
Contents
Page
Introduction 1
Theresa N.H. Lee
Neuronal Nicotinic Acetylcholine Receptor Diversity
4
James W. Patrick
Neurotransmitter and Drug Receptor Genes
14
George R. Uhl
The Diversity of the Dopamine Receptors 23
Olivier Civelli, James R. Bunzow, Qun-Yong Zhou, and
David K. Grandy
Cloned Dopamine Receptors: Targets in Therapy of
Drug Abuse 34
Philip Seeman
Cannabinoid Receptors: Which Cells, Where, How, and Why? 48
Lisa A. Matsuda, Tom I. Bonner, and Stephen J. Lolait
Studies on µ-Opioid-Binding Sites With Peptide Antibodies
57
Eric J. Simon, Theresa L. Gioannini, Yi-He Yao, and
Jacob M. Hiller
Molecular Cloning and Characterization of Neurotransmitter
Transporters
66
Randy D. Blakely
iii
Regulation of Gene Expression by Dopamine: Implications
in Drug Addiction
84
Orla M. Conneely, Ronan F. Power, and Bert W. O’Malley
Regulation of Opioid Gene Expression: A Model To Understand Neural
Plasticity
98
Michael J. Comb, Linda Kobierski, Hung Ming Chu, Yi Tan,
David Borsook, Karl Herrup, and Steven E. Hyman
Cellular and Molecular Analysis of Opioid Peptide Gene
Expression
113
Cynthia T. McMurray, Karen M. Pollock,
and James Douglass
The Prohormone and Proprotein Processing Enzymes PC1
and PC2: Structure, Selective Cleavage of Mouse POMC
and Human Renin at Pairs of Basic Residues, Cellular
Expression, Tissue Distribution, and mRNA Regulation
132
Nabil G. Seidah, Robert Day, Suzanne Benjannet, Normand
Rondeau, Alain Boudreault, Timothy Reudelhuber, Martin K.-H.
Schafer, Stanley J. Watson, and Michel Chrétien
List of NIDA Research Monographs
151
iv
Introduction
Theresa N.H. Lee
At the first National Institute on Drug Abuse (NIDA) technical review meeting
on “Molecular Approaches to Drug Abuse Research” held in August 1989,
one member of each of the three families of receptors and channels was
chosen to illustrate various strategies of gene cloning due to the limited number
of drug receptor genes cloned. These are the dopamine D2 receptor of the
G protein-coupled receptor family, the nicotinic acetylcholine receptor of the
ligand-gated channel family, and the potassium channel of the voltage-gated
channel family. Because of the extensive and innovative studies on the
nicotinic acetylcholine receptor, it was selected as an example to cover
postcloning research endeavors such as regulation of expression using
molecular genetics and transgenic mice as well as studies of structure and
function relationship using site-directed mutagenesis and other techniques.
The second NIDA technical review meeting on “Molecular Approaches to
Drug Abuse Research” was held on July 30 and 31, 1991, at the National
Institutes of Health. This technical review encompassed almost all the pivotal
research developments of the past 2 years in the field. What extraordinary
progress scientists in this field have made since the 1989 meeting! The
proceedings of this conference are presented in the following chapters of
this monograph.
As a logical extension of the agenda of the 1989 conference, the 1991
technical review began with Dr. James W. Patrick’s update on the diversity of
ligand/receptor interactions of neuronal nicotinic acetylcholine receptors based
on studies of functional oligomeric receptors. Presentations followed on the
successful cloning of genes of two other members of the ligand-gated channel
family—the glutamate (AMPA/Kainate) receptor and the -aminobutyric acid
(GABA) p-1 receptor by Dr. Stephen Heinemann and Dr. George R. Uhl,
respectively.
Perhaps as a reflection of the intense interest in the molecular cloning of the
opioid receptor genes, there have been an unprecedented number of genes
cloned in the G protein-coupled receptor family in these 2 years. These
scientists have made international news recently with their breakthroughs in
1
receptor gene cloning and subsequent remarkable studies. Some of the
examples presented in the 1991 conference were Dr. Olivier Civelli and Dr.
Philip Seeman on the molecular biology and pharmacology of dopamine
receptors D1, D2, D3, D4, and D5 and Dr. Lisa A. Matsuda on the cannabinoid
receptor. Dr. Eric J. Simon gave an update on the µ-opioid receptor. Even
though the cloning of the -opioid receptor gene was not conclusive at the
time of the conference, the recent cloning and expression of the long-awaited
prohormone and proprotein convertases PC1 and PC2 genes opened up
numerous avenues in the field (Dr. Nabil G. Seidah).
This technical review also coincided with another breakthrough in the field,
namely, the successful cloning of the gene for the dopamine transporter with
12 transmembrane domains. This work will undoubtedly help elucidate the
molecular mechanism of cocaine action. The leader of one of the groups that
accomplished this important task, Dr. Susan Amara, has also joined the NIDA
scientific community recently as a grantee. Although Dr. Amara was unable to
attend the conference, Dr. Randy D. Blakely spoke instead on the molecular
cloning and characterization of GABA and norepinephrine transporter genes.
This technical review would not have been complete without coverage of
the enormous contributions of NIDA scientists to our understanding of the
regulation of expression of opioid peptides. Dr. Michael J. Comb and Dr.
James Douglass provided two superb examples with their presentations on
this subject. From a different perspective, Dr. Orla M. Conneely discussed
the recent evidence from their laboratory that members of the steroid/thyroid
receptor family of transcription factors could be activated by dopamine and
dopamine D1 receptor agonists.
The 1991 technical review also commemorated the establishment of the
Molecular Biology and Genetics Program initiated 3 years earlier in the Division
of Preclinical Research of NIDA. This program now encompasses nearly every
important area employing molecular approaches to drug abuse research; this is
clearly illustrated in the technical review and this monograph. I am extremely
proud to point out that, of the 11 people contributing to this monograph, 8 are
members of the NIDA scientific community, and another 2 are in the process
of applying for support.
Drug abuse researchers interested in molecular approaches will find this
coming decade challenging and fruitful, with ground-breaking results expediting
understanding of the underlying basis of addiction to generate better strategies
for effective treatment, education, and prevention.
2
AUTHOR
Theresa N.H. Lee, Ph.D.
Program Director
Molecular Biology and Genetics Program
Biomedical Branch
Division of Preclinical Research
National Institute on Drug Abuse
Parklawn Building, Room 10A-19
5600 Fishers Lane
Rockville, MD 20857
3
Neuronal Nicotinic Acetylcholine
Receptor Diversity
James W. Patrick
INTRODUCTION
Society must deal with the dangerous dichotomy of drugs, a dichotomy that
arises from the fact that the drugs that provide the best treatment for many
illnesses also present tremendous opportunity for abuse. Furthermore, both
naturally occurring and synthetic drugs provide extremely powerful tools for
the study of biological systems. The use of these drugs as research tools has
resulted in a refinement of understanding of both the drugs and their biological
sites of action. This has, in turn, led to the development of ever more specific
and powerful drugs with subsequent new opportunities for abuse. This trend is
expected to continue because recent studies have revealed an unexpected
and, for the moment, bewildering diversity of drug receptors. This diversity
suggests that eventually one will be able to engineer exquisitely specific drugs
targeted to specific areas of the brain with minimal side effects. On the one
hand, one expects that this work will reveal new sites and mechanisms of
action of drugs and, unfortunately, will offer more opportunity for abuse through
the development of new classes of drugs. On the other hand, one hopes that
the insights gained in this work will result in the production of drugs with minimal
abuse potential or perhaps generate therapies to deal with abuse. There is
every reason to believe that new insights into receptors, new drugs, and rapidly
expanding access to the nervous system will lead to a new level of appreciation
and understanding of both the function of the brain and the uses and abuses of
drugs.
The rationale that underlies much of the research in this field is based on
the recent observation that there is a large family of genes that encode
neurotransmitter receptors and that almost all the ligand-gated ion channels
are members of this superfamily. Because these receptors are all members
of the same family they share structural and functional features. However,
they differ in many important ways, and these differences provide powerful tools
with which to probe their important common features. The family of genes that
compose the nicotinic receptors is studied to understand the structure, function,
4
and regulation of the nicotinic receptors. The information thus obtained has
provided the foundation for all other studies of ligand-gated ion channels and
will very likely continue to be important for understanding other receptors such
as the members of the glutamate, GABA, glycine, or serotonin receptor gene
families. Therefore, insights provided into the structure and function of the
neuronal nicotinic receptors will help one’s understanding of the ligand-gated
ion channels in general.
NICOTINIC ACETYLCHOLINE RECEPTORS
The nicotinic acetylcholine receptor has long been the prototypical ligand-gated
ion channel, and most of what is known about the structure and function of
these ion channels derives from studies of the muscle-type nicotinic receptor
isolated from the electric rays and fishes. Significant progress in this area has
come in the last 5 to 10 years following the application of the techniques of
molecular biology to the study of the muscle nicotinic receptor. These studies
led to the elucidation of the primary structure of the subunits of the receptor
(Ballivet et al. 1981; Noda et al. 1982, 1983a, 1983b; Sumikawa et al. 1982;
Claudio et al. 1983), its synthesis from RNA derived from cDNA clones (Mishina
et al. 1984), the determination of functional domains by mutagenesis (Mishina
et al. 1985), mapping of antibody binding sites (Tzartos et al. 1988, 1990),
the discovery of a new receptor subunit (Takai et al. 1985) and its role in
synaptogenesis at the neuromuscular junction (Methfessel and Sakmann
1986; Sakmann et al. 1985; Gu and Hall 1988), and the identification of
genomic sequences that control synthesis of receptor subunits (Gardner
et al. 1987). Understanding of the nicotinic receptor and the neuromuscular
junction in normal and diseased states has advanced dramatically as a result
of these studies.
The acetylcholine receptor at the neuromuscular junction was chosen for
these studies because good ligands were available and because the junction
was well studied and amenable to the types of experiments the author and his
colleagues thought were necessary. However, it seemed likely that a great
deal could be learned from the nicotinic receptors found at other nicotinic
cholinergic synapses. It also seemed likely that this information would be
important for understanding the receptor at the neuromuscular junction and
for understanding the role these receptors play in neurons. It has long been
known that cholinergic transmission at sympathetic ganglia is nicotinic, that the
nicotinic acetylcholine receptors on neurons differ from those found at the
neuromuscular junction, and that nicotinic receptors might play a very important
role in neurotransmitter release. The potential for understanding the nicotinic
receptors and ligand-gated ion channels in general, as well as the potential for
appreciating new roles for cholinergic function on neurons, led many
laboratories to the neuronal nicotinic acetylcholine receptors.
5
One approach was based on the idea that sequences encoding the muscle-type
nicotinic acetylcholine receptors would hybridize to, and thus identify, the
sequences encoding the neuronal nicotinic acetylcholine receptors. This
general approach led to the identification in the rat and chicken of a family
of genes encoding subunits of nicotinic acetylcholine receptors that are
expressed in the central nervous system (CNS) (for reviews, see Luetje et al.
1990a; Nordberg et al. 1989). Currently, the products of six different genes
(alpha2, alpha3, alpha4, alpha7, beta2, and beta4) are known to generate at
least seven different functional receptors (Nef et al. 1988; Deneris et al. 1988;
Wada et al. 1988; Boulter et al. 1986, 1987; Goldman et al. 1986; Duvoisin et
al. 1989; Couturier et al. 1990; Schoepfer et al. 1990). There are additional
members of this gene family (alpha5, alpha8, and beta3) whose gene products
have not yet been shown to be associated with a function (Schoepfer et al.
1990; Boulter et al. 1990; Deneris et al. 1989). The proteins encoded by these
nine genes have homologous extracellular, transmembrane, and cytoplasmic
domains and, in general, are classified as alpha or beta subunits. The alpha
subunits are identified by contiguous cysteines in the extracellular domain. The
beta (or nonalpha) subunits are identified by the lack of these cysteines and by
the ability of either beta2 or beta4 to substitute for the beta1 subunit in the
formation of a functional muscle-type nicotinic receptor.
The proteins derived from six of these genes associate in various combinations
to form functional receptors in the Xenopus oocyte. The alpha2, alpha3, and
alpha4 subunits each form functional receptors in combination with either beta2
or beta4. Receptors thus formed vary with respect to their single channel
properties (Papke et al. 1989) and pharmacology (Luetje and Patrick 1991;
Luetje et al. 1990b) depending on which of the three different alpha subunits
are present. Likewise, the substitution of a beta4 subunit for a alpha2 subunit
alters the response of the receptor to various agonists and antagonists
(Duvoisin et al. 1989; Luetje and Patrick 1991). There is also good evidence
that the complement of receptor subtypes present on a neuron changes during
development (Moss et al. 1989).
The receptor formed in the oocyte from alpha4 and beta2 subunits is probably a
pentamer comprising three beta subunits and two alpha subunits (Cooper et al.
1991). Although it is clearly possible to form receptors containing more than
one kind of alpha subunit (S. Helekar and J. Patrick, unpublished observations),
the diversity of oligomeric receptor structures formed in the CNS is not known.
Two additional members of the gene family have been identified in the chick
(Couturier et al. 1990; Schoepfer et al. 1990). These new members are alpha7
and alpha8, and the proteins encoded by these clones differ from the other
alpha subunits in several regards. The alpha7 subunit forms an acetylcholine-
6
gated ion channel in the absence of other added subunits (Couturier et al.
1990). Therefore, alpha7 appears to form homooligomeric receptors. The
functional receptors thus formed are blocked by alpha-bungarotoxin, unlike any
of the other neuronal receptors studied to date. Finally, they are derived from
genes with an intron/exon structure that is different from that of the other
neuronal receptor subunits (Couturier et al. 1990).
There are additional members of the gene family for which a function has
not yet been identified. Alpha5 is homologous to the other alpha subunits in
its overall architecture and in the presence of the two contiguous cysteines
in the extracellular domain (Boulter et al. 1990). However, this protein does
not form a functional receptor when RNA encoding it is injected into oocytes
in combination with any other receptor encoding RNA. It seems unlikely that
these clones fail to encode functional receptors as a consequence of some
cloning artifact that generated an incorrect sequence because the sequence
has been confirmed by analysis of genomic clones. The gene encoding
alpha5 is expressed in the CNS in a precise set of neuronal structures (Wada
et al. 1990). There has not yet been a systematic analysis of the contribution
this subunit might make as a third component of an oligomeric receptor.
Beta3 likewise fails to form functional receptors when injected in pairwise
combinations with the known functional receptors and, like alpha5 is
expressed in a well-defined set of neuronal structures.
In situ hybridization has shown that the members of this gene family are
expressed throughout the CNS (Wada et al. 1989, 1990). The beta2-encoding
RNA is found in almost all brain nuclei examined, and the other alpha- and
beta-encoding RNAs are expressed in specific but generally overlapping
subsets of nuclei. The beta4 subunit was first described as localized to the
medial habenula but is now known to be widely expressed in the central and
peripheral nervous systems (Moss et al. 1989). The alpha subunits are
expressed in discrete but overlapping sets of loci. However, these data do not
show the location of the expressed protein, and it is not yet known which
particular subunit combinations are located on dendrites, cell bodies, or axons.
In summary, molecular biological approaches to neuronal nicotinic receptors
have defined a gene family, documented the expression of this gene family in
the CNS, and demonstrated that various combinations of the proteins derived
from these genes form functionally different receptors. It is not yet known if all
the members of the gene family have been identified. Nor does one know the
full spectrum of combinations of subunits that exist in vivo or the roles that these
different subunit combinations play in the function and/or modification of
synapses.
7
It is the case, however, that neuronal nicotinic receptors differ in their
functional properties depending on which beta subunits are included in the
oligomer. Receptors containing a beta2 and an alpha3 subunit are sensitive to
the neuronal bungarotoxin (Boulter et al. 1987), whereas those in which an
alpha2 replaces the alpha3 subunit are 100-fold less sensitive (Wada et al.
1988). Receptors substituting an alpha4 are intermediate in sensitivity (Boulter
et al. 1987; Luetje et al. 1990b). This is consistent with the view that the ligand
binding site is on the alpha subunit. However, substitution of a beta4 for beta2
renders the alpha3-containing receptor insensitive to this toxin, suggesting that
the beta subunits either contribute to the toxin binding site or modify the
conformation of the alpha subunits (Duvoisin et al. 1989; Luetje et al. 1990b).
Different combinations of alpha and beta subunits are also distinguishable in
their responses to agonists. The relative sensitivities for acetylcholine, nicotine,
dimethylphenylpiperainium, and cytisine were determined for all six receptor
combinations that can be made from alpha2, alpha3, or alpha4 in combination
with either beta2 or beta4. These experiments were done in the Xenopus
oocyte where it was also established that the expressed muscle-type receptor
had a pharmacological profile indistinguishable from the cell line from which the
clones were derived. The results of these studies demonstrate that both the
alpha and the beta subunits contribute to agonist specificity. Receptors
containing a beta2 subunit differ in their response to acetylcholine and nicotine
depending on which alpha subunit is present. Nicotine is about thirtyfold more
effective on receptors containing alpha2 than on receptors containing alpha3,
suggesting that the alpha subunits determine agonist specificity. Cytisine is the
least effective agonist in all receptors containing a beta2 subunit. However,
substitution of the beta4 for the beta2 renders cytisine the best of the four
agonists. Although cytisine is the best agonist on beta4-containing receptors,
competition experiments suggest that cytisine is an antagonist on beta2-
containing receptors. These results demonstrate that both the alpha and
beta subunits contribute to both agonist and antagonist recognition and suggest
that one possible consequence of the expression of different combinations of
receptor subunits in the CNS is altered ligand affinity and specificity (Luetje and
Patrick 1991).
The neuronal nicotinic receptors differ from the muscle nicotinic receptors in
two additional interesting ways. The neuronal nicotinic acetylcholine receptors
are more permeable to calcium and in this property more closely resemble the
N-methyl-D-aspartate type of glutamate receptor than a muscle nicotinic
acetylcholine receptor (Vernino et al. 1992). The neuronal nicotinic receptors
are also modulated by external calcium ions. The author of this chapter and his
colleagues observed that increasing the external calcium ion concentration
resulted in large increases in the current produced by a given concentration of
8
receptors expressed in bovine chromaffin cells. The effect is not seen with
either barium or calcium, is not a consequence of activation of the calcium-
activated chloride channel, and is not a consequence of an increased
contribution of calcium to the current but rather a modulation by calcium of
the response of the receptor to agonist. This result distinguishes the neuronal
nicotinic receptors from the muscle nicotinic receptors, which, in contrast, show
a decrease in current in the presence of elevated extracellular calcium and
suggest a different mechanism for regulation of receptor function at synapses
in the CNS. Both the extent of regulation by external calcium and the
magnitude of the calcium permeability are determined by the particular
combination of subunits that make up the receptor. Both of these phenomena
could contribute to cholinergic neurotoxicity and could be particularly relevant
if the receptors were located presynaptically.
DISCUSSION
One consequence of the diversity of receptor subunits and of the receptors
they form is a diversity of responses to different ligands. This observation
suggests that efforts to more carefully define the properties of the ligands
that activate or block different receptors could have important results. It might
prove possible to define classes of agonists or antagonists that are exquisitely
specific for particular combinations of subunits. These ligands might have
several important uses. They might be valuable for dissecting the function
of particular cholinergic systems in the brain by providing the investigator
with sharp tools. They might also be powerful drugs to help overcome
addiction to nicotine. Alternatively, they might prove to be drugs that provide
some of the beneficial aspects of nicotine but with fewer of the harmful results
and prove useful in treating diseases in which misfunction of nicotinic function
is suspected, such as Alzheimer’s disease. This diversity of ligand binding
specificity is clearly a double-edged sword because, although it may allow the
creation of powerful tools, it may (as pointed out above) provide yet additional
drugs with abuse potential.
However, it seems reasonable that there is a window of opportunity in the
pharmacological diversity of the ligand-gated ion channels. There is the
potential for the design of drugs that would provide important access to
function in the CNS. It also seems likely that the magnitude of the window
is currently underestimated. There may be many more aspects of receptor
function that vary with subunit combination, and access to the diversity related
to these aspects may be important. For example, different receptor subunit
combinations might be differentially regulated by peptides or external ions,
allow passage of different combinations of ions, be subject to regulation by
different cytoplasmic mechanisms, or be found in different portions of the
9
neuron. An interesting result in this regard might be the presence of nicotinic
receptors in the presynaptic membrane where they might regulate release of
such neurotransmitters as dopamine or serotonin. The discovery of the
diversity of ligand-gated ion channels is recent, but the idea has been rapidly
assimilated. However, there remains the exploitation of this diversity to better
understand the brain and to design drugs to better deal with the various
diseases that affect the brain.
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Papke, R.L.; Boulter, J.; Patrick, J.; and Heinemann, S. Single channel currents
of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis
oocytes. Neuron 3:589-596, 1989.
Sakmann, B.; Methfessel, C.; Mishina, M.; Takahashi, T.; Takai, T.; Kurasaki,
M.; Fukuda, K.; and Numa, S. Role of acetylcholine receptor subunits in
gating of the channel. Nature 318:1538-543, 1985.
Schoepfer, R.; Conroy, W.; Whiting, P.; Gore, M.; and Lindstrom, J. Brain a-
bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this
branch of the ligand-gated ion channel gene superfamily. Neuron 5:35-48,
1990.
Sumikawa, K.; Houghton, M.; Smith, J.C.; Bell, L.; Richards, B.M.; and Barnard,
E.A. The molecular cloning and characterization of cDNA coding for the
alpha subunit of the acetylcholine receptor. Nucleic Acids Res 10:5809-
5822, 1982.
Takai, T.; Noda, M.; Mishina, M.; Shimizu, S.; Furutani, Y.; Kayano, T.; Ikeda,
T.; Kubo, T.; Takahashi, H.; Takahashi, T.; Kuno, M.; and Numa, S. Cloning,
sequencing, and expression of cDNA for a novel subunit of acetylcholine
receptor from calf muscle. Nature 315:761-764, 1985.
Tzartos, S.J.; Kokla, A.; Walgrave, S.L.; and Conti-Tronconi, B.M. Localization
of the main immunogenic region of human muscle acetylcholine receptor to
residues 67-76 of the alpha subunit. Proc Natl Acad Sci U S A 85:2899-
2903, 1988.
Tzartos, S.J.; Loutrari, H.V.; Tang, F.; Kokla, A.; Walgrave, S.L.; Milius,
R.P.; and Conti-Tronconi, B.M. Main immunogenic region of Torpedo
electroplax and human muscle acetylcholine receptor: Localization and
microheterogeneity revealed by the use of synthetic peptides. J Neurochem
54:51-61, 1990.
Vernino, S.; Amador, M.; Luetje, C.W.; Patrick, J.; and Dani, J. Calcium
modulation and high calcium permeability of neuronal nicotinic acetylcholine
receptors. Neuron 8:127-134, 1992.
Wada, E.; McKinnon, D.; Heinemann, S.; Patrick, J.; and Swanson, L.W. The
distribution of mRNA encoded by a new member of the neuronal nicotinic
acetylcholine receptor gene family (alpha5) in the rat central nervous system.
Brain Res 526:45-53, 1990.
12
Wada, E.; Wada, K.; Boulter, E.; Deneris, E.S.; Heinemann, P.J.; and Swanson,
L. The distribution of alpha2, alpha3, alpha4, and beta2 neuronal nicotinic
receptor subunit mRNAs in the central nervous system. A hybridization
histochemical study in the rat. J Comp Neurol 284:314-335, 1989.
Wada, K.; Ballivet, M.; Boulter, J.; Connolly, J.; Wada, E.; Deneris, E.S.;
Swanson, L.W.; Heinemann, S.; and Patrick, J. Functional expression of a
new pharmacological subtype of brain nicotinic acetylcholine receptor.
Science 240:330-334, 1988.
AUTHOR
James Patrick, Ph.D.
Professor and Head
Division of Neuroscience
Baylor College of Medicine
One Baylor Plaza
Houston, TX 77030
13
Neurotransmitter and Drug Receptor
Genes
George R. Uhl
INTRODUCTION
Understanding neurotransmitter and drug receptor genes has potential for
enhancing the molecular neurobiology of substance abuse in at least two
fashions. First, understanding the molecular mechanisms whereby drugs
and neurotransmitters affected by drugs interact with their initial biological
targets, their receptors, can enhance understanding of acute drug action.
Structure-function relationships can be approached by modifying both the
ligands and the receptors, enhancing possibilities for development of, for
example, antiabuse medications. Second, since receptor genes likely are
key to the mechanisms underlying substance abuse, they are promising
candidates for the population variants that could explain some of the individual-
to-individual differences in susceptibility to drug abuse in human populations.
Recent studies of genes encoding a novel -aminobutyric acid (GABA) receptor
and of variant dopamine D2 receptor genes in drug-using and drug-free human
populations clearly demonstrate these points.
NOVEL GABA RECEPTORS: GABA C?
Classical pharmacological, binding. and electrophysiological studies suggest
that GABA produces most of its activities through interactions with two major
receptor classes: (1) GABA A receptors, composed of subunits that form
GABA-gated chloride channels and bind bicuculline, muscimol, barbiturates,
and benrodiazepines and interact with ethanol, and (2) GABA B receptors
that are responsive to baclofen and can alter calcium fluxes. Thus, GABA A
receptors are principal sites for the action of three classes of abused
substances: ethanol, barbiturates, and benzodiazepines.
Studies of cloned GABA A receptor subunits reveal that mixtures of
and -subunits may form functional brain receptors. Variation in subunit
composition can alter the activities of the resultant receptors; the specific
14
receptor subunit profile expressed by a neuron thus determines its differential
cellular responsiveness to GABA or related drugs (Pritchett and Seeburg
1990; Luddens et al. 1990). With the diversity of the possible GABA receptors
that could be formed in vivo, the exact criteria for defining GABA A receptors
have become debatable: (1) Should classical pharmacologic criteria, such
as inhibition by the competitive GABA antagonist bicuculline, define these
receptors, or (2) should structural membership in the family of multimeric
ligand-gated chloride channels defined by cloning studies constitute the
defining feature?
Some GABA responses in visual pathways are mimicked by muscimol and
inhibited by picrotoxin but are insensitive to the competitive GABA A antagonist
bicuculline and to GABA B antagonists (Sivilotti and Nistri 1988, 1989, 1991;
Nistri and Sivilotti 1985). These receptors are also insensitive to barbiturates
and benzodiazepines. Miledi and coworkers have recently shown that GABA
responses conferred by retinal mRNA in the Xenopus oocyte system display
the same bicuculline and baclofen resistance (Polenzani et al. 1991). Based
on the pharmacologic criteria noted above, GABA C responses thus would
be neither GABA A nor GABA B. GABA responses that are insensitive to
both bicuculline and baclofen have been called GABA C by Johnston (1986).
However, electrophysiological features of GABA C responses are consistent
with their mediation by ligand-gated ion channels such as those characteristic
of GABA A receptors (Olsen and Venter 1986): Conceivably, bicuculline-
insensitive GABA responses could be conferred by a receptor highly
homologous to other known GABA A receptors.
We have recently cloned a cDNA for a receptor subunit, GABA p-1, whose
mRNA is highly expressed in retina. When expressed as a single subunit in
the Xenopus oocyte system, p-1 mRNA consistently and robustly confers
picrotoxin-sensitive GABA responses whose reversal potential indicates
changed chloride conductance. Although several GABA A receptor subunits
can form functional ligand-gated channels when expressed in various
combinations, such responses are typically variable and inconsistent unless
several subunits are coexpressed (Blair et al. 1988; Khrestchatisky et al.
1989; Shivers et al. 1990; Malherbe et al. 1990; Verdoorn et al. 1990).
GABA p-1 responses are strikingly insensitive to inhibition by bicuculline
(Shimada et al., in press). The GABA binding site of this receptor thus
shows substantial differences from the GABA binding sites on and
ß-homo-oligomeric receptors (Blair et al. 1988; Khrestchatisky et al. 1989;
Shivers et al. 1990; Malherbe et al. 1990; Verdoorn et al. 1990). This site’s
properties fit with those of the “unusual” bicuculline-resistant GABA receptor
in visual pathways, with GABA responses in Xenopus oocytes injected with
15
retina mRNA, and with the GABA C receptor defined by Johnston (Sivilotti
and Nistri 1989; Johnston 1986). Expression of the GABA p-1 cDNA as a
homo-oligomer thus creates a unique GABA binding site. When the primary
structure of this receptor cDNA is compared with other known ligand-gated
channels, its closest homology is with GABA A receptor subunits.
Nevertheless, its sequence is more divergent from the other classes of GABA
receptor subunits than they are from each other. If a GABA A receptor is
defined based on its membership in this family and based on its ability to form a
Iigand-gated channel, then the GABA p-1 receptor belongs in the GABA A
family. To the extent that GABA A receptors are defined based on a
pharmacologic feature, bicuculline sensitivity, this receptor falls into the class of
“unusual GABA receptors” or GABA C receptors (Sivilotti and Nistri 1991;
Johnston 1986).
Could this receptor be expressed as a p-1 homo-oligomer in the retina or
brain? The unique properties of the p-1 receptor are maintained when it is
coexpressed with either an or a ß-GABA A subunit (Shimada et al., in press).
Although studies defining which subunits are coexpressed by neurons are
necessary before the significance of such observations for in vivo receptor
function can be known, these results—and observations that the major
component of GABA responses obtained in Xenopus expression studies of
mRNA isolated from the retina is bicuculline insensitive—are consistent with
the p-1 subunit’s self-association in vivo.
The pharmacologic profile obtained from the expressed p-1 subunit suggests
that the resultant receptors could be termed GABA C. If further evidence
supports a self-associating role for this subunit, this role may be sufficiently
unique to demand this designation. In any case, these receptors’ properties
make them ideal molecular tools with which to investigate the features
necessary to confer ethanol, benzodiazepine, and barbiturate sensitivity on
ligand-gated chloride channel GABA receptors. By making chimeric receptors
containing specific regions of GABA A-subunits spliced onto the p-1
backbone, researchers have the opportunity to produce single subunits that
should express at high levels by themselves and gain sensitivity to barbiturates
and benzodiazepines with the addition of specific -sequences. Such
constructions will allow testing of hypotheses such as that advanced by
Pritchett and Seeburg (1990) that specific N-terminal amino acids are
important molecular features for benzodiazepine actions.
DOPAMINE D2 RECEPTOR ALLELES IN SUBSTANCE ABUSERS
Several substances that share the potential for abuse by humans also share the
ability to enhance dopamine activity in mesolimbic/mesocortical circuits thought
16
to be important for behavioral reward and reinforcement (Lippa et al. 1973;
Di Chiara and Imperato 1988; Wise and Rompre 1989). For example, cocaine’s
ability to inhibit reuptake of dopamine indicates a possible direct action for this
highly reinforcing drug in these dopaminergic circuits (Ritz et al. 1987;
Grigoriadis et al. 1989).
Blum and coworkers (1990) suggested that the “A1” allele of the dopamine
D2 receptor gene may display an association with alcoholism. This allele,
identified by a Taq I restriction fragment length polymorphism (RFLP) of the
human dopamine D2 receptor (Grandy et al. 1989), was present in 69 percent
of alcoholics but only 20 percent of nonalcoholics. However, Bolos and
colleagues (1990) found that the A1 allele frequency was not significantly
higher in 40 alcoholics than in 127 individuals from two other samples not
characterized with respect to alcohol use. A substantial genetic contribution
to susceptibility to alcoholism is supported by family, twin, and adoption
studies (Goodwin 1979; Cloninger et al. 1981; Cloninger 1987). A genetic
component of vulnerability to drug abuse is less clearly documented but
has been suggested in both twin and adoption studies (Cadoret et al. 1987;
Pickens et al. 1991). These considerations led to the examination of whether
subjects with substantial self-reported alcohol, other drug, or nicotine use also
display elevated A1 allelic frequencies.
Examining such an allelic association in drug abusers raises methodological
concerns relating to polysubstance abuse, reliability of subjects, and means
used to categorize factors such as extent of drug use and dependence.
Recognizing these difficulties and the importance of a possible association
between drug use and specific receptor gene alleles, O’Hara and colleagues
(submitted for publication) studied D2 receptor alleles in almost 400 individuals
volunteering for research protocols at the National Institute on Drug Abuse
Addiction Research Center or presenting to the Johns Hopkins hemodialysis
and genetics clinics to provide population controls.
The A1 allele was present at a higher frequency in blacks than in whites
(z=4.37, P<.00001) for all groups combined. The A3 allele was present in
15 of 206 black subjects but absent in the 225 white subjects (z=3.64, P<.001),
providing further evidence for a racial difference in D2 allelic status. No
association between ethnicity and presence of the A1 allele was found in
an analysis of Greek (7 of 25=28 percent A1 present) and Italian (8 of 25=32
percent A1 present) individuals.
Associations between A1 allele presence and drug use were examined
separately in blacks and whites. In whites, a trend toward an association
was found in comparisons of subjects with substantial (++ and +++) vs.
17
minimal (0 and +) total drug use (z=1.57, P<.06). Bartholomew’s test also
showed a trend for an increasing gradient in proportion of subjects with the A1
allele across the four levels of substance use (P<.10). Black subjects displayed
no apparent association for either comparison.
Allelic association was also examined for individual drugs. A1 allele presence
was higher for white heavy users (++ and +++) of most substances compared
with whites with the lowest overall substance use (0 on total use); none of
these differences reached statistical significance. However, since many of
the subjects used multiple substances, the comparisons are not between
independent groups. Black subjects displayed no association between A1
presence and use of any substance class.
The hypothesis that individual differences in substance abuse may be due, in
part, to different alleles of the dopamine D2 receptor gene arose from initial
work in alcoholics (Blum et al. 1990) and was strengthened by a compelling
biological rationale for interactions between abused drugs and brain dopamine
systems (Di Chiara and lmperato 1988; Wise and Rompre 1989).
The lack of strong association between D2 receptor gene alleles and
substance use evident in this study is consistent with estimates of the
heritable components of alcoholism and drug abuse (Devor and Cloninger
1989; Cadoret et al. 1987). One recent study of concordance rates for
alcoholism in twin populations suggests that between 20 and 30 percent of
the vulnerability to abuse or depend on these substances may be genetic in
origin (Pickens et al. 1991). Attempts to link familial alcohol susceptibility to
specific chromosomal markers and patterns of inheritance in families have not
been consistent with a single genetic locus (Gilligan et al. 1987; Aston and Hill
1990). The strong association between a single gene allele and alcoholism
found by Blum and coworkers (1990) would thus fit poorly with this extent of
heritability. The large environmental influences on expression of alcoholism
and Blum and colleagues’ study of unrelated individuals rather than defined
pedigrees also make the strength of their findings surprising.
The associations that Uhl and colleagues (1992) found reach only the margins
of statistical significance and are evident in only one race. Thus, they should
be approached with both interest and caution, although an association of this
magnitude would fit better with previous results concerning heritability of drug
abuse. Striking results (P<0.001) can be obtained when Uhl and colleagues’
(1992) data are combined with other published studies: 31 percent (78 of 253)
of white individuals lacking documented substance abuse and 46 percent (88 of
193) of whites with alcohol abuse/dependence or heavy substance use display
the A1 allele (Blum et al. 1990; Grandy et al. 1989; Bolos et al. 1990; Parsian et
18
al. 1991). Although alternative explanations must be considered (see below),
these results cannot rule out an association between a D2 receptor genotype
and substance use.
In contrast to the finding of a modest possible association between receptor
gene alleles and level of drug use, there was a highly significant effect of race
on allelic frequencies. The 20-percent A1 allele frequency in whites contrasts
with 37 percent for blacks. No white individual had an A3 allele, whereas the
frequency in blacks was 3 percent. These data agree with findings of higher
A1 allele presence in blacks than in whites (Blum et al. 1990) and no A3 alleles
in 167 whites (Bolos et al. 1990).
Gene allelic frequency differences among distinct white populations are well
documented for disease-related genes such as those causing thalassemia
(southern European predominance) and cystic fibrosis (northern European
predominance) (Cystic Fibrosis Genetic Analysis Consortium 1990; Orkin et
al. 1982). Population-to-population differences within Caucasian groups
presumably exist for many anonymous RFLP markers as well, although
analysis comparing Greek and Italian individuals fails to provide support
for such differences in D2 allelic distribution (O’Hara et al., submitted for
publication). The modest possible allelic association with substance use in
whites could result from disproportionate membership of heavy substance users
in ethnic groups with high A1 frequencies, but the broad populations from which
the different groups were drawn and O’Hara and colleagues’ (submitted for
publication) failure to find substantial differences in two Caucasian populations
make this hypothesis less likely.
The results of this study could be extended in several ways. Sequencing genes
cloned from A1 and A2 homozygotes could pinpoint base pair changes at the
polymorphic Taq I sites and address the possibility that other base pair changes
linked to these alleles might produce functional differences. Individuals with
A1, A2, and A3 genotypes could also be tested to identify differing physiological
and psychological responses to administered drugs. Finally, study of the
genotypes of additional white individuals with carefully ascertained ethnicity
and drug use, members of more kindreds displaying striking familial patterns
of substance use, and individuals meeting criteria for drug abuse/dependence
diagnoses could enhance the data presented here. Positive results of such
investigations would strengthen confidence that a D2 receptor allele confers
vulnerability to substance abuse and provides a biological marker allowing
targeted interventions for vulnerable individuals.
19
SUMMARY
Studies of the two neurotransmitter receptor genes described here illustrate the
rich possibilities that this area of molecular neurobiology holds for drug abuse
research. Since many of the 100 to 150 different neurotransmitters could have
multiple receptor genes, as many as 1,000 different genes or 1 percent of the
human genome might encode neurotransmitter receptors. Clearly, as the other
chapters in this monograph also indicate, the field is ripe with possibilities of
discovery with implications for drug action.
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subunits of the GABAA receptor form ion channels with properties of the
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Blum, K.; Noble, E.P.; Sheridan, P.J.; Montgomery, A.; Ritchie, T.;
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Goldman, D. Population and pedigree studies reveal a lack of association
between the dopamine D
2 receptor gene and alcoholism. JAMA 264:3156-
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Cadoret, R.J.; O’Gorman, T.; Troughton, E.; and Heywood, E. An adoption
study of genetic and environmental factors in drug abuse. Arch Gen
Psychiatry 43:1131-1136, 1987.
Cloninger, C.R. Neurogenetic adaptive mechanisms in alcoholism. Science
236:410-416, 1987.
Cloninger, C.R.; Bohman, M.; and Sigvardsson, S. Inheritance of alcohol
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Gilligan, S.B.; Reich, T.; and Cloninger, C.R. Etiologic heterogeneity in
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Goodwin, D.S. Alcoholism and heredity. Arch Gen Psychiatry 36:57-61, 1979.
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Reed, L.; Megenis, R.E.; and Civelli, O. The human dopamine D
2 receptor
gene is located on chromosome 11 at Q22-Q23 and identifies a TAQ 1
RFLP. Am J Hum Genet 45:778-785, 1989.
Grigoriadis, E.E.; Lew, A.A.W.; Sharkey, J.S.; and Kuhar, M.J. Dopamine
transport sites selectively labeled by a novel photoaffinity probe: 125l-Deep.
J Neurosci 9(8):2664-2670, 1989.
Johnston, G.A.R. Multiplicity of GABA receptors. In: Olsen, R.W., and Venter,
J.C., eds. Benzodiazepine/GABA Receptors and Chloride Channels.
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1986. pp. 57-71.
Khrestchatisky, M.; MacLennan, A.J.; Chiang, M.Y.; Xu, W.; Jackson, M.B.;
Brecha, N.; Sternini, C.; Olsen, R.W.; and Tobin, A.J. A novel a subunit in
rat brain GABAA receptors. Neuron 3:1745-753, 1989.
Lippa, A.S.; Antelman, S.M.; Fisher, A.E.; and Canfield, R.D. Neurochemical
mediation of reward: A significant role of dopamine? Pharmacol Biochem
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Luddens, H.; Pritchett, D.B.; Kohler, M.; Killisch, I.; Keinanen, K.; Monyer, H.;
Sprengel, R.; and Seeburg, P.H. Cerebellar GABAA receptor selective for
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Malherbe, P.A.; Draguhn, A.; Multhaup, G.; Beyreuther, K.; and Mohler, H.
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and Cloninger, C.R. Alcoholism and alleles in the human D
2 dopamine
receptor locus. Arch Gen Psychiatry 48:655-663, 1991.
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Clayton, P.J. Heterogeneity in the inheritance of alcoholism: A study of
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21
Polenzani, L.; Woodward, R.M.; and Miledi, R. Expression of mammalian
aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes.
Proc Natl Acad Sci U S A 88:4318-4322, 1991.
Pritchett, D.S., and Seeburg, P.H. -Aminobutyric acid, receptor -subunit
creates novel type II benzodiazepine receptor pharmacology. J Neurochem
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dopamine transporters are related to self-administration of cocaine. Science
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Shimada, S.; Cutting, G.; and Uhl, G. -Aminobutyric acid A or C receptor?
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P.R.; and Seeburg, P.H. Two novel GABAA receptor subunits exist in distinct
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Sivilotti, L., and Nistri, A. Complex effects of baclofen on synaptic transmission
of the frog optic tectum in vitro. Neurosci Lett 85:249-254, 1988.
Sivilotti, L., and Nistri, A. Pharmacology of a novel effect of -aminobutyric acid
on the frog optic tectum in vitro. Eur J Pharmacol 164:205-212, 1989.
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system. Prog Neurobiol 36:35-92, 1991.
Uhl, G.R.; Persico, M.D.; and Smith, S.S. Current excitement with D
2 dopamine
receptor gene alleles in substance abuse. Arch Gen Psychiatry 49:157-160,
1992.
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Functional properties of recombinant rat GABAA receptors depend upon
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40:191-225, 1989.
AUTHOR
George R. Uhl, M.D., Ph.D.
Chief
Laboratory of Molecular Neurobiology
Addiction Research Center
National Institute on Drug Abuse
Associate Professor of Neurology and Neuroscience
Johns Hopkins University School of Medicine
P.O. Box 5180
Baltimore, MD 21224
22
The Diversity of the Dopamine
Receptors
Olivier Civelli, James R. Bunzow, Qun-Yong Zhou, and David K.
Grandy
INTRODUCTION
Among all the neuronal pathways, the dopamine system is thought to have the
leading role in modulating drug addiction. This system relies on the interaction
of one neurotransmitter, dopamine, with several receptors.
Classically, dopamine was thought to exert its effects by binding to only two
G protein-coupled receptors, known as the D1 and D2 receptors (Kebabian
and Calne 1979). These receptors have been differentiated pharmacologically,
biologically, and physiologically and also by their anatomical distribution.
These two receptors exert their biological actions by coupling to and activating
different G protein complexes. The D1 receptor interacts with the Gs complex
to activate adenylyl cyclase, whereas the D2 interacts with Gi to inhibit cyclic
adenosine monophosphate (CAMP) production.
CLONING OF THE D2 DOPAMINE RECEPTOR
The cloning of the D2 dopamine receptor resulted from the use of a strategy
based on the sequence homology expected to exist among G protein-
coupled receptors. The ß2-adrenoreceptor coding sequence was used as
a hybridization probe to screen a rat genomic library under low-stringent
hybridization conditions. By screening the equivalent of three genomes,
90 positive clones were identified, from which 20 were characterized and
partially sequenced. This allowed the characterization of the clones encoding
the rat ß1-adreno (Machida et al. 1990), the serotonin 1a (Albert et al. 1989),
the muscarinic 4 (J.R. Bunzow and O. Civelli, unpublished observation), and
another clone, RGB-2, which, as described below, encodes the dopamine D2
receptor (Bunzow et al. 1988).
The RGB-2 clone was used to screen a rat brain cDNA library. One positive
clone, containing a 2.5-kb insert, was sequenced, and its corresponding
23
peptide sequence was determined. This clone encodes a 415-amino acid
protein with all the expected characteristics of a G protein-coupled receptor:
It has seven hydrophobic domains, 21 amino acid residues that are conserved
among all cloned G protein-coupled receptors and potential glycosylation and
phosphorylation sites, and a significant degree of sequence similarity with the
other receptors in this gene family.
The full-length RGB-2 cDNA was cloned into a plasmid containing the
metallothionein promoter, and this construct was cotransfected with pRSVneo
(a selectable marker conferring resistance to the antibiotic neomycin) into
mouse Ltk-cells. Stable transfectants were prepared and analyzed for their
ability to bind dopamine ligands (Bunzow et al. 1988). L-RGB2Zem-1
membranes bound D2 dopamine agonists and antagonists with the same
pharmacological profile as do rat striatal membranes. These studies used
the antagonist [3H] spiperone, whose binding was shown to be saturable and
of high affinity. [3H] spiperone binding to L-RGB2Zem-1 membranes was
displaced by several antagonists with the stereospecificity expected of a D2
receptor and with the same Kis as determined in rat striatal membranes.
D2 receptors are present on lactotroph cells of the anterior pituitary where
they regulate prolactin (PRL) secretion. The somatomammotroph cell line
GH4C1 is derived from a rat pituitary tumor, is devoid of endogenous dopamine
receptors, and is known to secrete PRL. GH4C1 cells were transfected with
the RGB-2 cDNA metallothionein construction. One clone, GH4ZR7, was
found to express high levels of RGB-2 mRNA (Albert et al. 1989). Since
the D2 receptor is expected to inhibit CAMP levels, vasointestinal peptide
(VIP) was used to stimulate endogenous CAMP production. Dopamine
inhibited both basal and VIP-stimulated CAMP levels in media from GH4ZR7
cells. Intracellular CAMP levels were also inhibited. The stereospecificity of
these inhibitions was demonstrated using isomers of sulpiride: The active
enantiomer (-)sulpiride blocked the inhibition, whereas (+)sulpiride had no
effect. To demonstrate that the changes in CAMP levels were the result of
an inhibition of adenylate cyclase, dopamine was added to membranes of
VIP- or forskolin-stimulated GH4ZR7 cells, and adenylate cyclase activity was
measured. Dopamine inhibited adenylate cyclase activity in a stereoselective
manner. Finally, the inhibition of PRL secretion by dopamine was assayed
in GH4ZR7 cells. VIP and thyrotropin-releasing hormone (TRH) are known
to enhance PRL release by a cAMP-dependent and a cAMP-independent
mechanism, respectively. Dopamine was able to inhibit PRL secretion
stimulated by both hormones. These inhibitions were reversed by the active
antagonist (-)sulpiride but not by (+)sulpiride. Therefore, the RGB-2 cDNA
encodes a D2 dopamine receptor that is functional.
24
CLONING OF THE DOPAMINE D1 RECEPTOR
The success of the homology approach in D2 receptor cloning opened the
door for the cloning of other dopamine receptors, in particular the D1 receptor.
The authors and colleagues took advantage of the polymerase chain reaction
(PCR)-based approach, which had been developed to clone several thyroid
G protein-coupled receptors (Libert et al. 1989). This approach consists of
synthesizing two sets of synthetic oligonucleotides that correspond to two
highly conserved regions among all the G protein-coupled receptors (found
generally in transmembrane domains III and VI). These oligonucleotides
are used as primers in a PCR reaction for specific amplification of cDNAs
containing complementary sequences. The cDNAs used for the D1 receptor
cloning were synthesized from rat striatum. To direct the PCR approach
toward the specific cloning of the D1 receptor, we added another technical
step. Because it is known that Gs-coupled catecholamine receptors have a
putative third cytoplasmic loop of 52 to 78 amino acids (Zhou et al. 1990), our
total population of PCR products was size fractionated, and products in the
expected range were sequenced. Of 24 PCR products, 7 encoded potential G
protein-coupled receptors, one of which showed structural features expected to
belong to Gs-coupled catecholamine receptors. This clone was used to screen
a rat cDNA and human and rat genomic libraries since most catecholamine
receptor genes lack introns in their coding regions (Lefkowitz et al. 1988).
The isolated clone was sequenced and shown to contain all the characteristics
expected of G protein-coupled receptors (i.e., share the highest degree of
similarity and prototypical structural features of the catecholamine receptors).
The absence of a glutamic residue found in the third transmembrane
domains of all the ß-adrenoreceptors and the size of the third cytoplasmic
loop suggested that the cloned receptor could be a Gs-coupled dopamine
receptor, namely the D1 receptor.
The demonstration that the cloned receptor was the D1 receptor was
accomplished by expressing the corresponding gene. First, the putative
D1 receptor human gene was expressed by transient expression in COS-7
cells. Membrane proteins from the transfected cells were tested for their
ability to bind D1 receptor ligands. The specific antagonist SCH 23390 was
found to have the highest affinity for the cloned receptor, and the overall
pharmacological profile was that of a D1 receptor binding site. The biological
activity of the cloned receptor was studied upon transient transfection in human
kidney 293 cells and analysis of dopamine stimulation of adenylyl cyclase
activity. The cloned receptor was shown to stimulate adenylyl cyclase activity
according to a pharmacological profile expected for the D1 receptor. Therefore,
we concluded that we had cloned the D1 dopamine receptors.
25
DIVERSITY OF THE DOPAMINE RECEPTORS
As discussed above, pharmacological analyses had agreed on the existence of
only two dopamine receptors (Hess and Creese 1987; Creese 1986; Leff and
Creese 1985). With the cloning of these two receptors, new tools were at hand
to further understanding of the dopamine system. In 2 years, several studies
using molecular biological approaches have shown that the classical view of the
dopamine system was incomplete.
Two Forms of the D2 Receptor
The discovery that not one but two dopamine D2 receptor forms exist was
reported in 1989 (Selbie et al. 1989; Grandy et al. 1989; Dal Toso et al. 1989;
Giros et al. 1989; Monsma et al. 1989; Chio et al. 1990; Miller et al. 1990;
O’Malley et al. 1990) and showed that the two D2 receptor forms exist in
human, rat, and bovine cells. These two forms differ in 29 amino acid residues
located in the putative third cytoplasmic loop of the receptor. The short form is
the one originally cloned (as described above); the long form is new and was
discovered either by screening cDNA libraries or by PCR analyses.
Several data were obtained about the 29 amino acid addition. First, the
additional residues do not modify the affinity or the profile of the D2 receptor
for antagonists (Grandy et al. 1989; Giros et al. 1989). Second, they do not
affect significantly the ability of the receptor to inhibit CAMP production (Dal
Toso et al. 1989) as could have been expected from their location in the third
cytoplasmic loop. Third, it was found that the 29-amino acid addition contains
two potential glycosylation sites, but thus far, nothing is known about their
importance (Grandy et al. 1989). Fourth, it was found that although the two
forms of the D2 receptor coexist in all tissues analyzed, their ratio varies. The
short form is the least abundant; its concentration is very low in the pituitary but
represents about half of the D2 receptor mRNA in the pons or medulla (Giros et
al. 1989; O’Malley et al. 1990). Fifth, the generation of the two forms of D2
receptor was shown to be the result of an alternative splicing event occurring
during the maturation of the D2 receptor pre-mRNA (Grandy et al. 1989; Dal
Toso et al. 1989; O’Malley et al. 1990), which was demonstrated by the
discovery of an 87-bp exon encoding the additional amino acid residues.
These studies also led to the description of the organization of the D2 receptor
gene, with the coding part of the D2 receptor encoded by seven exons, one of
which (exon 5 in Grandy et al. 1989) is alternatively spliced.
In summary, the two D2 receptor forms have not been shown to differ in their
pharmacological or biological activities. They are generated by alternative
splicing and coexist in a tissue-specific ratio. Any differences in their biological
significance have yet to be demonstrated.
26
New Dopamine Receptors
Probably the most striking discovery to emerge from the cloning of G protein-
coupled receptors is their diversity. Every class of G protein-coupled receptor
studied by recombinant DNA techniques has been proven more complex than
had been characterized pharmacologically (Bonner et al. 1987; Schwinn et al.
1990; Emorine et al. 1989). In view of the diversity of physiological responses
modulated by dopamine receptors, there was ample reason to believe that the
dopamine receptor class would also follow this trend. The success of the
homology approach in cloning the D2 receptor and the availability of dopamine
receptor probes led to the search for new, undescribed dopamine receptors.
D3 Receptor. Through the combination of cDNA library screening, PCR
extension, and genomic library screening, a cDNA was isolated that encodes
for a novel receptor related to the D2 receptor, thereafter coined the D3
receptor (Sokoloff et al. 1990). This receptor shares 75 and 41 percent of its
putative transmembrane sequences with the D2 and D1 receptors, respectively.
Moreover, it is encoded by a gene that contains five introns in its coding region
and whose organization is similar to that of the D2 receptor. The D3 receptor
structure is also highly similar of that of the D2 receptor, in that it contains a
large third cytoplasmic loop of similar size to that of the long form of the D2
receptor. However, the organization of the gene in this loop does not allow for
alternatively spliced forms. The D3 receptor also contains the residues found
to be important for catechol and amine groups recognition in the catecholamine
receptors. Altogether, the structure of the D3 receptor suggested a close
relationship to the D2 receptor, which was confirmed by pharmacological
analyses.
When expressed in eucaryotic cells (COS-7 or CHO cells), the D3 receptor
was shown to have a pharmacological profile reminiscent of that of the D2
receptor. It binds D2 ligands (not D1 or other catecholamine ligands), although
its affinity to most neuroleptics was 10- to 100-fold less than that of the D2
receptor. However, the D3 receptor was found to bind two particular
antagonists, (+)AJ76 and (+)UH232, with three to five times more affinity
than the D2 receptor. These antagonists are thought to have a higher
specificity for the dopamine presynaptic receptors or autoreceptors.
Interestingly, the binding of dopamine to the D3 receptor was not affected
by the addition of guanylnucleotides, which block G protein-coupled receptors
in their high affinity state and are used to measure G protein coupling. This
result might be explained by the absence of suitable G proteins in the
transfected cells (COS-7 and CHO cells) or by a low modulation of dopamine
binding by guanylnucleotides at the D3 receptor. The latter was not shown to
modulate CAMP formation in CHO cells, in contrast to the other dopamine
receptors.
27
The tissue distribution of the D3 receptor was determined by Northern blot and
in situ hybridization analyses. It not only was found to overlap but also to differ
from that of the D2 receptor. The D3 receptor is absent in the pituitary and is
expressed at low levels in the neostriatum, whereas high densities of the D2
receptor are present in both. The distribution of the D3 receptor overlaps with
the D2 receptor in the olfactory tubercules and the hypothalamus. Moreover,
the D3 receptor is expressed at high levels in the islands of Calleja and in the
nucleus accumbens, regions that are part of the limbic system and where the
concentrations of D2 receptor are relatively lower. In addition, the D3 receptor
was found to colocalize with the D2 receptor in presynaptic cells that produce
dopamine in the substantia nigra and in the ventrotegmental areas.
D4 Receptor. By analyzing the mRNA population of the neuroepithelioma
SK-N-MC cells with D2 receptor cDNA probes under conditions of low
stringency, the existence of a D2-related mRNA was detected (Van Tol et al.
1991). The corresponding cDNA and gene were sequenced and found to
encode another novel dopamine receptor, the D4 receptor. In its putative
transmembrane domains, the D4 receptor is 41, 52, and 51 percent identical
to the D1, D2, and D3 receptors, respectively, and it contains the residues
necessary for catecholamine recognition. Its putative third cytoplasmic loop
is shorter than that of the D2 receptor. At the genomic level, the D4 coding
sequence is separated by four introns that are positioned similarly to those
of the D2 receptor. In addition, a 52-bp repeat borders both sides of the third
intron. This unusual intron-exon junction does not contain conventional splice
sites and allows for potentially variable alternative splicing events without
changes in protein sequence.
The D4 receptor was expressed in COS-7 cells, and its pharmacological
profile was determined. Most of the tested agonists and antagonists displayed
affinities for the D4 receptor similar to or lower than that for the D2 receptor.
However, the D4 receptor binds one particular antipsychotic, clozapine, with
an affinity tenfold higher than either the D2 or D3 receptors.
Clozapine is a particularly interesting antipsychotic agent whose action is not
associated with the motor control side effects that plague other neuroleptics.
However, clozapine has its own side effects. Most deleterious is that it causes
agranulocytosis in a few cases, which has prevented its general use. In any
case, the discovery of a dopamine receptor specific for a neuroleptic that
might not affect the centers for motor control in the central nervous system is
important. A preliminary analysis of the D4 receptor tissue location indicated
that the D4 receptor is expressed in the mesocorticolimbic system rather than in
the nigrostriatal systems (Van Tol et al. 1991), adding credit to the hypothesis
that stimulation of this receptor has little impact on control of movement. These
28
observations suggest that the D4 receptor is most likely the primary target
mediating the antipsychotic action of clozapine.
Other Dopamine Receptors. There are indications that the diversity in
dopamine receptors does not stop at the D4 receptor. The existence of
another D2-like receptor has been reported (Todd et al. 1989). This receptor
was expressed upon transfection of rat genomic DNA into mouse fibroblasts
and was identified by its ability to bind iodinated spiperone. This receptor
has a pharmacological profile closely resembling that of the cloned D2 receptor,
yet it has a sequence different from that of the cloned D2 receptor as shown by
the inability of its mRNA to hybridize to three sets of oligonucleotides specific to
the D2 sequence. Stimulation of this receptor by dopamine leads to an
increase in intracellular calcium concentrations that appears not to be mediated
through G protein coupling.
Other D1-like receptors have also been described. D1-like receptors have
been detected in renal tissue (Felder et al. 1989). These are linked to the
activation of phosphatidylinositol-specific phospholipase C. These D1-like
receptors are different from the cloned D1 receptor because the latter is not
expressed in renal tissue (Zhou et al. 1990; Dearry et al. 1990). A D1-like
receptor that couples to inositol phosphate production has also been detected
by expression in Xenopus oocytes (Mahan et al. 1990). This receptor is
encoded by a mRNA found in the rat striatum but is of different size than the
one encoding the cloned D1 receptor.
There are also indications that yet other dopamine receptors might exist.
Pharmacological and biological data discussing the existence of putative
receptors have been discussed (Andersen et al. 1990). Moreover, several
laboratories have recently cloned a new D5 or D1b receptor (D.G. Grandy,
personal communication, 1991; H.H.M. Van Tol, personal communication,
1991; M.G. Caron, personal communication, 1991).
CONCLUSIONS AND PERSPECTIVES
Based on pharmacological studies, the dopamine receptors have been
classified into two subtypes, the D1 and D2 receptors. The cloning of the
dopamine receptors has revealed that, although the number of dopamine
receptors is larger than expected, they can still be classified under the
broad categories of D1- and D2-like receptors. The D1-like receptors are
encoded by genes that have no intron in their coding sequence, share more
than 50 percent identity with the D1 receptor in the sequences part of the
transmembrane domains, have a pharmacological profile that resembles that
of the D1 receptor and that binds efficiently the antagonist SCH 23390, and
29
stimulate adenylyl cyclase activity. The D2-like receptors have similar
characteristics when compared with the D2 receptor—they efficiently bind
spiperone and inhibit adenylyl cyclase activity.
The complexity of the dopamine receptor family is not surprising in view of the
complexity of the other receptor families that are part of the G protein-receptor
supergene family (Bonner et al. 1987; Schwinn et al. 1990; Emorine et al.
1989). How many dopamine receptors will be found is unknown. However, as
presently understood, there are two widespread and quantitatively predominant
dopamine receptors, the classical D1 and D2 receptors. The other dopamine
receptors are present in significantly lower amounts in restricted localizations,
and they can be related to the two major dopamine receptors through their
pharmacological profile. Therefore, most of what has been known about
dopamine agonists’ and antagonists’ actions has to be reevaluated in view of
the existence of the different dopamine receptors. This underscores the impact
that the cloning of the D2 receptor will have on the field of dopamine receptors.
Finally, the discovery of new dopamine receptors is also interesting from an
evolutionary point of view. One neurotransmitter interacts with a variety of
receptors, suggesting that nature has spent a large effort in developing the
targets in mechanism of synaptic transmission, a fact that could increase
understanding of higher brain function.
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ACKNOWLEDGMENT
This work was supported by National Institute of Mental Health grant
MH-45614.
AUTHORS
Olivier Civelli, Ph.D.
Associate Professor
Department of Cell Biology and Anatomy
Scientist
Vollum Institute for Advanced Biomedical Research
James R. Bunzow, M.S.
Senior Research Associate
Vellum Institute for Advanced Biomedical Research
32
Qun-Yong Zhou, Ph.D.
Department of Molecular Biology and Biochemistry
David K. Grandy, Ph.D.
Senior Research Associate
Vollum Institute for Advanced Biomedical Research
Oregon Health Sciences University
Portland, OR 97201
33
Cloned Dopamine Receptors: Targets
in Therapy of Drug Abuse
Philip Seeman
INTRODUCTION
Most self-rewarding behaviors operate through the brain dopamine system.
This generalization also holds for drug abuse. For example, many common
drugs of abuse, including amphetamine, cocaine, LSD, nicotine, ethanol, and
opiates, promote the release of dopamine in vivo (Di Chiara and lmperato
1988). Moreover, the acute overdoses of some of these drugs of abuse (e.g.,
amphetamine, cocaine, or LSD, but not ethanol or opiates) often are treated
by dopamine receptor-blocking medications such as haloperidol or other
neuroleptics.
EFFECTIVE THERAPY TARGETS: TRANSPORTERS OR RECEPTORS?
In clinical psychiatry and neurology, most medications that are used to alleviate
various illnesses block neurotransmitter transporter sites or receptor sites. In
general, the more effective medications are those that block receptors directly.
Medications that block the transmitter uptake sites (or transporter sites),
although useful, generally have been less effective clinically than those that
block or stimulate receptors directly.
For example, imipramine or chlorimipramine are used in the treatment of
clinical depression. These compounds inhibit the uptake or transport of
norepinephrine into nerve terminals. These medications generally require
about 2 or 3 weeks before clinical improvement occurs. Moreover, the
magnitude of the overall efficacy of these medications is unclear, because
many studies indicate that admitting a depressed patient into a hospital
alleviates depression in 60 percent of patients. This compares with about
70 percent improvement when using both medication and hospitalization of
depressed patients.
The mechanism thought to explain the action of these antidepressant drugs is
that, by inhibiting the uptake of norepinephrine, the drugs increase or prolong
34
the release of the transmitter, which in turn leads to a reduction of
ß-adrenoceptors in the postsynaptic neurones. Thus, the ß-adrenoceptors
are indirectly and slowly affected by the transporter-inhibiting drugs.
A more rapid and effective antidepressant action may be obtained by
stimulating the ß-adrenoceptors directly by means of ß-adrenoceptor agonists.
Although this agonist-type strategy has been found experimentally to be
rapid and effective in alleviating depression, the cardiovascular side effects
currently preclude routine treatment by agonist therapy.
However, fluoxetine is an important example of a successful transporter
blocker that alleviates depression relatively rapidly in about one-third of
depressed patients. The effect becomes apparent within 1 week of treatment.
A second example of presynaptic therapy affecting the transporter site is in
Parkinson’s disease. Dopamine uptake inhibitors, including benztropine,
are no longer used in treating this illness. However, agonists that stimulate
dopamine D1 or D2 receptors are effective in Parkinson’s disease.
Presynaptic receptor therapy is also less effective clinically. For example,
compounds such as BHT 920 and 3-PPP act on presynaptic dopamine
receptors to inhibit the release of dopamine. Although such inhibition
does confer neuroleptic-like effects clinically (e.g., against psychosis or
schizophrenia), the clinical action alleviates only about 40 percent of psychotic
patients, compared with an improvement of 35 percent of such patients without
neuroleptics, and approximately 70- to 90-percent improvement with dopamine
receptor-blocking neuroleptics.
GOAL: TARGETING DIFFERENT DOPAMINE RECEPTORS IN BRAIN
PSYCHOMOTOR REGIONS
Although receptors are effective clinical targets, recent medications are not
sufficiently receptor selective to yield clinical actions free of unwanted side
effects. The recent discoveries of multiple receptors for each transmitter now
permit the design and development of medications selective for a specific
subtype of receptor.
Thus, designing a neuroleptic that selectively targets D2-like receptors in
nonmotor brain regions will obviate motor side effects of such a neuroleptic in
psychotic patients. This appears to be the case with clozapine, a neuroleptic
that is selective for the D4 dopamine receptor (Van Tol et al. 1991).
35
TYPES OF DOPAMINE RECEPTORS
The first type of dopamine receptor reported, now termed D1, was identified by
its ability to respond to dopamine and to stimulate adenylate cyclase (Kebabian
et al. 1972). The DNA for this receptor has been cloned (Sunahara et al. 1990;
Zhou et al. 1990; Dearry et al. 1990); the amino acid sequence of D1 is shown
in figure 1.
The second type of dopamine receptor, now termed D2, was identified by its
affinity for nanomolar concentrations of antipsychotic drugs (Seeman et al.
1974, 1975a, 1975b, 1976, 1984, 1987; Titeler et al. 1978; Seeman and
Niznik 1990). The DNA for D2 has been cloned (Bunzow et al. 1988; Grandy
et al. 1989; Martens et al. 1991); the amino acid sequence for D2 is shown in
figure 1.
Additional dopamine receptors have been found recently by homology probing,
that is, by probing genomic DNA or cDNA with oligonucleotides containing
bases similar or identical (homologous) to D1, D2, and related catecholamine
receptors.
This approach yielded short and long forms of D2 (Giros et al. 1989; O’Dowd
et al. 1990), a D3 dopamine receptor (Sokoloff et al. 1990; Giros et al. 1990),
a D4 dopamine receptor (Van Tol et al. 1991), and a D5 dopamine receptor
(Sunahara et al. 1991).
In addition, this approach yielded truncated forms of these receptors, such as
a D3 receptor with its third transmembrane portion deleted and a D3 receptor
with its second outer (or extracellular) portion deleted (Giros et al. 1991). The
sequences of these truncated D3 receptors, named D3 (TM-del) and D3 (O2-
del), respectively, are shown in figure 1.
Moreover, the method of homology probing has revealed at least two human
pseudogenes of D5 (Nguyen et al. 1991).
Of the amino acid sequences shown in figure 1, 13 amino acids are
homologous in at least 75 various membrane-located receptors (for various
amines, peptides, and hormones) that are all GTP sensitive. These 13 amino
acids, which are common to all the G-linked receptors, are illustrated by arrows
in figure 2 and by solid black triangles in figure 1.
36
FIGURE 1. Amino acid sequences of dopamine receptors. D1 human is from Sunahara et al. 1990; D1 rat from
Zhou et al. 1990; D2 human from Grandy et al. 1989 and Dal Toso et al. 1989; D2 rat from Bunzow et al.
1988; D2 frog from Martens et al. 1991; D3 human from Giros et al. 1990; D3 rat from Sokoloff et al.
1990; D3 rat (with transmembrane segment 3 deleted) and D3 rat (with extracellular segment 2 deleted)
from Giros et al. 1991; D4 human from Van Tol et al. 1991; and D5 human from Sunahara et al. 1991.
The solid black triangles indicate the 13 amino acids that are found to be identical (homologous) in more
than 75 G-linked receptors for various amines, peptides, and hormones.
FIGURE 1. continued
FIGURE 1. continued
FIGURE 2. Illustrating the rat dopamine D2 (short) receptor within the
membrane. The arrows indicate the 13 amino acids that are
homologous in more than 75 G-linked receptors for amines,
peptides, and hormones. Dopamine is assumed to attach its
hydroxyl groups to the serine residues in the fifth transmembrane
segment.
CLONED DOPAMINE RECEPTOR PHARMACOLOGY
The sensitivities of the various dopamine receptors to agonists and antagonists
are listed in table 1. For each of these sites to be termed a dopamine receptor,
it is essential that dopamine be the most potent endogenous transmitter to
inhibit binding; otherwise, the site is not a dopamine receptor, by definition.
All the binding sites shown in the table meet this criterion, because epinephrine,
norepinephrine, and serotonin were all weaker than dopamine in inhibiting the
binding of each radioligand.
40
TABLE 1. Sensitivities of dopamine receptors to agonists and antagonists
Various
Human
Species
Rat Human
Rat
Human
Site or Clone
D1
Tissue D2
D2 short D2 long D2 short
D2 long
D3
D4 hybrid D5
Tissue/cell Cos-77,13,14 Striatum Ltk-1,8
CHO
293 cell Ltk-LT8,12
CHO
Cos-7 Cos-714
ant. pit.6GH4ZR72.5
Cos-7
9
Ligand *Sch239827
*Spip
*Sch2339013,14
K, nM
Agonists
K, nM
Cos-7
11
293
10
*Spip1.2 †l-Sul
*Spip *Dom
8†
I-Sul
*Spip12 *Sch2339014
*Spip5
*Spip
12
*Dom8
*Rac
9
†
I-Sul
11
*Spip
10
K, nM K, nM K, nM K, nM K, nM K, nM
K, nM
ADTN-(+)
Apomorphine
Bromocriptine
Dopamine
Dopamine+GN
Epinephrine
Fenoldopam
(-)Norepinephrine
NPA
Pergolide
PHNO-(+)
Quinpirole-(-)
Serotonin
SKF 38393
SKF 76783
4,60014
2107
2,34014
>55,0007
20;60
50,00013
1,81614
1,36314
14,0007
9,69014
877
64514
Hi:1.76
Hi:~26
1.9
6
Hi:7.56
Hi:2.86
~6,0006
Hi:0.46
243
250
9
1455.3312.612 203
7.43
Hi:7311 Hi:2.811 24,8009253
17,00011,7053273
Hi:1.26
Hi:4.86
~10,0006
Hi:1576
2130.623
57635.13
9,56035,0003
Hi:24
909
Hi:4.1
363
340 454
Hi:28 228
Hi:49-450
321 15
1,760 12,000
6.5
1,136
918
79
46 >20,000
4,180 3,000
1,800 100
530
TABLE 1. continued
Site or Clone
Antagonists
Butaclamol-(+)
Chlorpromazine
Clozapine
Eticlopride
Fluphenazine
Haloperidol
*lodosulpnde
Ketanserin
Pimozide
Prochlorperazine
Radopride
*Radopride
Remoxipride
SCH 23390
SKF 83566
Spiperone
*Spiperone
Sulpiride-S
Do.
Sulpiride-R
Thioridazine
YM-09151-2
*YM-09151-2
Human
D1
Various
Species Rat
Tissue D2 D2 short D2 long
0.9;3
7314
141
14
18,00014
2114
2714
190
7
>72,0007
0.1;0.4
0.314
2207
36,00014
20,45414
100
14
0.96
36
100±204
0.096
1±0.55
1.5±0.54
2086
46
24
1.94
300±904
1,6906
0.076
0.056
186
15±54
0.066
0.950.812
2.83
1.5
9
168556313812
0.8-3 0.5-0.8
0.613
>1,0001
2.43
4.73
1.651.83
7148
0.09620.0693
0.095
5.511 9.23
15.954.811
115
11
103
11
3.33
0.095
Human
Rat
Human
D2 short D2 long
D3
D4 hybrid
D5
18
10.512
3.29
9138
0.0538
055
10
.0510
469
3112
0.0912
6.13
1803
9.83
1.23
3.73
353
3.53
0.613
253
4223
7.83
40
27
37
133
9
250
2.1
19,000
46
14
5.1
48
148
43 2,500
237
3,690
3,560
0.06
0.08
52
0.3
0.4
4,500
77,270
12
0.09
28,636
300
TABLE 1. continued
KEY: *=tritium; †l-Sul=[1251]iodosulpiride; ant. pit.=anterior pituitary; GN=guanine nudeotide; Dom=domperidone; Halo=haloperidol; Hi=high-
affinity state of D2; hybrid=hybrid of gene and cDNA from SKNMC neuroblastoma; NPA=N-propylnorapomorphine; Rac=raclopride;
Spip=spiperone
1Bunzow et al. 1988
2Albert et al. 1990
3Sokoloff et al. 1990
4Seeman 1990
5P. Seeman, unpublished data
6Seeman and Niznik 1988
7Dearry et al. 1990
8Grandy et al. 1989
9Stormann et al. 1990
10Dal Toso et al. 1989
11Giros et al. 1989
12Van Tol et al. 1991
13Sunahara et al. 1990
14Sunahara et al. 1991
The D1-like receptors are D1 and D5. They are approximately equally sensitive
to all agonists except dopamine. The dopamine dissociation constant (K value)
shown in the table for D1 is 2,340 nM and for D5 is 228 nM. Both these values
refer to the low-affinity state of D1 and D5, because under these experimental
conditions the Cos-7 cells did not reveal the high-affinity state. Because D5
is approximately 10 times more sensitive to dopamine, this suggests that D5 is
more readily activated than D1 in vivo. Thus, D5 may provide the major
background tone in the nervous system, or it may be recruited during massive
discharges of dopamine from nerve terminals, as might occur during episodes
of drug abuse.
Although the expressed D1 and D5 receptors did not reveal the high-affinity
state in tissue culture under these conditions, it is known that the high-affinity
state of D1 in brain tissue has a K of about 0.7 nM for dopamine, compared
with a value of about 10 nM for the high-affinity state of D2 for dopamine. Thus,
the D1/D5 receptors are about one order more sensitive to dopamine than the
D2-like receptors (that is, D2, D3, and D4) (see also Gehlert et al. 1992). The
reason for emphasizing this point is that the high-affinity state of these receptors
is the functional state (George et al. 1985).
As exemplified for clozapine in the table, this drug has about a tenfold higher
affinity for D4 than for D2 or D3. This supports the principle of developing
receptor-selective medications in the future.
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Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Bunzow, J.R.; Server,
A.C.; and Civelli, O. Cloning of the cDNA and gene for a human D2
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4
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ACKNOWLEDGMENT
This chapter was prepared with support from National Institute on Drug Abuse
grant DA-07223-01.
46
AUTHOR
Philip Seeman, M.D., Ph.D.
Professor
Departments of Pharmacology and Psychiatry
University of Toronto
Medical Sciences Building
Eight Taddlecreek Road
Toronto, Ontario M5S 1A8
CANADA
47
Cannabinoid Receptors: Which Cells,
Where, How, and Why?
Lisa A. Matsuda, Tom I. Bonner, and Stephen J. Lolait
INTRODUCTION
In both laboratory animals and humans exposed to marijuana or
cannabimimetic compounds, the majority of effects appear to be mediated
by the central nervous system. Although -tetrahydrocannabinol
-THC)
has been known to be the primary psychoactive component of marijuana
since the mid-1960s (Gaoni and Mechoulam 1964), a receptor-mediated
mechanism for marijuana’s actions has been recognized only within the past
several years (Howlett et al. 1990). The existence of a cannabinoid receptor
implies that an endogenous “cannabinoid” ligand also must exist. However,
the identity of this ligand is unknown. In addition, a lack of readily available
cannabinoid receptor antagonists has further hindered the elucidation of the
physiological and pathological significance of cannabinoid receptors. Recent
cloning and identification of a cDNA that encodes a G protein-coupled
cannabinoid receptor not only provides solid evidence for a cannabinoid
receptor protein but also provides nucleic acid sequence information that
can be used to localize the mRNA for this receptor (Matsuda et al. 1990).
By using the technique of in situ hybridization histochemistry (ISHH), the
neuroanatomical localization of cannabinoid receptor mRNA identifies
populations of brain cells that potentially could mediate cannabinoid-induced
effects. Furthermore, comparing the localization of the mRNA for the receptor
with the localization of cannabinoid receptor proteins (Herkenham et al. 1991a)
reveals clues regarding the circuitry of the cells expressing these receptors.
LOCALIZATION OF RECEPTOR mRNA IN RAT BRAIN
The expression of the cannabinoid receptor gene was visualized in coronal
tissue sections of adult rat brain using ISHH with a 35S-tailed 48-base
oligonucleotide probe, SKR6-1. This probe complements sequence from the
rat cDNA, SKR6 bases 349-396 (Matsuda et al. 1990). In transfected cells,
this cDNA encodes a receptor that mimics the cannabinoid receptor previously
characterized in both neural cell lines and brain tissues in vitro (Howlett et al.
48
1986; Bidaut-Russell et al. 1990). In response to -THC and CP 55940,
a potent synthetic nonclassical cannabinoid analog, cyclic AMP (CAMP)
production in SKR6-transfected cells decreases in a dose-dependent,
stereoselective, and pertussis toxin-sensitive manner (figure 1) (Matsuda et al.
1990). Moreover, the gene for the human homolog of this receptor encodes a
protein that binds 3H-CP 55940, a potent, synthetic cannabinoid analog, in a
saturable and specific manner (Felder et al. 1991).
In sections from adult rat brain, a regionally specific and unique localization
pattern for the cannabinoid receptor mRNA was found (Matsuda et al.,
submitted for publication). In general, labeling intensities were highest in
forebrain regions (olfactory areas, caudate nucleus, hippocampus) and in
FIGURE 1. Cannabinoid-induced inhibition of calcitonin gene-related peptide
(CGRP, 250 nM)-stimulated CAMP production. Intact Chinese
hamster ovary cells stably expressing the SKR6 CDNA were
exposed to various concentrations of CP 55940 or -THC for 5
minutes. Data points represent the average percent inhibition
of CAMP (±SEM) from three experiments, each performed in
triplicate. Chemical structures of CP 55940 and -THC are
shown. -THC was obtained from the National Institute on Drug
Abuse; CP 55940 was donated by the Pfizer Research Corp.
49
the cerebellar cortex (figure 2). Although the localization of cannabinoid
receptor mRNA was quite similar to that of the cannabinoid receptor protein
(Herkenham et al. 1991a), numerous discrepancies were evident. In most
cases, mismatches between cannabinoid receptors and receptor mRNA likely
resulted from the expression of receptor proteins in the axons and/or terminals
of projection neurons. Although receptor and mRNA discrepancies could
result from comparing ISHH labeling with binding data obtained with a ligand
that recognizes multiple cannabinoid receptor subtypes, the ligand used to
localize cannabinoid receptors (
3H-CP 55940) appears to recognize a single
cannabinoid receptor subtype (Devane et al. 1988; Herkenham et al. 1990).
Indeed, Herkenham and colleagues (1991b, 1991c) have reported experimental
evidence for the localization of cannabinoid receptors to projection target areas
of both striatal and cerebellar neurons.
In the midbrain and hindbrain, ISHH labeling generally was uniform (of similar
intensity) among all labeled cells within a given region or anatomical nucleus,
and intensities varied from moderate to very low for different regions (data not
shown). In the forebrain, the intensity of ISHH labeling varied from very low to
very high. Several forebrain areas displayed a nonuniform labeling pattern in
which heavily labeled cells were present with cells labeled to a more moderate
extent. This nonuniform labeling was clearly evident in the hippocampus, where
cells displaying very high intensity labeling were scattered throughout Ammon’s
horn and in a monolayer subjacent to the granular layer of the dentate gyrus;
moderately labeled cells included those in the pyramidal cell layer and those
in the hilar region of the dentate gyrus (figure 3). Other forebrain regions—
anterior olfactory nuclei (figure 2, panel a), nucleus of the lateral olfactory tract,
basolateral amygdaloid nucleus (data not shown), horizontal limb of the nucleus
of the diagonal band (figure 3, panel b), cerebral cortex (figure 2, panels b and
c)—also displayed cells that were labeled to a high intensity. Although the
physiological significance of high levels of mRNA likely will depend on the
density of cannabinoid receptors on these cells, the mechanism(s) involved
in this range of gene expression would be of interest.
Possible explanations for high amounts of receptor mRNA include an increased
rate of transcription of the receptor gene and/or a decreased rate of transcript
degradation. If transcription rate varies (degradation rates remaining constant),
high amounts of mRNA could result from different promoters and cell-specific
induction mechanisms. Although splicing variants in 5’-untranslated sequences
have been identified by comparisons of the human cannabinoid receptor
gene (T.I. Bonner, M.J. Brownstein, C.C. Felder, C. Chen, and L.A. Matsuda,
unpublished manuscript), the SKR6 cDNA, and a human cannabinoid receptor
cDNA (Gerard et al. 1990), more detailed information concerning the sites
that regulate cannabinoid receptor gene expression is not yet available. In the
50
FIGURE 2. ISHH labeling of cannabinoid receptor mRNA in adult rat brain. Panels a through dare negative film
images of coronal sections taken at various levels through the brain; labeling by the SKR6-1 probe
appears as white on a dark background. Abbreviations: Fr, frontal cortex; AO, anterior olfactory
nucleus; Cg, cingulate cortex; Sl, intermediate septal nucleus; C, caudate nucleus; MS, medial
septal complex; DB, nucleus of the diagonal band; Pir, piriform cortex; Hi, hippocampus; PrC,
precommissioned nucleus; Sth, subthalamic nucleus; Cb, cerebellum; Sol, nucleus of the solitary
tract; IO, inferior olive.
FIGURE 3. Expression of the cannabinoid receptor gene in areas associated
with memory. Images were produced in a manner similar to those
pictured in figure 2 but were taken at higher magnifications.
Panel a: hippocampus, CA1, pyramidal cell layer of field CA1 of
Ammon’s horn; CA3, pyramidal cell layer of field CA3 of Ammon’s
horn: DG, dentate gyrus (arrow features the hilar region of the
dentate gyrus). Panel b: medial septal complex, MS, medial
septum; VDB, vertical limb of the nucleus of the diagonal band;
HDB, horizontal limb of the nucleus of the diagonal band. Panel
c: mammillary body, LM, lateral nucleus.
52
situation where mRNA stablity may vary (constant rates of transcription), less
mRNA would accumulate in cells in which the cannabinoid receptor mRNA is
more readily degraded. Conclusive information regarding this possibility is also
not available; however, cDNA clones with different lengths of 3’-untranslated
sequence have been described and appear to be the result of two distinct
polyadenylation sites (Matsuda et al. 1990). This finding may be relevant
since motifs in sequences 3’ of the coding region appear to influence message
stability in many eukaryotic genes (Shaw and Kamen 1986; Brawerman 1987).
Clearly, additional research is needed to determine the mechanism(s) by which
certain subpopulations of cells express high levels of cannabinoid receptor
mRNA.
In animals, cannabimimetic activity is routinely assessed by drug discrimination,
specific motor responses (dog ataxia, rodent catalepsy) (Razdan 1986), or
an increase in pain thresholds (Johnson and Melvin 1986). High levels of
receptor mRNA in the caudate nucleus and cerebellum (figure 2) are
consistent with the marked effects that cannabinoids exert on the motor
behavior of laboratory animals. Potential sources for cannabinoid-induced
analgesia include numerous regions that displayed low-to-moderate amounts
of receptor mRNA, including the cingulate cortex (moderate), intralaminar
thalamic nuclei (moderate), central grey (low), nucleus raphe mangus (low),
pontine reticular formation (low), and paragigantocellular reticular nucleus
(moderate) (data not shown).
In humans, memory deficits are some of the most consistently reported
effects of marijuana (Miller and Branconnier 1983). Clear labeling observed
in the rat forebrain suggests several potential sites in the human brain that
could mediate this effect, including the hippocampus, medial septal complex,
lateral nucleus of the mammillary body (figure 3), and the amygdaloid complex
(data not shown)—regions that traditionally have been associated with various
aspects of memory functions. Similarly, labeling was detected clearly in rat
forebrain regions that correspond to those that could mediate marijuana-
induced effects on human appetite and mood (hypothalamus, amygdaloid
complex, anterior cingulate cortex [data not shown]).
In contrast to the effects on mood and cognition, marijuana-induced
autonomic effects in humans are mild (Hollister 1986). However, in animals,
cannabimimetics decrease respiration, heart rate, and blood pressure
(Dewey 1986). These cardiovascular and respiratory responses are likely
due to the moderate levels of cannabinoid receptor mRNA that were found
in numerous midbrain and hindbrain regions (the pontine nucleus, the
paragigantocellular reticular nucleus, the lateral reticular nucleus, the area
postrema [data not shown], nucleus of the solitary tract [figure 2]). Although
53
the cannabinoid-induced effects on autonomic functions are rarely lethal in
humans, further study of the cells that express the cannabinoid receptor gene
in the midbrain and hindbrain may reveal an important physiological role(s)
for the cannabinoid receptor and its endogenous ligand.
SUMMARY
Localization of the mRNA for this receptor has identified many regions of the
rat brain in which the gene for this receptor is active. Several of these regions
are consistent with the cannabinoid- or marijuana-induced effects that occur
in both laboratory animals and humans. However, other labeled regions are
not easily associated with well-known effects of marijuana (Matsuda et al.,
submitted for publication). Although great progress has been achieved in
elucidating the mechanism of action of cannabis in recent years (Howlett et al.
1990), much remains to be discovered about the expression of cannabinoid
receptors in the brain and exactly how this receptor influences numerous brain
functions.
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Herkenham, M.; Lynn, A.B.; de Costa, B.R.; and Richfield, E.K. Neuronal
localization of cannabinoid receptors in the basal ganglia of the rat. Brain
Res 547:267-274, 1991b.
Herkenham, M.; Lynn, A.B.; Little, M.D.; Johnson, M.R.; Melvin, L.S.; de Costa,
B.R.; and Rice, K.C. Cannabinoid receptor localization in brain. Proc Natl
Acad Sci U S A 87:1932-1936, 1990.
Hollister, L.E. Health aspects of cannabis. Pharmacol Rev 38:1-20, 1986.
Howlett, A.C.; Bidaut-Russell, M.; Devane, W.A.; Melvin, L.S.; Johnson, M.R.;
and Herkenham, M. The cannabinoid receptor: Biochemical, anatomical
and behavioral characterization. Trends Neurosci 13:420-423, 1990.
Howlett, A.C.; Qualy, J.M.; and Khachatrian, L.L. Involvement of G
i in the
inhibition of adenylate cyclase by cannabinoid drugs. Mol Pharmacol
29:307-313, 1986.
Johnson, M.R., and Melvin, L.S. The discovery of nonclassical cannabinoid
analgetics. In: Mechoulam, R., ed. Cannabinoids as Therapeutic Agents.
Boca Raton, FL: CRC Press, 1986. pp. 121-145.
Matsuda, L.A.; Bonner, T.I.; and Lolait, S.J. Localization of cannabinoid
receptor mRNA in rat brain. J Comp Neurol, submitted for publication.
Matsuda, L.A.; Lolait, S.J.; Brownstein, M.J.; Young, A.C.; and Bonner, T.I.
Structure of a cannabinoid receptor and functional expression of the cloned
cDNA. Nature 346:561-564, 1990.
Miller, L.L., and Branconnier, R.J. Cannabis effects on memory and the
cholinergic limbic system. Psychol Bull 93:441-456, 1983.
Razdan, R.K. Structure-activity relationships in cannabinoids. Pharmacol Rev
38:75-149, 1986.
Shaw, G., and Kamen, R. A conserved AU sequence from the 3’ untranslated
region of GM-CSF mRNA mediates selective mRNA degradation. Cell
46:659-667, 1986.
ACKNOWLEDGMENTS
The work described in this chapter was supported in part by grants provided
by the National Alliance for Research on Schizophrenia and Depression and
the Stanley Foundation, USA (SJL). M.J. Brownstein synthesized the
oligonucleotide probes. W.S. Young III, M. Herkenham, and C.R. Gerfen
provided technical guidance and also assisted in confirming identified brain
regions.
AUTHORS
Lisa A. Matsuda, Ph.D.
Assistant Professor
Department of Psychiatry and Behavioral Sciences
55
Medical University of South Carolina
171 Ashley Avenue
Charleston, SC 29425
Tom I. Bonner, Ph.D.
Research Biologist
Stephen J. Lolait, Ph.D.
Guest Researcher
Laboratory of Cell Biology
National Institute of Mental Health
National Institutes of Health
Building 36, Room 3A17
Bethesda, MD 20892
56
Studies on µ-Opioid-Binding Sites With
Peptide Antibodies
Eric J. Simon, Theresa L. Gioannini, Yi-He Yao, and Jacob M.
Hiller
PURIFICATION OF A µ-OPIOID-BINDING PROTEIN AND ITS
CHARACTERIZATION
The purification to homogeneity of a µ-opioid-binding protein (OBP) from
bovine striatal membranes and its characterization by its ability to bind
opioid antagonists saturably, reversibly, and with high affinity was reported
previously by Gioannini and colleagues (1985). Briefly, the purification
involved two major steps: (1) affinity chromatography on a then-novel
derivative of naltrexone, 6-desoxy-aminoethylaminonaltrexone, coupled to the
carboxyl side chain of CH-Sepharose, followed by (2) lectin chromatography
on wheat germ agglutinin agarose. A single band of apparent molecular weight
(mol wt) of 65 kD was obtained on SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). The purification achieved was 60,000- to 70,000-fold, close to
the theoretical value for a protein of this size with a single binding site. The
purified OBP binds opioid antagonists with high affinity, but it binds against
agonists with low affinity, presumably because it is uncoupled from the Gi
protein to which it is normally coupled. The evidence that OBP is a µ-binding
opioid protein is as follows:
1.
OBP is isolated on an affinity matrix that contains a ligand that binds
µ-receptors preferentially.
2.
Binding of opioid antagonists is displaced by the µ-selective ligand D
Ala2Gly-I5 enkephalin (DAGO), albeit at high concentrations, but not by
selective -ligands.
3.
OBP binds the µ-specific antagonist 3H-cyprodime (Schmidhammer et
al. 1989). At saturating concentrations of ligand, OBP binds as much
cyprodime as bremazocine does, suggesting that all the binding to OBP
of bremazocine, a universal ligand, is to µ-sites.
57
4.
Finally, the molecular weight of OBP (65 kD) is the same as that observed
when 125l-ß-endorphin is cross-linked to opioid-binding sites in tissues and
a cell line, which were shown to be µ-sites by specific displacement with
µ-ligands (Howard et al. 1985, 1986).
The presence of disulfide bridges (-S-S-) that contribute to the secondary
structure of OBP is indicated by the difference in mobility of OBP in SDS-PAGE
under nonreducing vs. reducing conditions (Gioannini et al. 1985). As shown
in figure 1, treatment of OBP with increasing concentrations of reducing
reagent dithiothreitol (DTT) produced a stepwise shift from an apparent
molecular weight of 53 to 65 kD (Gioannini et al. 1989). The importance
of disulfide bonds, not only for secondary structure but also for function, is
reflected in the sensitivity of opioid ligand binding to inhibition by DTT. The
major opioid receptor types differed in their sensitivity to DTT as follows:
µ> with -sites being virtually resistant to even very high concentrations
of DTT (Gioannini et. al. 1989). The inhibition produced by DTT is reversible,
was observed to a much lesser degree with antagonist ligands than with
agonists, and was due to a reduction in affinity (increase in kD) rather than in
the number of receptor sites (Bmax). The possibility that these disulfide bridges
may have a role in receptor activation by agonist ligands is being explored.
MICROSEQUENCING OF PURIFIED OBP AND GENERATION OF
ANTISERA AGAINST PEPTIDE SEQUENCES
The availability of pure OBP encouraged the authors to attempt to determine
a portion of its primary sequence. Direct sequencing of OBP proved
unsuccessful, indicating that OBP is an N-terminally blocked protein and
must be fragmented to obtain peptides for amino acid sequencing, which
greatly increases the quantities of purified protein required. Two peptide
fragments, 21 and 13 amino acids in length, respectively, were generated by
chemical cleavage of OBP with CNBr followed by isolation of the peptides on
reverse phase high-performance liquid chromatography. The amino acid
sequences obtained were not found in databases of known protein sequences.
Polyclonal antibodies have been generated against portions of the peptides by
injection of rabbits with the synthetic peptides coupled to thyroglobulin, followed
by appropriate booster injections. The interaction of these antibodies with
purified OBP, bovine brain regions, and several cell lines are discussed below.
INTERACTION OF PEPTIDE ANTIBODIES WITH PURIFIED OBP
The peptide fragments used for antibody production and the antibodies
generated against each are indicated in table 1.
58
FIGURE 1. The effect of increasing DTT concentrations on the apparent
molecular weight of purified µ-OBP. Purified µ-OBP was
radioiodinated and separated from free iodine. Samples were
treated with the indicated concentrations of DTT and subjected
to SDS- PAGE.
SOURCE: Gioannini et al. 1989. Copyright 1989 by John Wiley & Sons, Ltd.
Reprinted by permission of John Wiley & Sons, Ltd.
All the antibodies were able to immunoprecipitate a major portion of 125l-
labeled purified OBP after incubation with the antibody followed by incubation
with protein A. The amount precipitated after correction for background
(radioactivity precipitated by normal rabbit serum [NRS] or irrelevant immune
sera) ranged from 40 percent for the weakest antibody (Ab 161) to more than
60 percent for the strongest, Ab 162 and Ab 165, at dilutions of 1:200.
Background of the assay accounted for 5 to 7 percent of added radioactivity.
Ab 162 and Ab 165 can immunoprecipitate 30 percent of 125l-OBP even at a
1:1,000 dilution. The protein precipitated by these antibodies was dissociated
from the complex and subjected to SDS-PAGE. Autoradiography of the gel,
59
TABLE 1. Antibodies generated against peptide sequences derived from
purified OBP
Antibody Fragment
165, 166, 6639 N-terminal 12 amino acids of peptide 1
163
C-terminal 7 amino acids of peptide 1
161, 162 N-terminal 10 amino acids of peptide 2
carried out under reducing conditions, showed a radioactive band with an
apparent molecular weight of 65 kD (i.e., the molecular weight of OBP).
Sequential treatment of OBP with antibodies, derived from the two different
peptides, indicated that initial treatment with either antibody removed all
immunoprecipitable antigen (i.e., no further immunoprecipitation occurred with
the second antibody). These results confirm that the two peptides isolated and
sequenced from fragmented OBP are derived from the same protein.
The interaction of OBP with the antipeptide antibodies also was examined in
immunoblots. An immunoreactive protein corresponding to a 65 kD mol wt
was detected with each antibody examined. The strongest signal was
produced with antibodies against the N-terminal portion of peptide 1 (Ab 165,
166, 6639) at a 1:100 dilution. The 65-kD band obtained in immunoblots with
OBP can be blocked by preincubation of the antisera with the appropriate
peptide (100 µM), but not by other peptides. No signal was detected when
OBP was immunoblotted against nonimmune serum. It is noteworthy that no
signal was detected unless OBP was reduced with DTT. Apparently, the
presence of the disulfide bridges and the resulting secondary structure of the
protein prevent access of the antibodies to the epitopes.
IMMUNOBLOTS OF TISSUES AND CELL LINES WITH ANTISERA AGAINST
OBP PEPTIDES
The authors decided to determine whether the peptide antibodies were capable
of producing positive immunoblots with extracts of tissues containing high
concentrations of µ-opioid receptors. The first tissue examined was an extract
of bovine striatal membranes, the source of purified OBP. lmmunoblotting with
peptide antisera gave a band at the appropriate molecular weight of 65 kD.
The positive signal at 65 kD mol wt seen in immunoblots with both digitonin
and CHAPS extracts of bovine striatal membranes prompted us to examine
cell lines and other bovine brain tissues for positive signals in immunoblots
60
with the antipeptide antibodies. lmmunoblots of SDS-solubilized bovine tissues
with the various antibodies indicated the presence of immunoreactive protein
(65 kD mol wt). The signal could be blocked by preincubation of antisera with
the appropriate peptide (50 to 100 µM). The tissues that reacted with antibody,
in addition to striatum, were frontal cortex, hippocampus, and thalamus, all
regions known to have moderate-to-high levels of µ-opioid receptors. Pons
and white matter produced no or a barely detectable response, which correlates
with their low levels of opioid receptors. The sensitivity of Ab 165, which
produces the strongest signal, is evidenced by its ability at a dilution of 1:100
to detect µ-opioid-binding material equivalent to 0.001 percent of the 30 to 50
µg of protein loaded per sample (300 to 500 pg of OBP) in an immunoblot.
lmmunoblots with the cell line SK-N-SH, which contains predominantly
µ-binding sites, produced a strong positive reaction at a position comparable
to that seen with purified OBP and brain tissue extracts (65 kD). The
immunoreactive protein detected in NG1O8-15 cells, a cell line that is reported
to contain only -receptors, migrated to a position slightly lower than that seen
in the SK-N-SH cells or with purified OBP (apparent molecular weight of
approximately 58 kD). In both cell lines, the response could be blocked by
preincubation of the serum with 100 µM concentration of peptide. Two
negative control cell lines, HELA cells and C6 glioma cells, produced no
detectable response. The detection of a response with the NG108-15 cells
suggests cross-reactivity of the antibodies with -receptors, although the
presence of small amounts of µ-receptors, hitherto not detected by binding
assays, could not be ruled out.
The ability of the antipeptide antibodies to react with native receptors was
investigated by examining the effect on opioid ligand binding and by evaluating
the extent to which active receptors can be removed from solution by
immunoprecipitation. None of the antibodies inhibited binding of opioid ligands
to either membrane-bound or soluble receptors. No depletion of receptors
was detected in the supernatants after immunoprecipitation with any of the
antibodies. It was concluded that the antibodies recognize only denatured
receptors. Not unexpectedly, the short amino acid sequences to which the
antibodies were made may not be accessible to the antibody in the native
receptor or may assume a secondary structure not recognized by the antibody.
All the above results, with antisera generated against microsequence from the
purified OBP, support the authors’ contention that we have purified an opioid-
binding site—in particular, the immunoblots of tissues and cell lines that give
bands of the appropriate molecular weight. Moreover, there is good correlation
between positive signals and levels of opioid receptors in the tissues or
cultures.
61
CROSS-REACTIVITY OF PURIFIED OBP WITH ANTISERA GENERATED
AGAINST BOVINE RHODOPSIN
Biochemical and physiological evidence indicates that all three major types of
opioid receptors, µ, negatively modulate adenylate cyclase and,
therefore, are coupled to guanine nucleotide regulatory proteins (G proteins).
This suggests that opioid receptors belong to the large family of receptors for
hormones, neurotransmitters, and peptides that effect signal transmission by
activating a G protein. Analysis of the amino acid sequences of many proteins
of this class has revealed some significant structural features common to all
members of this family, the most striking of which is the presence of seven
hydrophobic domains thought to span the cell membrane. The following results
further support the hypothesis that OBP belongs to this class of G-coupled
proteins.
Antibodies generated against membrane-associated rhodopsin and against five
specific amino acid sequences in rhodopsin were used. In immunoblots against
OBP, two antibodies, one against membrane-associated rhodopsin and one
against a sequence in the carboxyl terminal tail (CT1), reacted strongly,
whereas an antibody against the 1-2 loop (first cytoplasmic loop between
transmembrane domains 1 and 2) reacted weakly. Weiss and colleagues
(1987) had previously studied the interaction of purified ß-adrenergic receptor
from S49 lymphoma cells with this same series of antibodies under identical
reaction conditions. The pattern of reactivity of these antibodies with OBP
was the same as that previously reported by Weiss and coworkers (1987)
for purified ß-adrenergic receptor. These researchers had shown that
preincubation of antiserum CT1, an antiserum against a sequence in the
C-terminal tail of rhodopsin, with the peptide (rhodopsin 325-343) used to
generate it, was able to diminish greatly the signal obtained in immunoblots of
the ß-adrenergic receptor. This supports the specificity of the immune cross-
reaction. A rhodopsin peptide (324-348), which contains the sequence (325-
343) used to generate the CT1 antiserum, was made available to the authors’
laboratory. This peptide, when preincubated with antiserum CT1 at 100 µM
concentration, completely abolished the positive signal in immunoblots with
OBP. A rhodopsin peptide (rhodopsin sequence 331-348), which lacked the
first six amino acids of the CT1 sequence, was ineffective in reducing the
intensity of the 65-kD signal.
To verify that rhodopsin antibodies recognize the same protein as the peptide
antibodies, OBP was immunoprecipitated by Ab 165. An immunoblot of the
proteins remaining in the supernatant after immunoprecipitation showed a
strong diminution of the signal (65-kD band) obtained with the rhodopsin
antibody CT1, relative to a control supernatant from “immunoprecipitation” with
62
NRS. The protein immunoprecipitated with Ab 165 was eluted from the antigen-
antibody complex and examined in an immunoblot against CT1 . A positive
signal was observed at 65 kD mol wt, whereas no signal was detected with the
NRS control. This experiment indicates that the protein recognized by the
rhodopsin antibodies is the same as that recognized by OBP-derived peptide
antibodies.
The results reported here, in conjunction with those of Weiss and colleagues
(1987), indicate that the three proteins—bovine rhodopsin, S49 lymphoma
ß-adrenergic receptor, and OBP purified to homogeneity from bovine striatal
membranes—share common epitopes. Since there seems to be relatively little
amino acid sequence homology in the areas used for antibody production (at
least between rhodopsin and the ß-adrenergic receptor), structural features,
perhaps at the level of secondary and/or tertiary structure, along with limited
amino acid homology, may be responsible for the immunological cross-
reactivity. Weiss and coworkers (1987) have reported evidence that three
amino acids, K, N, and P in positions 325-327, may be important for recognition
of antibody CT1 by rhodopsin and by ß-adrenergic receptor. Current data
suggest that this also may be true for OBP.
The results presented constitute evidence, beyond that previously obtained,
that µ-opioid-binding sites are members of the family of G protein-coupled
receptors and are likely to show the typical seven membrane-spanning domain
structure of these proteins, when their complete amino acid sequence becomes
known. It should be noted that similar results have been obtained by W.A. Klee
and coworkers (personal communication, October 1991). These researchers
found that -receptors labeled with the affinity label 3H-FIT and purified from
NG108-15 cells in culture were immunoprecipitated by an antiserum generated
against a peptide sequence from the C-terminal tail of rhodopsin. This finding
lends credence to ours and suggests that µ- and -opioid receptors both share
antigenic sites with rhodopsin.
UPDATE ON ATTEMPTS TO CLONE THE µ-OBP
Oligonucleotides have been prepared based on the amino acid sequence of the
isolated peptides. Bovine brain and striatal cDNA libraries have been screened
with the labeled oligonucleotide probes, and clones have been obtained. Partial
sequencing revealed that the clones did not contain the full probe used to
detect them; that is, they were false-positive clones. This is undoubtedly due to
the considerable degeneracy of the authors’ sequences, which contain many
amino acids coded by four or six codons. Screening is currently in progress
using different oligonucleotides. Clones are being sequenced to see if they
contain the sequence present in the probe. If so, sequencing of the complete
63
cDNA will be carried out. The resulting structure should give an idea whether it
is the opioid receptor, although proof must await transfection of suitable cells
and expression of an active binding site with the appropriate ligand specificity.
REFERENCES
Gioannini, T.L.; Howard, A.D.; Hiller, J.M.; and Simon, E.J. Purification of an
active opioid binding protein from bovine striatum. J Biol Chem 260:15117-
15121, 1985.
Gioannini, T.L.; Liu, Y.-F.; Park, Y.-H.; Hiller, J.M.; and Simon, E.J. Evidence
for the presence of disulfide bridges in opioid receptors essential for ligand
binding. Possible role in receptor activation. J Mol Recognition 2(1):44-48,
1989.
Howard, A.D.; de la Baume, S.; Gioannini, T.L.; Hiller, J.M.; and Simon, E.J.
Covalent labeling of opioid receptors with radio-iodinated human beta-
endorphin. J Biol Chem 260:10833-10839, 1985.
Howard, A.D.; Gioannini, T.L.; Hiller, J.M.; and Simon, E.J. Identification of
distinct binding site subunits of mu and delta opiate receptors. Biochemistry
25:357-360, 1986.
Schmidhammer, H.; Burkard, W.P.; Eggstein-Aeppli, L.; and Smith, C.F.C.
Synthesis and biological evaluation of 14-Alkoxymorphinans. (-)-N-
Cyclopropylmethyl-414-dimethoxy-morphinan-6-one, a selective µ-opioid
receptor antagonist. J Med Chem 32:418-421, 1989.
Weiss, E.R.; Hadcock, J.H.; Johnson, G.L.; and Malbon, C.C. Antipeptide
antibodies directed against cytoplasmic rhodopsin sequences recognize the
beta-adrenergic receptor. J Biol Chem 262:4319-4323, 1987.
ACKNOWLEDGMENTS
The work described in this chapter was supported by National Institute on Drug
Abuse grant DA-00017 (EJS).
Dr. Gary Johnson (National Jewish Center for Immunology and Respiratory
Medicine, Denver, CO) and Dr. Ellen Weiss (University of North Carolina,
Chapel Hill, NC) supplied the rhodopsin antisera. Dr. Anatol Arendt and Dr.
Paul Hargrave (University of Florida) supplied the rhodopsin peptides for the
blocking experiments. Dr. Catherine Strader (Merck Sharp and Dohme,
Rahway, NJ) isolated the peptides from purified OBP. Dr. Lawrence Taylor, Dr.
Huda Akil, and Dr. Stanley Watson (University of Michigan Medical School)
generated antisera against the peptides.
64
AUTHORS
Eric J. Simon, Ph.D.
Professor
Departments of Psychiatry and Pharmacology
Yi-He Yao, Ph.D.
Assistant Research Scientist
Department of Psychiatry
Jacob M. Hiller, Ph.D.
Research Associate Professor
Department of Psychiatry
New York University Medical Center
550 First Avenue
New York, NY 10016
Theresa L. Gioannini, Ph.D.
Research Assistant Professor
Department of Psychiatry
New York University Medical Center
Associate Professor
Department of Natural Sciences
Baruch College
City University of New York
New York. NY 10010
65
Molecular Cloning and Characterization
of Neurotransmitter Transporters
Randy D. Blakely
INTRODUCTION
For precise chemical signaling between neurons and target cells, the
magnitude and duration of neurotransmitters in synaptic spaces must be
tightly regulated. Two principal and distinct mechanisms are classically
recognized as being responsible for rapid transmitter inactivation. Transmitter
can be either enzymatically metabolized, as with acetylcholinesterase hydrolysis
of acetylcholine (Taylor 1991), or actively accumulated into presynaptic terminals
and/or surrounding glia (Iversen 1967; Schousboe 1981; Hendley 1984; Cooper
et al. 1986; Horn 1990; Nicholls and Atwell 1990). First demonstrated in the
periphery by Axelrod and colleagues while studying the fate of intravenous
radiolabeled catecholamine (Axelrod 1971), rapid transport across neuronal
and glial membranes has been extensively characterized for many central
neurotransmitters. In addition to norepinephrine (NE), the neurotransmitters
dopamine (DA), serotonin (5-HT), L-glutamic acid (Glu), -aminobutyric acid
(GABA), and glycine (Gly) each can be actively transported across brain
membranes in a region-dependent manner consistent with the localization
of neuronal terminals releasing these compounds. Choline availability is the
rate-limiting process in acetylcholine biosynthesis. Therefore, it is no surprise
that even cholinergic neurons utilize an active transport process to actively
recapture synaptic choline following acetylcholine hydrolysis (Kuhar and Murrin
1978). The physiologic importance of transmitter inactivation by reuptake
carriers is particularly apparent when examining the clinical and societal impact
of agents with a high selectivity for blocking monoamine transport, including
cocaine, amphetamines, and many antidepressants (Ritz et al. 1987; Galloway
1988; Richelson 1990). Furthermore, the ability of synaptic transporters to
accumulate neurotransmitter-like toxins, including 6-hydroxydopamine, 1-methyl-
4-phenylpyridinium (MPP+), 5,6-dihydroxytryptamine, and AF64A, among
others, suggests that these proteins have a role in the establishment of
selective neuronal vulnerability to exogenous agents. Thus, neurotransmitter
transporters represent critical targets for both therapeutic and pathologic
alterations of synaptic function.
66
Active transport of neurotransmitters (and choline) is driven by the
transmembrane Na+ gradient (Kanner and Schuldiner 1987), established
by Na+/K+ adenosinetriphosphatase (ATPase), in a process analogous to
the active transport of glucose across brush border epithelia (Stein 1986).
However, unlike intestinal glucose transport, additional ions are required for
transport of many neurotransmitters, such as intracellular K+ and extracelluar
Cl-. Membrane vesicle (Kanner and Schuldiner 1987) and whole cell patch
clamp (Nicholls and Atwell 1990) studies indicate that the effects of these ions
on transport are unrelated to their effects on transmembrane potential (Vm),
but rather indicate that there is parallel movement of these ions during each
translocation cycle. These energetic properties, along with clear pharmacologic
differences, distinguish these transporters from the intracellular vesicular
carriers used to package neurotransmitters for exocytosis (Cooper et al. 1986;
Kanner and Schuldiner 1987). Specific radiolabeled antagonists, including
[3H]nisoxetine for the NE transporter (Tejani-Butt et al. 1990), [
3H]GBR12909
and [
3H]GBR12935 for the DA transporter (Anderson 1989), [
3H]citalopram
for the 5-HT transporter (D’Amato et al. 1987), and [
3H]hemicholinium-3 for
the choline transporter (Sandberg and Coyle 1985), have been used to label
transporters, both in broken cell preparations and in brain sections, permitting
both pharmacologic and anatomic analyses. These tools have confirmed the
enrichment of transporter sites on presynaptic terminal membranes and, at
least for the choline transporter (Saltarelli et al. 1987), have revealed alterations
in carrier density on plasma membranes in response to depolarization.
Despite decades of kinetic and pharmacologic studies on the behavior of
neurotransmitter transporters in membrane preparations, structural data
relevant to questions of mechanism and regulation have been difficult to obtain.
One initial assumption is that all transporters utilizing cotransported Na+ ions,
from Escherichia coli (E. coli) to man, are derived from a common ancestral
gene, with sequence identity illustrative of common tasks such as Na+ binding
or translocation, and sequence divergence responsible for additional ionic and
pharmacologic specificities. Homologous, facilitated glucose transporters
(which lack the ability to move sugar uphill against a concentration gradient)
are found throughout phylogeny as members of an extended gene family
(Henderson 1990). The intestinal Na+/glucose (Hediger et al. 1987, 1989)
and the E. coli Na+/proline (Nakao et al. 1987) transporters are encoded by
single genes and bear sequence similarities absent from the family of facilitated
carriers, revealing the presence of a distinct gene family encoding Na+/
symporter proteins. One possibility was that neurotransmitter transporters
would bear detectable sequence conservation with this latter gene family.
However, attempts to use the limited homology of the Na+/glucose and Na+/
proline transporters to identify novel carriers expressed in the rodent central
nervous system met with no success (R.D. Blakely, unpublished data),
suggesting a novel gene family might encode neurotransmitter transporters.
67
EXPRESSION OF NEUROTRANSMlTER TRANSPORTERS IN XENOPUS
LAEVIS OOCYTES
In the absence of sequence information suitable for molecular cloning of
transporter cDNAs by conventional hybridization strategies, Blakely and
colleagues (1988, 1991a) turned to the Xenopus laevis oocyte expression
system to characterize and perhaps clone transporter mRNAs. Following
the injection of poly(A)+RNA, Xenopus laevis oocytes actively accumulated
many different transmitter substrates, including Glu, GABA, Gly, 5-HT, DA,
and choline (Blakely et al. 1988). Transport of these substrates was time-
and temperature-dependent, and uptake was abolished with the removal of
extracellular Na+. Regional enrichment of transporter mRNAs followed well
the distribution of soma predicted to synthesize each carrier. In addition,
pharmacologic sensitivities of oocyte-expressed transporters were similar
(Blakely et al. 1991a) if not identical to those observed in brain membrane
preparations (table 1). Using mRNA prepared from different brain regions
during development, Blakely and colleagues (1991a) revealed a postnatal
rise in Glu, GABA, and Gly transporter transcript abundance paralleling the
postnatal rise in brain transport activity for these substrates. Most important
in the consideration of expression cloning strategies was that mRNAs encoding
Glu, GABA, and Gly carriers could be size-fractionated on sucrose density
gradients, with minimal loss of activity relative to unfractionated mRNA and
with a narrow distribution of peak activities (figure 1, example of Glu transporter
size fractionation). These data supported the assumption that neurotransmitter
transporters were likely to be encoded by single, separable mRNAs and,
therefore, could be cloned utilizing expression as a functional screen for the
presence of a single transporter clone in a cDNA library.
MOLECULAR CLONING AND EXPRESSION OF A COCAINE- AND
ANTIDEPRESSANT-SENSITIVE NOREPINEPHRINE TRANSPORTER
DERIVED FROM A HUMAN NEUROBLASTOMA (SK-N-SH) CELL LINE
Despite the demonstrated ability of the Xenopus laevis oocyte expression
system to reconstitute neurotransmitter transporters, there were several
drawbacks precluding its use for the cDNA cloning of these carriers. Seasonal
variability in oocyte viability and expression levels, coupled with an inability
to produce a high “signal-to-noise” ratio for monoamine transporters (Blakely
et al. 1988), dictated the design and implementation of an alternative strategy
(figure 2). This method, based on the ability of COS cells to replicate episomal
copies of transfected plasmids bearing SV40 replication origins (Gluzman
1981), has been used in various forms to clone cDNAs for several membrane
proteins, including several peptide receptors (Aruffo and Seed 1987; Sims et
al. 1988; D’Andrea et al. 1989; Munro and Maniatis 1989). Pacholczyk and
68
TABLE 1. Inhibitor sensitivity of oocyte uptake activities induced by adult rat
Substrate
L-Glu
mRNAs
RNA Source
Forebrain
Cerebellum
GABA
Gly
Choline
DA
Inhibitor Concentration Percent Inhibition
Dihydrokainate
1 mM 61.0 ± 6.9
Dihydrokainate
100 mM 16.0 ± 5.3
D-aspartate
100 µM
91.7 ± 15.5
Nipecotic acid
100 µM 10.0 ± 1.1
Spinal cord Dihydrokainate
1 mM 63.0 ± 8.1
Cerebellum D-aspartate
100 µM
8.2 ± 1.4
Nipecotic acid
100 µM
81.2 ± 9.0
Spinal cord Nipecotic acid
100 µM
75.7 ± 10.1
Spinal cord Nipecotic acid
100 µM 0.3 ± 0.1
Spinal cord Hemicholinium-3
10 µM 82.2 ± 4.0
Midbrain Nomifensine
10 µM
98.3 ± 12.3
Adrenal Nomifensine
10 µM
79.4 ± 6.8
NOTE:
Xenopus laevis oocytes were injected with 40 ng poly(A+) RNA and
incubated at 18 °C for 2 to 3 days prior to assay. Uptake assays (22 °C)
with different [3H]-labeled substrates were performed as described in
Blakely and colleagues (1991a) with or without inhibitors at the
concentrations indicated. Mean transport inhibition (±SEM) relative to
assays conducted with substrates alone is presented.
SOURCE:
Blakely, R.D.; Clark, J.A.; Pacholczyk, T.; and Amara, S.G. Distinct,
developmentally regulated brain mRNAs direct the synthesis of
neurotransmitter transporters. J Neurochem 56:860-871, 1991a.
Copyright 1991 by Raven Press, Ltd. (New York).
colleagues (1991) reasoned that the uptake of an iodinated analog of NE,
meta-iodobenzylguanidine (mlBG), could be visualized in cells successfully
transfected with the NE transporter (NET). Prior to embarking on this
method, they characterized the transport of mlBG in SK-N-SH cells, an
adrenergic neuroblastoma cell line possessing a high-affinity NE uptake
system (Richards and Sadee 1986), and found the substrate to be
69
FIGURE 1.
Size-fractionation of Glu transporter mRNA
SOURCE: Blakely, R.D.; Clark, J.A.; Pacholczyk, T.; and Amara, S.G.
Distinct, developmentally regulated brain mRNAs direct the
synthesis of neurotransmitter transporters. J Neurochem 56:860-
871, 1991a. Copyright 1991 by Raven Press, Ltd. (New York).
accumulated in a desipramine-sensitive manner as reported (Smets et al.
1989) and imageable autoradiographically. Because the technique could
be performed on large tissue-culture pans, Pacholczyk and colleagues (1991)
70
FIGURE 2. COS cell expression system for cDNA cloning of Na+/L-NET. AV
MLP-adenovirus major late promoter.
were able to screen large pools of cDNA library plasmids derived from SK-N-SH
mRNA. Plasmid DNA derived from scraped cells overlying “hot spots” on the
film was extracted by the Hirt lysis procedure, and the method was repeated
several times on a smaller scale. Finally, a single plasmid was isolated, which
was capable of producing a functional mlBG transporter in transfected
COS cells, with transport activity abolished when autoradiographic uptake
71
experiments were conducted in the presence of the NE transport-specific
inhibitor desipramine (T. Pacholczyk, unpublished data).
To demonstrate that the single cloned cDNA directed the synthesis of a
transporter with the expected ionic and pharmacologic sensitivities of the native
human NET (hNET), Pacholczyk and colleagues (1991) cloned the cDNA
insert downstream of a T7 RNA polymerase promoter in pBluescript SKII(-)
and transfected the construct into transformed human fibroblasts (HeLa)
infected with a vaccinia virus encoding T7 RNA polymerase (Fuerst et al.
1986). Blakely and coworkers (1991b) had demonstrated that this expression
method gave faithful, rapid, and high-level expression of cloned Na+/glucose
and Na+/GABA transporters, thus providing a convenient transient expression
method for analysis of the putative hNET. Assays performed on HeLa cells
12 hours after transfection revealed the generation of a high level of Na+-
dependent NE transport activity; analysis of substrate-dependence revealed
the induction of a single, saturable activity with an NE Kt of 457 nM (figure 3).
Assays conducted with a wide range of transporter and receptor antagonists
revealed the NE uptake (1) to be markedly sensitive to selective NE transport
inhibitors as compared with DA or 5-HT transport inhibitors, (2) to be potently
antagonized by both cocaine and D-amphetamine, and (3) to be insensitive to
inhibitors of either amino acid and sugar transport or to adrenergic and
adrenergic receptor antagonists (figures 4a and 4b, table 2). As expected,
reserpine, an inhibitor of the vesicular NE transport inhibitor (Kanner and
Schuldiner 1987), also failed to block NE uptake in transfected cells, ruling out
any possibility for ectopic expression of vesicular carrier. Thus, all the readily
testable pharmacological features of Uptake 1 (Iversen 1967) appear to be
encoded by this single cDNA. Given that multiple RNAs are not required to
synthesize a functional carrier, these findings also suggest that the membrane-
imbedded transporter may be monomeric in composition, as has been argued
for the transporter Lac permease (Costello et al. 1987), or may exist as a
homomultimer, as envisaged with the Na+/glucose transporter (Stevens et al.
1990).
Sequence analysis of the isolated NET cDNA reveals a large open reading
frame encoding a highly hydrophobic 617-amino acid peptide with a predicted
molecular weight of ~69 kD. Hydrophobicity analysis (Kyte and Doolittle 1982)
reveals ~12 regions capable of forming transmembrane domains. Although this
motif of 11 to 13 transmembrane domains superficially resembles the pattern
observed with both the Na+/glucose and E. coli Na+/proline symporters,
sequence analysis fails to reveal significant conservation in primary sequence.
Rather, shared sequences evident with the cloned rat and human Na+/GABA
transporters (GAT1) (Guastella et al. 1990; Nelson et al. 1990) reveal the
presence of a new transporter gene family, united by the function of their
72
FIGURE 3. Transport of L-NE in transfected HeLa cells
SOURCE: Pacholczyk et al. 1991. Reprinted by permission from Nature
350:350-354. Copyright © 1991 Macmillan Magazines Ltd.
(London).
substrates as neurotransmitters. Alignment of the predicted amino acids for
GABA and NE carriers reveals 46-percent identity, which rises to 68 percent,
allowing for conservative substitutions. Sequences predicted to lie within or
adjacent to predicted transmembrane regions contain many of the conserved
residues. One striking stretch, lying between putative transmembrane domains
1 and 2, possesses 19 out of 20 residues absolutely conserved. A comparison
of their virtually superimposable hydrophobicity profiles gives a clear indication
that the GABA and NE transporters are likely to assume similar secondary
structures in the plasma membrane (figure 5). As with GAT1, a large
hydrophilic loop bearing three consensus sites for N-linked glycosylation is
observed between transmembrane domains 3 and 4. When paired with the
absence of a detectable signal sequence for membrane insertion, this feature
suggests an initial model with both NH2- and COOH-termini in the cytoplasm,
as depicted in figure 6.
73
FIGURE 4. Inhibitor sensitivity of transfected Na+/L-NET
SOURCE: Pacholczyk et al. 1991. Reprinted by permission from Nature
350:350-354. Copyright © 1991 Macmillan Magazines Ltd.
(London).
74
TABLE 2. Inhibitor sensitivity of L-NE uptake in pNET-transfected HeLa cells
Inhibitor KI (nM) Hill (nH)
Mazindol
1.36
Desipramine 3.88
Nomifensine 7.68
Nortriptyline
16.5
D-amphetamine
56.1
lmipramine 65.4
Amitriptyline
100
GBR 12909
133
DA
139
Cocaine
140
Paroxetine 312
Benztropine 822
Citalopram >1,000
Propranolol 10,000
5-HT >10,000
Yohimbine >10,000
Prazosin >10,000
Hemicholinium-3 >10,000
Nipecotic acid >10,000
Phloridzen >10,000
Reserpine >10,000
0.98
1.22
1.17
0.95
1.00
1.08
0.75
1.06
0.86
1.25
0.75
0.82
—
—
—
—
—
—
—
—
—
NOTE: HeLa cells (200,000 to 300,000/well) infected with a T7 RNA
polymerase-containing vaccinia virus were transfected with pNE
(100 ng) and incubated with 20 nM [2,5,6,3H]-L-NE (New England
Nuclear) ±inhibitors for 15 minutes at 37 °C. K
I values and Hill
coefficients reflect mean estimates from triplicate determinations
of complete uptake inhibition curves, adjusting for substrate
concentration after Cheng and Prusoff (1973).
SOURCE: Adapted from Pacholczyk et al. 1991. Reprinted by permission
from Nature 350:350-354. Copyright © 1991 Macmillan Magazines
Ltd.
High-affinity, antidepressant-sensitive NE uptake activity is enriched in
projections of brain stem noradrenergic neurons, particularly from the locus
coeruleus, as well as in terminals of noradrenergic sympathetic neurons and
in the neural crest-derived adrenal medulla. Northern hybridizations with
labeled NET reveal a 5.8-kb RNA localized to rat brain stem, rat adrenal gland,
75
FlGURE 5. Hydrophobicity comparison of GABA and NET proteins encoded
by cloned GAT1 and hNET CDNAS.The transformation is as
described by Kyte and Doolittle (1982) with a window of 19
residues. Hydrophobic values are positive in this display with
shaded regions indicating the 12 putative transmembrane
regions.
rat PC-12 cells, and the human SK-N-SH cells (Pacholczyk et al. 1991). A
more widespread 3.6-kb band is also observed in brain regions and cell lines
and may represent a splicing variant of NET or a related and cross-hybridizing
transcript. Because desipramine-sensitive NE uptake has been reported in
primary cultures of neonatal rat astrocytes, this latter species may also
represent a more broadly distributed glial carrier. Also, although both PC-12
and SK-N-SH cells exhibit many noradrenergic traits, their phenotypes can
be quite heterogeneous depending on culture conditions, and thus, multiple
transcripts may also reflect the mixed character of these cells.
In summary, a single 1,983-bp cDNA clone encoding a 617-amino acid
protein is sufficient to transfer upon nonneuronal cells the ability to accumulate
NE with high affinity, in a Na+-dependent manner, and with marked sensitivity
76
FIGURE 6. Hydrophobicity-based structural model of the human Na+/L-NET
SOURCE: Adapted from Pacholczyk et al. 1991. Reprinted by permission
from Nature 350:350-354. Copyright © 1991 Macmillan Magazines
Ltd.
to specific antidepressants, cocaine, and amphetamine. As these latter agents
have profound effects on human behavior, exhibiting both therapeutic and
abuse potential, identification of a clone for this transporter may provide a
direct route to the rational design of novel therapies. In addition, the availability
of a cDNA encoding the NET should allow for an examination of the degree to
which structural alterations in the NE carrier gene underlie hereditary patterns
of major affective disorders, hypertension, and drug abuse.
EVALUATION OF DIVERSITY WITHIN THE NEUROTRANSMITTER
TRANSPORTER GENE FAMILY
The presence of conserved amino acid sequences between GAT1 and NET
suggests that other neurotransmitter transporters, such as those for DA, 5-HT,
Glu, Gly, and choline, are likely to be members of this family. Indeed, given the
77
major pharmacologic differences between the transporters encoded by GAT1
and NET, it is likely that the observed 46-percent identity between these two
proteins represents the lower limit of sequence similarity to be observed as
new members are uncovered. This would seem true for the monoamine
transporters, where considerable antagonist overlap exists among NE, DA,
and 5-HT uptake systems (Hendley 1984; Cooper et al. 1986; Richelson 1990).
One conventional approach to the identification of related gene products is the
screening of cDNA libraries by hybridization at reduced stringency. Using this
approach, however, Nelson and colleagues (1990) were unable to identify
homologs of GAT1 , suggesting that additional family members might exhibit
too little overall identity with GAT1 to permit elucidation in this manner.
Recently, with the introduction of the polymerase chain reaction (PCR) (Saiki
et al. 1988), it became possible to use short stretches of dispersed sequence
identity to identify and clone novel members of an extended gene family (Gould
et al. 1989; Libert et al. 1989; Kamb et al. 1989). In the implementation of this
strategy, Peek and colleagues (1991) have designed degenerate, inosine-
substituted oligonucleotides based on the sequence identity present between
GAT1 and NET in the regions connecting transmembrane domains 1 and 2
as well as that observed in transmembrane 6. With these oligonucleotides
as primers, Peek and colleagues (1991) amplified rat and human cDNAs and
subcloned products of a size similar to that found between these sequences
in NET. Sequence analysis of the cloned amplification products revealed
multiple, distinct gene products all bearing sequence identity to NET and
GAT1. Several of these products bear considerable sequence identity to NET
(60 to 80 percent), and others exhibit only the level of identity (40 to 50 percent)
observed when comparing NET and GAT1 over these regions. Although these
amplification fragments represent partial cDNA clones, they can be used to
determine the anatomical localization of the endogenous transcripts by Northern
and in situ hybridizations and can be used as high-stringency probes for the
identification of their full-length functional sequences by conventional library
hybridization. Northern analysis of each of these products revealed several
that are broadly distributed across the rodent central nervous system and
perhaps difficult to associate with the distribution of a particular neurotransmitter
system. However, several fragments hybridize in a regionally selective manner.
For example, one clone is enriched in forebrain regions, whereas another is
highly concentrated in thalamic, midbrain, and brain stem regions. Particularly
striking is the hybridization of two clones selectively to midbrain and brain stem
regions. Peek and coworkers (1991) performed in situ hybridization analysis of
these clones and found one of these to be localized to the substantia nigra and
ventral tegmental area, whereas the other is confined to the midbrain and brain
stem raphe complex, identifying these two clones as likely candidates for the
DA and 5-HT transporter cDNAs, respectively.
78
Utilizing oligonucleotides derived from the 5’-end of the PCR fragment
encoding the 5-HT transporter candidate, Blakely and colleagues (1991c)
isolated a series of overlapping clones from a rat brain stem cDNA library.
Sequence analysis of one of these clones reveals the presence of a large
open reading frame in register with that of NET. After subcloning the
bacteriophage insert into a T7-promoter-bearing plasmid, Blakely and
colleagues (1991c) transfected this cDNA into HeLa fibroblasts infected
with T7 polymerase-vaccinia virus. After 8 hours, transfected fibroblasts
exhibit marked expression of 5-HT transport, which can be abolished by the
selective 5-HT transport antagonist fluoxetine (figure 7). Thus, like NET, the
5-HT transporter (SERT) appears to be encoded by a single RNA (Blakely
et al. 1991c). The availability of a cDNA clone encoding this carrier should
permit a refined analysis of the structural basis of antidepressant interactions
with nerve terminals. Sequence comparisons with NET and GAT1 reveal
FIGURE 7. Expression of 5-HT transport in HeLa cells transfected with SERT
cDNA
79
highly conserved residues likely to be important for common tasks, including
substrate translocation and Na+ binding. In addition, with regulation of the
5-HT carrier reported to be altered in depressed humans (Paul et al. 1981;
Meltzer et al. 1981), the availability of a cDNA clone encoding this carrier
should lead to a direct search for structural changes associated with the
human SERT homolog in affective disorders. Recently, the first cloning of
a functional rat brain DA transporter cDNA was also achieved (Kilty et al.
1991; Shimada et al. 1991), bearing sequence identity to the substantia nigra
localized PCR species described above. Together, the elucidation of the
5-HT and DA transporters broaden understanding of the structural basis for
neurotransmitter transporter diversity and provides new tools for the molecular
analysis of transporter gene expression and regulation in brain disorders and
drug abuse.
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AUTHOR
Randy D. Blakely, Ph.D.
Assistant Professor and Woodruff Neuroscience Investigator
Department of Anatomy and Cell Biology
Emory University School of Medicine
Atlanta, GA 30322
83
Regulation of Gene Expression by
Dopamine: Implications in Drug
Addiction
Orla M. Conneely, Ronan F. Power, and Bert W. O’Malley
INTRODUCTION
Cocaine is thought to produce pleasure by increasing dopamine transmission
in mesocortical and mesolimbic dopaminergic tracts. Increased dopaminergic
transmission results from an inhibition of the dopamine reuptake transporter
by cocaine, which also inhibits reuptake of both serotonin and norepinephrine
(Ritz et al. 1988). The reward-seeking behavior produced by cocaine self-
administration is similar to that produced by electrical self-stimulation of
dopaminergic pathways. The observation that chronic cocaine administration
decreases electrical self-stimulation reward indices suggests that a down-
regulation of dopaminergic reward areas may accompany addiction (Gawin
and Ellinwood 1988).
The molecular mechanisms underlying long-term neuroadaptive changes that
accompany chronic cocaine abuse have yet to be established. However, it
is becoming increasingly evident that neuronal adaptation to repeated
perturbation of neurochemical signals involves changes in gene expression.
Examples of such genomic regulation of neuronal adaptation include the
regulation of gene expression by serotonin during long-term facilitation
(Barzilai et al. 1989) and the development of kindling by repeated electrical
stimulation of the hippocampus (Jonec and Wasterlain 1979; Shin et al. 1990).
Therefore, it seems plausible that the effects of chronic cocaine abuse would
be reflected at a level of altered gene expression in dopaminergic and/or
dopaminoceptive cells. In this case, elucidation of the mechanisms by which
dopamine transduces a neurotransmitter signal to changes in gene expression
would be expected to yield valuable information on the molecular nature of
neuroadaptation to repeated cocaine administration. To this end, the authors
have identified a class of transcription factors that are activated by dopamine.
Activation of these factors results in a dopamine-dependent induction of
transcription of specific target genes. The activated factors are members
of the steroid/thyroid receptor superfamily of transcription factors.
84
THE STEROID/THYROID RECEPTOR FAMILY OF TRANSCRIPTION
FACTORS
The steroid/thyroid receptor family constitutes a subclass of transcription
factors that regulate and coordinate complex events in development,
differentiation, and homeostasis. These proteins control behavioral and
physiological responses by regulating the expression of specific gene networks
in response to diverse stimuli (for a review, see Evans 1988). Members of
the above family of transcription factors contain three conserved regions of
amino acid sequence (McDonnell et al. 1987). The conserved regions consist
of a DNA-binding domain responsible for specific binding of the transcription
factor to upstream regulatory DNA sequences within the promoter region
of responsive target genes (Evans 1988) and two additional regions of
conservation located in the ligand-binding domain of the ligand-activated
members of this family (McDonnell et al. 1987). The steroid receptors were
the first characterized members and serve as prototypes for this class of
transcription factors (Hollenberg et al. 1985; Greene et al. 1986; Conneely
et al. 1987). These receptors are soluble proteins that upon specific binding
to their cognate steroid hormone become active transcription factors capable
of regulating the expression of specific target genes (Evans 1988). In addition
to the steroid receptors, the ligand-activated members of this family include
receptors for retinoic acid (Giguere et al. 1987), vitamin D
3 (Baker et al.
1988), and thyroid hormone (Weinberger et al. 1986); more than 20 additional
members have been identified. They have been termed “orphan receptors”
since no ligand has been identified that binds to these proteins. However,
recent evidence has demonstrated that several members of the steroid/
thyroid receptor superfamily can be activated in the absence of ligand by
phosphorylation (Glineur et al. 1990; Denner et al. 1990). This observation
raises the possibility that the orphan receptors may be activated by direct
ligand binding and/or by phosphorylation via indirect signaling pathways.
One such orphan receptor, chicken ovalbumin upstream promoter-transcription
factor (COUP-TF) (Wang et al. 1989), participates in the regulation of several
genes, including the chicken ovalbumin gene (Wang et al. 1987), the rat insulin
II gene (Hwung et al. 1988), and the proopiomelanocortin gene (Drouin et al.
1989). Although COUP-TF is widely distributed in tissues throughout the body,
including the brain, at least one of its functions appears to be the regulation of
retinal cell development since the Drosophila counterpart of this protein is
required for the determination of photoreceptor cell fate (Mlodzik et al. 1990).
The present study was initiated in an attempt to identify chemical signals that
would lead either directly or indirectly to the activation of COUP-TF. In the
context of the studies described below, functional COUP-TF expression is
measured as COUP-TF-dependent activation of a specific target gene.
85
DOPAMINE REGULATES COUP-TF-DEPENDENT GENE TRANSCRIPTION
To search for compounds that activate COUP-TF, the authors replaced the
DNA-binding domain of COUP-TF with that of the tissue-specific progesterone
receptor (figure 1). Since the enhancer DNA specificity is provided by the
DNA-binding domain, the resulting chimera is expected to direct COUP-TF-
dependent transcription of progesterone-responsive target genes (Beato
1991). This strategy was employed to prevent competition in the assay from
endogenous COUP-TF in transfected cells. Expression plasmids containing
the chimeric COUP-TF construct (pADFCOUP) and a progesterone-responsive
target gene (PRETKCAT) were cotransfected into a progesterone receptor
negative monkey kidney cell line, CV1. The target gene used in these studies
contained two copies of a progesterone-responsive enhancer element (PRE)
located upstream of the herpes simplex virus thymidine kinase promoter linked
to a chloramphenicol acetyl transferase (CAT) gene.
FIGURE 1. Strategy for generation of the chimeric COUP protein.
Schematic representations of cPR and COUP-TF are shown.
Prog designates the progesterone-binding domain of cPR.
The
chimeric COUP-TF (FCOUP) contains the NH2 and COOH-
terminal domains of COUP-TF fused to the DNA-binding domain
of cPR. The cPR cDNA (Conneely et al. 1987) was digested
with HindIII at nucleotide 1801, repaired, and ligated to Bsml-
digested, blunt-ended human COUP-TF cDNA (hCOUP) to fuse
the cPR DNA-binding domain to the COOH-terminus of COUP-
TF. A polymerase chain reaction was used to generate a Sacl
site in the COUP-TF cDNA immediately 5’ to the DNA-binding
domain. Sacl-digested COUP-TF cDNA then was ligated to Sacl-
digested PR cDNA to fuse the N-terminus of COUP-TF to the PR
DNA-binding domain.
SOURCE: Data from Power et al. 1991a
86
More than 150 compounds and tissue extracts were tested for their ability to
activate COUP-dependent CAT gene expression. Cells were maintained in
serum-free media, and individual compounds were added immediately after
transfection. The cells were harvested after 48 hours, and extracts were
assayed for expression of CAT activity. The results of these experiments
demonstrated that the neurotransmitter, dopamine, was capable of stimulating
COUP-TF-dependent activation of CAT expression. The data shown in figure 2
demonstrate that concentrations of dopamine as low as 3 mM were capable of
stimulating CAT gene activity fivefold to tenfold (lanes 2 to 7). Dopamine did
not stimulate CAT expression in cells transfected with a parent expression
plasmid that lacked the chimeric COUP-TF coding sequences (lane 12). This
finding indicated that activation of gene expression did not result from a general
increase in transcriptional activity in transfected cells but was dependent on the
expression of a chimeric COUP transcription factor.
The activation of COUP-TF by dopamine does not result from direct ligand
binding because dopamine binding assays demonstrated no direct binding
of COUP-TF to the neurotransmitter. This observation suggested that the
activation of COUP-TF resulted from an indirect signaling pathway. Dopamine
action is mediated by its interaction with several membrane-bound dopamine
receptors. These G-coupled receptors are of two subtypes, D1 and D2,
distinguished by their ability to stimulate, D1, and inhibit, D2, adenylyl cyclase,
respectively (Kebabian and Calne 1979). Analysis of the ability of selective
agonists of these receptor subtypes to activate COUP-dependent gene
expression demonstrated that the dopamine receptor agonist -ergocryptine
mimicked dopamine in its ability to activate CAT expression (lanes 8 to 11).
These data suggested that a dopamine-receptor-mediated signaling pathway
was responsible for the activation of the COUP-TF chimera.
CV1 CELLS POSSESS A DOPAMINE-SENSITIVE ADENYLYL CYCLASE
The presence of dopamine receptors in renal tissue has been documented
(Felder et al. 1989). To determine whether a dopamine-dependent signaling
pathway of the D1 receptor subtype is operative in CV1 cells, the authors
analyzed these cells for expression of a dopamine-sensitive adenylyl cyclase.
The results are shown in figure 3. Dopamine elicited a twofold increase in cyclic
AMP (CAMP) levels in membranes prepared from these cells. Maximum
stimulation was achieved with 100 mM dopamine. Thus, a dopaminergic
responsive cyclase system is intact in CV1 cells.
COUP-TF IS ACTIVATED BY PHOSPHORYLATION
The above data taken together suggested that the activation of COUP-TF by
dopamine may be a phosphorylation-mediated event. Phosphorylation has
87
FIGURE 2. Induction of CAT gene expression by dopamine (D), and
ergocryptine (E). pADFCOUP (5 µg) and PRETKCAT (5 µg)
were cotransfected into CV1 cells as described (Conneely et al.
1987). Cells were cultured for 2 days in serum-free media
supplemented with Nutridoma (Boehringer Mannheim:
Indianapolis, IN) in the absence (lane 1) or presence of varying
concentrations of dopamine (lanes 2 to 7) and -ergocryptine
(lanes 8 to 11). The media was replaced after 24 hours, and
fresh compounds were added. As a control, cells were
transfected with P91023 (B), which lacked COUP-TF coding
sequences, and with PRETKCAT and cultured in the presence
of 100 µM dopamine (lane 12). CV1 cell extracts were prepared
for CAT assays as described (Conneely et al. 1987), and the
assays were performed for 12 hours with 50 µg of protein extract
The results shown are representative of at least six separate
experiments in which duplicate points were performed. The
variation in signals between duplicate points in any one
experiment was not more than 5 percent. In all experiments the
positions of [
14C]chloramphenicol (C) and the 1- and 3-acetylated
forms of [
14C]chloramphenicol are indicated (1AC and 3AC,
respectively).
SOURCE: Data from Power et al. 1991a
88
FIGURE 3. Dopamine-sensitive adenylyl cyclase in CV1 cells. Production of
CAMP in homogenates from untransfected CV1 cells cultured in
Nutridoma-supplemented media was measured as a function of
increasing dopamine concentration. Adenylyl cyclase assays
were performed in duplicate for each point. Homogenization of
ceils and adenylyl cyclase assays were performed exactly as
described (Toro et al. 1987).
SOURCE: Data from Power et al. 1991b
89
previously been shown to play a role in activation of several transcription
factors, including members of the steroid/thyroid receptor family (Glineur et al.
1990; Denner et al. 1990). Therefore, we examined the ability of 8-bromo-cyclic
adenosine monophosphate (8-Br-cAMP) and the protein phosphatase 1 and 2A
inhibitor, okadaic acid, to mimic dopamine in the activation of CAT gene
expression. The result shown in figure 4 demonstrates that these agents also
stimulate COUP-TF-dependent transcription in the authors’ assay system and
thus support the hypothesis that the dopamine activation of COUP-TF is a
phosphorylation-mediated process.
DOPAMINE STIMULATES LIGAND-INDEPENDENT ACTIVATION OF
SEVERAL MEMBERS OF THE STEROID/THYROID RECEPTOR FAMILY
The ability to activate COUP-TF by a signaling pathway stimulated by dopamine
prompted an examination of the classical steroid receptor members of the same
family to determine whether they can be activated by dopamine in the absence
of ligand. The results of these assays are shown in figure 5.
The authors selected several members of this family and found that the
progesterone (cPRA, cPRB), estrogen (hER), and vitamin D
3 (hVDR) receptors
also were activated by dopamine and okadaic acid. Again, CV1 cells were
transfected with the appropriate receptor expression vector and a specific
reporter gene (HREtKCAT) containing the appropriate receptor responsive
enhancer element (HRE) located upstream of the thymidine kinase promoter
and a CAT gene. The level of activation attained with dopamine was
comparable to that attained with the natural ligand for these receptors (figure
5, panels A to D). Interestingly, not all steroid receptors were activated by
this neurotransmitter under our test conditions. CV1 cells cotransfected with a
vector for expression of the human glucocorticoid receptor pRShGRa (Giguere
et al. 1986) and the PRE/GRETKCAT reporter plasmid were transcriptionally
responsive to dexamethasone but were unresponsive to either dopamine or
okadaic acid (panel E). Likewise, cells transfected with a vector for expression
of the human mineralocorticoid receptor pRShMR (Arriza et al. 1987) showed
the expected increase in CAT gene expression in response to aldosterone
and dexamethasone but showed only a marginal response to dopamine and
okadaic acid (panel F). It has been shown that the progesterone receptor can
be activated by 8-Br-cAMP in the absence of hormonal ligand (Denner et al.
1990). The data above are consistent with the notion that the activation of
several members of this family by dopamine occurs through phosphorylation
of these transcription factors via a dopamine-dependent intracellular signaling
pathway. However, confirmation of this mechanism will require direct
identification of a dopamine-dependent phosphorylated site on these
transcription factors.
90
FIGURE 4. Activation of COUP-TF-mediated transcription by
phosphorylation. Cells transfected with pADFCOUP (5 µg)
and PRETKCAT (5 µg) were either untreated (basal) or treated
with 8-Br-cAMP (10-3M) or okadaic acid (OA) (5x10-8M) as
described (Denner et al. 1990).
SOURCE: Data from Power et al. 1991a
91
FIGURE 5. The effect of dopamine on the transcriptional activity of steroid
hormone receptors. Expression vectors (5 µm) for the chicken
progesterone receptor A and B forms (cPRA, cPRB) (Conneely
et al. 1987), human glucocorticoid receptor (hGR) (Giguere et al.
1986), and human mineratocorticoid receptor (hMR) (Felder et
al. 1989) were cotransfected with the reporter plasmid PRE/
GRETKCAT (5 µm) (Jantzen et al. 1987). The human estrogen
receptor (hER) expression construct (2 µm) was cotransfected
with an estrogen receptor-responsive reporter, ERE E1bCAT
(5 µm). The human vitamin D
3 receptor construct (1 µm)
(McDonnell et al. 1989) was cotransfected with a reporter
92
plasmid VDRETKCAT (4 µm) containing three copies of the
vitamin D receptor response element fused to the herpes simplex
virus thymidine kinase promoter and the bacterial CAT gene.
Transfected cells were either untreated (basal) or treated with
10-7M of the relevant ligands for each receptor; progesterone
(P4), estradiol (E2), 1,25(OH)2D3 (VITD3), dexamethasone (DEX),
and atdosterone (ALDO). Cells also were treated with the
indicated concentrations of dopamine (D) and okadaic acid
(OA; 5X10-8M). CAT activity was determined 42 hours
posttransfection.
SOURCE: Data from Power et al. 1991b
DOPAMINE REGULATION OF GENE EXPRESSION IS REGULATED BY A
RECEPTOR OF THE D1 SUBTYPE
To examine the role of D1 and D2 receptors in the mediation of dopamine-
dependent activation of gene expression, we tested the ability of selective
D1 and D2 agonists to mimic the dopamine activation of progesterone
receptor-dependent gene transcription. CV1 cells, transfected with the
cPRA expression vector and the reporter plasmid PRETKCAT, were
treated with either progesterone, dopamine, the selective D1 receptor agonist
SKF38393, or the selective D2 receptor agonist quinpirole. The relative
amounts of CAT activity expressed after treatment of the cells with each
compound are shown in figure 6. The D1-selective agonist SKF38393
stimulated CAT-gene expression to a level even greater than progesterone
or dopamine. However, no stimulation of CAT activity was obtained afler
treatment with the selective D2 receptor agonist quinpirole. Thus, the
activation of the steroid receptors by dopamine appears to result from a
signaling pathway that is mediated by a D1-adenylyl-cyclase-linked receptor.
DISCUSSION
The authors have demonstrated that the neurotransmitter dopamine can
stimulate an intracellular signaling pathway that results in activation of
members of the steroid/thyroid receptor family of transcription factors.
The ability of dopamine D1 receptor agonists to selectively mimic dopamine
in the activation of these factors, together with the demonstration of a
dopaminergic-responsive cyclase system in CV1 cells, suggests that the
pathway is mediated by a dopamine receptor of the D1 subtype. Furthermore,
the activation of COUP-TF and the steroid receptors examined in this study
appears to be mediated by phosphorylation. Receptors activated by dopamine
93
FIGURE 6. Regulation of progesterone receptor-mediated gene transcription
by dopamine receptor agonists. CV1 cells were transfected with
the cPRA expression vector and the PRETKCAT reporter as
described in the figure 2 legend. Transfected cells were either
untreated (basal) or treated with progesterone (P4; 10-7M),
dopamine (400 µM), the selective D1 dopamine receptor agonist
SKF38393 (100 µM), or the selective D2 dopamine receptor
agonist quinpirole (Quin; 20 µM). CAT activity was determined 42
hours posttransfection. Each result represents a mean value
obtained from duplicate experiments.
94
also are activated by 8-BR-cAMP and the phosphatase inhibitor okadaic acid,
whereas other members of the same family of receptors that are not activated
by dopamine also are not activated by 8-Br-cAMP and okadaic acid.
The data obtained in this study provide the first demonstration of a link between
a dopamine-stimulated signaling pathway and transcription factor activation.
The activation of members of the steroid/thyroid receptor family of transcription
factors by dopamine provides a means by which dopamine can regulate gene
expression in dopaminoceptive cells. Furthermore, the data suggest that the
same signaling pathways that mediate short-term cellular responses to
dopamine stimulation also may mediate long-term neuroadaptive responses
to dopamine by reprograming genomic expression. In this regard, additional
studies will be required to substantiate the physiological relevance of this
pathway, to identify which members of the steroid/thyroid receptor family are
expressed in dopaminoceptive cells, and to identify the specific target genes
that are regulated by dopamine.
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AUTHORS
Orla M. Conneely, Ph.D.
Assistant Professor
Ronan F. Power, Ph.D.
Research Associate
Bert W. O’Malley, M.D.
Professor and Chairman
Department of Cell Biology
Baylor College of Medicine
Texas Medical Center
One Baylor Plaza
Houston, TX 77030
97
Regulation of Opioid Gene Expression:
A Model To Understand Neural
Plasticity
Michael J. Comb, Linda Kobierski, Hung Ming Chu, Yi Tan,
David Borsook, Karl Herrup, and Steven E. Hyman
PLASTICITY OF OPIOID GENE EXPRESSION: A MODEL TO ANALYZE
MECHANISM
Over the past several years, it has become clear that neurotransmitter
regulation of gene expression is a critical mechanism underlying neural
plasticity (Golet et al. 1986; Montarolo et al. 1986; Goodman 1990; Sheng
and Greenberg 1990; Morgan and Curran 1991; Comb et al. 1987). The role
of peptide neurotransmitters in this process has received much attention
because they act as important regulators of neural signaling and their synthesis
and expression is dependent on the transcription of precursor genes in the
nucleus. This direct biosynthetic dependence on gene transcription endows
peptide transmitters with special “adaptive” properties that distinguish them
from classical small-molecule neurotransmitters (Comb et al. 1987). Perhaps
the most interesting and best studied example of this process in brain is the
activity-dependent switch in opioid peptide expression in hippocampus (Gall
1988; White et al. 1987; White and Gall 1987; Morris et al. 1988). Electrical
or chemical stimulation of the rodent perforant pathway, the major input to
hippocampus from entorhinal cortex, turns on expression of proenkephalin
and turns off expression of prodynorphin in granule cells of the dentate gyrus.
These changes in opioid precursor result in dramatic and longlasting changes in
proenkephalin and prodynorphin peptides (Gall 1988; White et al. 1987) in
mossy fibers and mRNAs (White et al. 1987; White and Gall 1987; Morris et
al. 1988; Sonneberg et al. 1989) in granule cell bodies. Because enkephalins
have a profound excitatory effect on target hippocampal pyramidal cells, this
activity-driven switch may exert major control over hippocampal excitability
and may be intimately involved in the molecular mechanisms underlying
plastic changes in hippocampal signaling.
98
As opioid peptides are critical regulators of neural pathways mediating pain,
reward, motivation, and hormone release, understanding how their synthesis
and expression is regulated takes on great practical as well as theoretical
significance. With the realization that neurotransmitters regulate endogenous
opioid biosynthesis has come the understanding that drugs that interfere with
or alter neurotransmission also may influence the expression of endogenous
opioids. Hence, neurotransmitters and drugs of abuse acting through synaptic
connections in brain may alter gene expression in the postsynaptic neuron.
Such drug-induced changes in gene expression may underlie important
components of addiction, withdrawal, and drug-seeking behaviors, which
have been particularly difficult to understand at a mechanistic level.
IMMEDIATE EARLY GENE REGULATION IN BRAIN
Seizures and electrical stimulation also trigger a rapid and transient RNA
induction of several different immediate early genes (IEGs) within granule cells
of the dentate gyrus. For example, RNA encoding different components of the
AP-1 transcription factor complex: c-Fos, c-Jun, and JunB are coinduced
(White and Gall 1987; Sonneberg et al. 1989; Saffen et al. 1988; Cole et al.
1989; Wisden et al. 1990) together with proenkephalin mRNA in hippocampal
granule cells by seizure and electrical stimulation of the perforant pathway.
The time course of IEG induction precedes the induction of proenkephalin
RNA (White and Gall 1987; Sonneberg et al. 1989). These observations have
led to the hypothesis that IEGs may act as “third messengers” to initiate and
coordinate programs of gene expression by regulating the expression of
appropriate “target” genes, for example, proenkephalin. This argument is
strengthened by the observation that binding sites for many of these factors
have been found within a compact region of the proenkephalin promoter
(figure 1) known to mediate synaptic and second-messenger regulation (Comb
et al. 1986, 1988; Hyman et al. 1988; Nguyen et al. 1990; Chu et al. 1991;
Kobierski et al. 1991). In addition, these proteins are known transcription
factors; the authors show herein that different components of the AP-1 complex
have different effects on proenkephalin transcription. Regulation of transcription
by the AP-1 complex at the proenkephalin-inducible enhancer represents the
first neural model system to understand IEG target gene regulation and
promises to outline general mechanisms that may regulate the synthesis
of other key neural signaling molecules. The recent discovery that drugs
of abuse such as cocaine (Young et al. 1991), amphetamine (Graybiel et al.
1990), and morphine (Chang and Harlen 1990) also activate the expression
of IEGs suggests that this process may represent an adaptive response of
the brain to drugs of abuse.
99
FIGURE 1. Human proenkephalin gene promoter sequence
TRANSSYNAPTIC REGULATION OF PROENKEPHALIN TRANSCRIPTION
Studies over the past 5 years have defined a short 50-base-pair region of
the human proenkephalin promoter that is necessary and sufficient for
transcriptional regulation by adenosine 3':5'-cyclic phosphate (CAMP)-,
12-O-tetradecanoyl-phorbol ester (TPA)-, and CA++-dependent intracellular
signaling pathways. Extreme conservation of this sequence between human
and rat genes reinforces its critical role in proenkephalin expression and
regulation. Two functional elements were localized within this region and
shown to interact synergistically to mediate second-messenger regulation
(Comb et al. 1986, 1988; Hyman et al. 1988; Nguyen et al. 1990; Chu et al.
1991; Kobierski et al. 1991). Previous studies have identified and characterized
transcription factors that interact with these DNA elements. NF-1 (Chu et
al. 1991) proteins interact with the CRE-1 element, and AP-1 and AP-4
interact with the CRE-2 element (Comb et al. 1988; Hyman et al. 1988;
Nguyen et al. 1990; Chu et al. 1991; Kobierski et al. 1991). Recent
identification and characterization of the CREB/ATF transcription factors
(Hai et al. 1989; Gonzalez and Montminy 1989) prompted the authors to
examine the role of these factors in the regulation of proenkephalin
transcription. DNA binding studies (in vitro) suggested that these factors
interact poorly with the proenkephalin-inducible enhancer as homodimers
and focused our attention on the AP-1 (Fos/Jun) family of transcription factors,
which bind the enhancer with high affinity (Golet et al. 1986).
Work from the authors’ laboratory (Comb et al. 1988; Kobierski et al. 1991)
and Tom Curran’s laboratory (Sonneberg et al. 1989) has demonstrated
that the AP-1 transcription factor binds the proenkephalin CRE-2 element
and that in vitro-translated c-Jun and c-Fos bind the CRE-2 element as Fos/
Jun heterodimers. Our laboratory has also shown that two other Jun
members, JunD and JunB, bind the CRE-2 element and have opposite
effects on proenkephalin transcription (Kobierski et al. 1991). In this chapter,
discussion iS restricted to the role of the AP-1 factors mediating second-
messenger regulation of proenkephalin transcription at the CRE-2 element.
Previous studies have also demonstrated that CAMP rapidly activates
proenkephalin transcription, with increases in steady-state mRNA levels
apparent within 15 minutes after the addition of forskolin to C6 glioma cells
(Kobierski et al. 1991). RNA levels peak at 1.5 to 2 hours following forskolin
addition and then rapidly decline to undetectable levels at between 4 and 8
hours. Activation is rapidly followed by a refractory period during which the
proenkephalin gene is unresponsive to further stimulation of the CAMP pathway.
The forskolin-dependent rapid activation is not affected by protein synthesis
inhibitors: however, protein synthesis inhibitors block the decline in mRNA
101
levels leading to super induction (Kobierski et al. 1991). In addition, forskolin
treatment of C6 glioma cells leads to rapid induction of c-Fos and JunB mRNA
and a reduction in the level of JunD RNA.
CHARACTERIZATION OF JUND, JUNB, AND C-JUN INTERACTIONS WITH
CRE-2
Three observations have focused the authors’ efforts on identifying members
of the AP-1 complex that bind the CRE-2 element and mediate inducible
expression: (1) Multiple copies of this element reconstitute the most essential
features of CAMP-, TPA-, and Ca++-inducible regulation; (2) ATF/CREB
molecules bind this element poorly if at all; and (3) AP-1 complexes bind the
CRE-2 element with high affinity. To test the ability of Fos/Jun molecules to
stimulate or repress proenkephalin transcription, we have developed expression
vectors and a cotransfection assay to determine the effect of expression of
various Fos/Jun molecules on proenkephalin expression. Full-length cDNAs
encoding c-Jun, JunB, c-Fos, and JunD have been introduced into a Rous
sarcoma virus expression vector. Expression vectors are then cotransfected
with pENKAT-12 into F9 cells in the presence or absence of a plasmid
expressing high levels of the cAMP-dependent protein kinase (PKA).
Results of this type of analysis have focused our attention on two different
Jun molecules, JunD and JunB, which have opposite effects on proenkephalin
transcription. JunD strongly transactivates proenkephalin transcription in a
manner that is totally dependent on the catalytic subunit of the CAMP-
dependent PKA (figure 2). JunB has no effect in the presence or absence
of PKA and acts to repress JunD-dependent stimulation of proenkephalin
transcription (figure 3). Finally, c-Jun strongly activates proenkephalin
transcription in a constitutive fashion that is not influenced by PKA (see
figure 2). Activation and repression by JunD, JunB, and c-Jun map to the
CRE-2 element by cotransfection using plasmids containing multicopy elements
and point mutations. We have extended these observations to several other
cell lines, including C6 glioma and SK-N-MC cells, that express proenkephalin.
These findings clearly demonstrate that transactivation of proenkephalin by
JunD is completely dependent on the catalytic subunit of the cAMP-dependent
PKA. In addition, as each of these molecules is expressed in the granule cell of
the hippocampus, JunD constitutively (Wisden et al. 1990) and JunB and c-Jun
in a highly inducible fashion (Saffen et al. 1988; Cole et al. 1989; Wisden et al.
1990), it is likely that these molecules play a critical role in the activation and
repression of proenkephalin gene expression in the nervous system.
102
FIGURE 2. Different effects of Jun proteins on proenkephalin transcription.
Plasmids (5 µg each) expressing JunD (RSVJunD), JunB
(RSVJunB), and c-Jun (RSVc-Jun) were cotransfected into
F9 cells together with a proenkephalin/CAT fusion gene
(pENKAT-12 5 µg) in the presence or absence of a plasmid
expressing the catalytic subunit of the cAMP-dependent PKA
(pMTCalpha 5 µg). In addition, a plasmid, pRSVßGal (10 µg),
was also included in each transfection, and ß-Gal expression
was used to normalize CAT expression between transfection
experiments. Data presented are from one experiment but are
representative of at least five different experiments.
CHARACTERIZATION OF JUND AND JUNB BINDING AT THE
PROENKEPHALIN ENHANCER
To examine the binding of JunD and JunB to the CRE-2 element, RNA
encoding JunD, JunB, and c-Fos was transcribed and then translated
individually and in combinations as described below in rabbit reticulocyte
lysates. Control lysates show no specific binding to a 30-base-pair
oligonucleotide, spanning the proenkephalin enhancer from -110 to -80.
However, lysates programed with JunD RNA produce a shifted band,
which is specifically self-competed but not competed by AP-2 or AP-4
oligonucleotides and is very weakly competed by a CRE-1 oligonucleotide.
Both TRE and CRE oligonucleotides compete effectively for JunD DNA
103
FIGURE 3. JunB represses PKA-dependent activation of proenkephalin by
JunD. Plasmids were cotransfected into F9 cells as described in
figure 2. In each case, 5 µg of DNA was used except where
indicated for pRSVJunB.
binding. In addition, mutations within the 3’-end of CRE-2, which have the
most profound effect on second-messenger regulation of proenkephalin
transcription (Comb et al. 1988), also produce the most severe effect on
JunD binding.
In contrast, binding of in vitro-translated JunB to the enhancer is not
observed. Because JunB is known to dimerize with JunD and c-Fos (Ryder
et al. 1989; Nakabeppu et al. 1988), we also analyzed the interaction of these
complexes with the CRE-2 element. Consistent with previous reports using
AP-1 oligonucleotides, binding of JunB and JunD to the CRE-2 element is
dramatically stimulated in the presence of c-Fos.
CLASSIC CRE AND TRE ELEMENTS ALSO MEDIATE PKA-DEPENDENT
TRANSCRIPTION VIA JUND
Because the proenkephalin enhancer contains a hybrid TRE/CRE-like element,
CRE-2, we tested the ability of JunD to transactivate “classic” TRE and CRE
elements in a PKA-dependent fashion by transiently transfecting F9 cells
with plasmids containing the somatostatin CRE, or the collagenase TRE,
104
together with pRSVJunD and pMTCaneo. JunD strongly transactivates the
somatostatin CRE element and transactivates the collagenase TRE to a
somewhat lesser extent. These results indicate that JunD can act at both
CRE and TRE elements to stimulate transcription in a PKA-dependent fashion.
ANALYSIS IN TRANSGENIC ANIMALS
To extend the analysis of mechanisms underlying activity-dependent regulation
of proenkephalin transcription to the intact nervous system, we have made
transgenic animals expressing proenkephalin/LacZ fusion constructs. The
first construct analyzed contains 3 Kb of human proenkephalin 5'-flanking
sequences and 1.5 Kb of 3’-flanking sequences driving expression of the
LacZ reporter. Expression of this construct has been examined in three
independent transgenic lines. This construct also contains the well-
characterized CAMP-, TPA-, and Ca++-inducible enhancer located within
the first 110 bases of 5’-flanking DNA (Comb et al. 1986) and is shown to
direct correct tissue-specific expression to all (with only two known exceptions,
striatum and olfactory tubercle) sites within the adult reproductive (Borsook
et al., in press) and nervous systems. Although some variation between
founder animals is apparent, there is a remarkable degree of tissue-specific
transgene expression observed in the adult mouse brain.
In brain, as in the reproductive system, ectopic expression of the transgene
is not seen. As illustrated in table 1, expression on the transgene in various
brain regions appears to be remarkably precise as determined by X-Gal
staining. LacZ expression has been most carefully examined in founder
#3 where X-Gal staining is seen in the spinal cord, thalamus, hypothalamus,
hippocampus, entorhinal cortex, cerebral cortex, amygdala, dorsal horn of
the periaqueductal gray matter, ventral tegmental area, pons and medulla
oblongata, and many other brain regions. As expected, the major sites
of hypothalamic transgene expression are the paraventricular nucleus
and ventromedial nucleus. High-level expression is also seen in the
central nucleus of the amygdala in excellent agreement with previous
immunohistochemical studies of cholchicine-treated rats. In addition,
correct tissue-specific expression is seen in laminae I and II (substantia
gelatinosa) of the dorsal horn of the spinal gray matter and is also observed
in scattered cells throughout laminae IV and V-VII. A comparison between
LacZ expression and previously reported expression of proenkephalin peptides
and mRNA is also shown in table 1. Taken together, these remarkable results
suggest that the 30 Kb proenkephalin/LacZ fusion gene directs expression of
LacZ to the vast majority of diverse sites where proenkephalin is normally
expressed in the adult. The one anomaly in brain expression is striatum.
Little or no expression of LacZ is seen in striatum of each of the three
independent founder lines examined. The missing or low-level expression
105
TABLE 1. Comparison of transgene LacZ expression vs. proenkephalin
mRNA (in situ) and peptide immunoreactivity
Structure
ß-Gal* In situ
lmmunoreactivity
Telencephalon
Olfactory bulb
Cingulate cortex
Entorhinal cortex
Olfactory tubercle
Caudate-putamen
Lateral septum
Bed n. stria termin
Diagonal band Broca
Preoptic area
Amygdala central
Medial
Cortical
Anterior
Lateral
Dentate gyrus
Hippocampal pyramidal
Diencephalon
Hypothalamus anterior
Perifornical
Lateral
Suprachiasmatic
Paraventricular
Ventromedial
Thalamus lateral geniculate
Periventricular
Mesencephalon and
rhombencephalon
Periaqueductal gray
Ventral tegmental
Locus coeruleus
Pontine reticular
Raphe magnus
Gigantocellular reticular formation
Raphe pallidus
Cerebellum (Golgi II)
Spinal cord (I, II, V, X)
++
—
+++
—
—
++
+
++
+
++++
+++
+++
++
+
+
+
++
+++
++
—
++
++++
+++
+
+++
++
+
++
+++
+++
+++
+
—
ND
+++
+++
++++
++++
++
+
+
+
++++
+++
+++
++
+
+
+
++++
+++
++
ND
++
++++
++
+
+
+++
+
+
+++
++
+
+++
++
+++
+++
+++
+++
++
++
++
+
+++
+
+++
+
+
+
+
++
+++
+
+/-
++
+
++
+
++
+++
+
+
+++
++
+
—
+++
KEY (relative intensity of expression): -=none detected; ND=not determined; and
increasing intensity=from + to ++++
*ß-Gal staining in mouse; in situ data and immunohistochemical data from rat,
adapted from Fallon and Leslie (1986)
106
in striatum and olfactory bulb suggests that additional element(s) may be
necessary for expression at these sites. Alternatively, these elements
may be present, yet are more sensitive to the site of chromosomal integration.
To further investigate this possibility, the authors are producing and analyzing
additional independent founder animals.
SUMMARY AND CONCLUSION
The recent finding that neurotransmitters and drugs that affect
neurotransmission have important influences on gene expression suggests
that drug-induced alterations in gene expression may underlie many
long-term effects of addictive drugs, for example, dependence and
drug-seeking behaviors. These long-term adaptive responses to opiate
drugs have been particularly difficult to understand at a mechanistic level.
Data presented here indicate that the gene encoding the opioid precursor
proenkephalin is highly regulated by neural activity, second-messenger
pathways, and PKA. These observations raise the possibility that drugs
of abuse (e.g., opiates acting through opiate receptors) may act at the
genetic level to modulate the expression of endogenous opiates and that
these effects may underlie one component of the brain’s long-term adaptive
response to exogenous opiates. The transgenic animals described above
can be used to investigate opiate drug-induced changes in proenkephalin
gene expression, allowing rapid analysis of changes in proenkephalin gene
expression in highly restricted populations of neurons in a fashion previously
impossible. In addition, by analyzing the effects of specific enhancer mutations
on tissue-specific and transsynaptic regulation of proenkephalin expression,
transgenic models will permit mechanistic investigations within the intact
nervous system that cannot otherwise be undertaken.
Investigation of mechanisms underlying this process requires the analysis of
intracellular signaling pathways, responsive DNA regulatory elements, and the
transcription factors transducing synaptic signals into gene regulation. In the
studies described herein, we demonstrate that AP-1 complexes consisting of
different Jun proteins differentially regulate proenkephalin transcription at the
CRE-2 element. c-Jun constitutively activates proenkephalin transcription,
whereas JunD activates in a fashion completely dependent on the activation of
second-messenger pathways and the cAMP-dependent PKA. JunB alone has
no effect on proenkephalin gene expression, yet this molecule effectively blocks
activation mediated by JunD and, hence, may act as a repressor. These data
are consistent with a model (figure 4) in which preexisting JunD mediates the
rapid cAMP-dependent activation of the proenkephalin enhancer, whereas IEGs
such as JunB or c-Fos mediate the protein synthesis-dependent inactivation.
Because c-Jun activates proenkephalin transcription constitutively, induction
107
FIGURE 4. Model for activation and repression of proenkephalin transcription by AP-1 complexes.
Neurotransmitters or drugs acting through neurotransmitter receptors activate second-messenger
pathways and PKA. Activated PKA rapidly dissociates to the nucleus and phosphorylates substrates,
leading to activation of preexisting JunD/CREB-like transcription factors, which in turn rapidly activate
proenkephalin transcription. These factors also activate IEGs such as c-Jun and JunB, which may
further stimulate or repress proenkephalin transcription.
of c-Jun may lead to a further and prolonged activation of proenkephalin gene
expression. Hence, the ratio of c-Jun to JunB induction may determine whether
proenkephalin is repressed or further activated.
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ACKNOWLEDGMENTS
The work described in this chapter was supported in part by U.S. Public Health
Service grants DA-05706 and DA-00151 (MJC).
AUTHORS
Michael J. Comb, Ph.D.
Assistant Professor
Yi Tan, M.D.
Research Fellow
Laboratory of Molecular Neurobiology
Massachusetts General Hospital
Charlestown, MA 02129
Linda Kobierski, B.A.
Graduate Student
Hung Ming Chu, Ph.D., M.D.
Graduate Student
Program in Neuroscience
Harvard Medical School
Boston, MA 02129
David Borsook, M.D., Ph.D.
Instructor
Department of Anesthesia
Massachusetts General Hospital
Harvard Medical School
Boston, MA 02129
111
Karl Herrup, Ph.D.
Associate Professor
Department of Developmental Neurobiology
E.K. Shriver Center
200 Trapelo Road
Watham, MA 02254
Steven E. Hyman, M.D.
Assistant Professor
Laboratory of Molecular Neurobiology
Department of Psychiatry
Massachusetts General Hospital
Charlestown, MA 02129
112
Cellular and Molecular Analysis of
Opioid Peptide Gene Expression
Cynthia T. McMurray, Karen M. Pallock, and James Douglass
INTRODUCTION
The molecular mechanisms underlying behavioral and physiological events
associated with the intake of addictive drugs such as cocaine, amphetamine,
and opiates are poorly understood. The problem of physical dependence to
and withdrawal from these drugs is challenging to approach experimentally,
as the establishment of these states likely involves a complex cascade of
biochemical and electrical events. Such events involve the diverse and
widely distributed array of central nervous system (CNS) neurotransmitters,
receptors, ion channels, and transporters and extend to complex autonomic
and sensorimotor neural networks. In addition, various events associated
with drug-seeking behavior and addiction are also influenced by genetic,
species, and environmental factors, which only serve to further complicate
experimentation in this area.
There is growing evidence that specific changes in neuronal transcription
patterns result from the administration of narcotic drugs. For example, it
has been reported that the administration of morphine significantly
decreases striatal levels of proenkephalin mRNA in the rat (Uhl et al. 1988),
and chronic naloxone treatment increases expression of both proenkephalin
and protachykinin in the rat striatum (Tempel et al. 1990). In striatum,
prodynorphin gene expression is increased following chronic administration
of the indirect dopamine antagonists methamphetamine and cocaine (Sivam
1989; Hanson et al. 1987). Thus, it appears that administration of narcotic
drugs can induce localized alterations in the synthesis of mRNA encoding
striatal peptide neurotransmitters. More recently, evidence has been presented
suggesting that the aforementioned transcriptional alterations may be mediated
by rapid, transient changes in expression of specific immediate early genes,
such as c-fos. For example, amphetamine and cocaine can induce drug-
specific activation of the c-fos gene in striosome-matrix compartments and
limbic subdivisions of the striatum (Graybiel et al. 1990). Furthermore, this
induction appears to involve the D1 dopamine receptor system (Young et al.
113
1991). Thus, to continue to gain insight into the nuclear events associated
with substance abuse, researchers must understand in greater detail the basic
mechanisms that regulate the expression of pertinent transcriptionally active
genes in the CNS, such as those mentioned above.
REGULATED EXPRESSION OF THE RAT PRODYNORPHIN GENE
Distribution and Physiological Effects of Dynorphin Peptides
The prodynorphin precursor encodes the dynorphin family of opioid peptides
(Kakidani et al. 1982; Civelli et al. 1985). Northern blot and in situ histochemical
analysis have determined that the prodynorphin gene is transcriptionally active
in a wide variety of cell types within the central and peripheral nervous systems
(Akil et al. 1984; Civelli et al. 1985). Such regions include the hypothalamus,
striatum, hippocampus, midbrain, brain stem, and cerebral cortex, as well as
neurons within the spinal cord and gut. Prodynorphin transcripts are also
present at relatively high levels in endocrine tissues, such as the anterior
pituitary, adrenal, testis, ovary, and uterus.
The opioid receptor subtype exhibiting specificity for prodynorphin-derived
peptides is the -opioid receptor (for review, see Mansour et al. 1988). The
receptor belongs to the G protein-linked family of receptors and is also coupled
to calcium conductances. This latter characteristic serves to biochemically
distinguish the -receptor from the µ- and -opioid receptor subtypes, which
are coupled to potassium conductances. Studies utilizing radiolabeled ligands
specific for -receptor binding have determined that this species of opioid
receptor is widespread throughout the CNS and endocrine system.
As with the opioid peptides derived from the proopiomelanocortin and
proenkephalin precursors, it has been difficult to assign precise physiologically
relevant functional roles to those peptides derived from the prodynorphin
precursor. However, numerous studies have suggested that the dynorphin
peptides serve to regulate a wide variety of physioIogical and behavioral
responses (Mansour et al. 1988). These roles include the mediation of
visceral analgesia, effects on appetite suppression, mediatron of hypotensive
cardiovascular responses, inhibition of vasopressin secretron and possibly
additional renal functions, involvement in modulation of motor seizure
thresholds and intensity and involvement in recovery from spinal cord injury
and stroke.
A role for dynorphin peptides in narcotic tolerance mechanisms
also has been sugested.
114
Transcriptional Regulation of the Rat Prodynorphin Gene In Vivo
The cloning of rat prodynorphin cDNA and genomic DNA (Civelli et al. 1985;
Douglass et al. 1989) has afforded the opportunity to study regulation of
prodynorphin gene expression in distinct neuronal cell types following specific
surgical or pharmacological manipulations. The spinal cord and hippocampus
represent those neural systems in which the most dramatic alterations of
prodynorphin gene expression have been observed to date.
In the spinal cord, prodynorphin transcripts and peptides are localized to
laminae I-II and V-VI, suggestive of a functional role in endogenous pain
recognition and control. Following the onset of unilateral inflammation of the
hindlimb, prodynorphin biosynthesis is substantially elevated in the region of
the spinal cord receiving sensory input from the affected limb (ladarola et al.
1988; Naranjo et al. 1991). Prodynorphin mRNA levels rise substantially within
the first 24-hour period, and maximal stimulation (eightfold to ninefold increase)
is observed between 3 and 5 days after the onset of inflammation. By day 14,
prodynorphin mRNA levels have approached control values. This time course
of induction and subsequent decline closely parallel that of hindlimb edema and
hyperalgesia. Spinal cord DynA1-8 levels also rise approximately threefold
during the inflammatory period, consistent with an increase in both the rate of
synthesis and release of dynorphin peptides from spinal cord neurons. These
data suggest the active participation of dynorphin-containing spinal cord
neurons in the modulation of sensory afferent input during peripheral
inflammatory pain states.
Opioid peptides derived from the prodynorphin precursor are also present
at high levels in the rat hippocampus. In this brain structure, dynorphin
immunoreactivity exhibits a restricted distribution, being localized to the
granule cell/mossy fiber axonal system (McGinty et al. 1983). Over the past
several years, numerous groups have documented striking changes in the
levels of hippocampal prodynorphin mRNA following the onset of seizure
activity. In one study, the acute effects of kainic acid (KA) administration were
examined (Douglass et al. 1991). Following a single subcutaneous injection
of KA (8 mg/kg), hippocampal prodynorphin mRNA levels rose thirteenfold to
fourteenfold within 3 hours, began to decline at 12 hours, and by 48 hours were
at or below control values. Although mRNA levels were dramatically stimulated,
hippocampal DynA1-8 levels remained at values significantly below control at all
time points monitored. This observation suggests that KA treatment also results
in a prolonged stimulation of release of dynorphin peptides from hippocampal
neurons.
115
Interestingly, other paradigms that induce seizure activity, such as
electroconvulsive shock (Xie et al. 1989a), prepyriform cortical kindling (Xie et
al. 1989b), and hippocampal kindling (Morris et al. 1987), serve to significantly
reduce hippocampal prodynorphin mRNA levels to 25 to 40 percent of control
values. Thus, it appears that the prodynorphin gene is capable of being both
positively and negatively transcriptionally regulated in the granule cells of the
dentate gyrus.
Characterization of the Rat Prodynorphin Gene
The studies described above document changes in prodynorphin mRNA
levels in various neuronal cell types and systems. Presumably, these changes
are the result of alterations in the rate of transcription of the prodynorphin
gene. The authors have isolated and characterized the rat prodynorphin gene
(Douglass et al. 1989) to gain an understanding of the molecular events that
mediate transcriptional regulation of the gene. Such analysis has led to the
identification of the rat prodynorphin mRNA CAP site and promoter element
and to the initial characterization of putative transcripitional regulatory elements.
As diagramed in figure 1, the rat prodynorphin gene is composed of four
exonic domains. Exons 1 and 2 encode 5’ untranslated regions of the mRNA,
whereas exon 3 encodes 15 bases of 5’ untranslated region and the first 23
amino acids of the prohormone. Exon 4 encodes the remaining 204 amino
acids of the prohormone, including the three leucine-enkephalin moieties, as
well as the entire 3’ untranslated region. In the CNS, exons 1 through 4 are
spliced together to generate a mature 2,400-base transcript. However, in the
testis (and perhaps adrenal, as well), exon 2 is not contained within the smaller
2,300-base transcript (Garrett et al. 1989). Thus, the 5’ untranslated region
beginning 15 bases from the translational start site is unique in the two species
of prodynorphin mRNA. Sucrose gradient polysome analysis of striatal and
testicular prodynorphin mRNA reveals that both species of mRNA are present
on high molecular weight polysomes, suggesting that the mRNA is efficiently
translated in both tissues (Garrett et al. 1989). The functional significance of
this alternate splicing event involving prodynorphin mRNA is currently unknown.
Analysis of the Rat Prodynorphin Gene Promoter
In vivo studies have documented that rat prodynorphin mRNA levels in specific
neuronal cell types can be dramatically altered following neural stimulation.
These changes presumably reflect alterations in the rate of synthesis of the
prodynorphin transcript. Thus, signals received by prodynorphin-expressing
neurons must be relayed to the cell nucleus, resulting in altered levels of
transcription of the prodynorphin gene. Such transcriptional effects are
116
FIGURE 1. Structure of the rat prodynorphin gene. Alternate splicing of rat prodynorphin precursor mRNA occurs to
generate different forms of mature mRNA in the brain and testis.
presumably mediated through precise interactions between specific DNA
binding proteins (i.e., trans-acting factors) and their target recognition sequence
elements near the prodynorphin promoter region (i.e., cis-acting elements). As
a first step in characterizing such interactions, the authors have determined the
nucleotide sequence of approximately 3.8 kb of rat genomic DNA located 5’ to
the prodynorphin mRNA CAP site. Figure 2 diagrams the location of potential
DNA regulatory elements near the promoter region of both the rat and the
human (Horikawa et al. 1983) prodynorphin genes, as identified by computer
sequence analysis.
For the rat prodynorphin gene, the nucleotide sequence TAGCGTCAG is
present in what constitutes the 5’ untranslated region of the transcript at
position +62. This element is highly homologous to the cyclic AMP (CAMP)
response element present in several neuroendocrine peptide genes (Goodman
1990). At position -102 is the sequence GCCAAT, representing the consensus
binding site for the enhancer protein, EBP20 (for review of trans-acting protein
factors, see Jones et al. 1988). Two potential AP-2 binding sites (AP-2
site=CCCCAGGC) are located at positions -973 and -1826; AP-2 is a 52-kD
protein that has been shown to mediate both phorbol ester and CAMP-
dependent transcriptional effects. The sequence GGGGCGG at position -1281
represents a potential binding site for the transcriptional activator protein, SP1.
Last, the sequence TGCGTCAG at position -1543 represents a sequence
identical to that found within the human proenkephalin promoter, referred to as
ENKCRE-2. This element is essential for both basal and regulated enhancer
function and binds the AP-1 complex with high affinity (Comb et al. 1988). The
same sequence element is also present at the human c-fos promoter, mediating
cell type-specific transcriptional properties (Velcich and Ziff 1990). The human
prodynorphin promoter also contains consensus binding sequences for AP-2,
SP1, and EBP20, although the sites are not present at the same location
relative to the mRNA CAP site when compared with the rat gene. However,
the nucleotide sequence from approximately -500 to +100 is highly conserved
between rat and human, suggesting that uncharacterized regulatory sequence
elements may be localized to this region.
To begin to determine if any of the aforementioned sequence elements are of
functional significance, various regions of the rat prodynorphin promoter have
been placed 5’ to the bacterial chloramphenicol acetyl transferase (CAT)
coding region and expressed transiently in CV1 cells. Analysis of several
different plasmid constructs is shown in figure 3. A minimal promoter fragment
representing nucleotides -122/+135 has relatively weak promoter activity, and
low levels of CAT activity are produced. This activity serves as a relative
reference point for analysis of other prodynorphin promoter/CAT plasmid
constructs. When the promoter region is extended in the 5’ direction to -598
118
FIGURE 2. Comparison of rat and human prodynorphin promoter regions. Pertinent restriction enzyme sites
are noted. Also noted are the locations of nucleotide sequences that have perfect homology with
characterized binding sites of specific DNA binding proteins. CAP sites, TATA elements, 5’ ends of
characterized cDNA clones, and exon/intron junctions are also shown for both genes. Lastly, the region
exhibiting the greatest degree of nucleotide sequence conservation between the two species is shaded.
FIGURE 3. Basal prodynotphin promoter CAT activity levels.
Characterization of the ability of rat prodynorphin genomic
DNA fragments to serve as transient transcriptional promoters.
Nucleotides representing 5’ and 3’ ends of rat prodynorphin
genomic DNA fragments (containing the mapped CAP site at
position +1, and the TATAAA promoter element at -28) ligated
proximal to the CAT structural gene are shown. The resulting
plasmids were transfected into CV1 cells, and cellular CAT
activity levels measured. Following standardization, CAT activity
levels were normalized against those for the minimal promoter
construct. The sequence and location of potential regulatory
elements are also noted.
or -930, again, low levels of CAT activity are observed. However, when the
promoter region is extended to -1250, -1504, or -1860, relative levels of CAT
activity are induced, 10-, 35-, and 60-fold, respectively. These data suggest
that positive transcription sequence elements, such as those described above,
are located in this region of genomic DNA. In addition, a positive transcription
sequence element appears to be present in the 5’ untranslated region of the
120
prodynorphin transcript between nucleotides +60 and +135, as there is an
approximately sixfold reduction in CAT activity when comparing the -1860/+135
promoter region to the -1860/+60 fragment. This positive effect may be
mediated through the cAMP-responsive element (CRE)-like element present
at +62. Thus, positive sequence elements both 5’ and 3’ to the CAP site may
act in concert to strengthen transcription from the promoter.
To begin to determine if positive-acting elements in the 5’ region of the promoter
can function outside of their natural context, the -1860/-1504 and -1504/-1250
fragments were placed in front of the minimal -122/+135 promoter. Surprisingly,
relative CAT activity levels were approximately 600- and 300-fold elevated,
respectively, when compared with -122/+135 CAT levels. Two possible
explanations for this observation are that negative transcription sequence
elements are present in the region of genomic DNA from -1250 to -122 or that
the spacing of the positive transcription elements relative to the promoter plays
a role in their efficacy.
HAIRPIN FORMATION WITHIN THE ENHANCER REGION OF THE HUMAN
PROENKEPHALIN GENE
The proenkephalin gene encodes the polyprotein precursor to the enkephalin
family of opioid peptides. The human proenkephalin gene promoter has been
extensively characterized (Comb et al. 1988), and a wide variety of DNA binding
proteins are able to interact with high degrees of affinity and specificity with the
promoter region from nucleotides -110 to -70. Site-specific mutational analysis
has determined that two specific sequence elements designated as ENKCRE-1
(-104 TGGCGTA -98) and ENKCRE-2 (-92 TGCGTCA -86) play a critical role
in the transduction of signals transmitted from cell surface receptors to the
proenkephalin nuclear transcription complex (Comb et al. 1988). Analysis of
the promoter region containing ENKCRE-1 and ENKCRE-2 reveals that these
elements are contained within a 23bp imperfect palindrome and that each
strand has the potential to form not only a duplex structure but also hairpin
structures (figure 4). A unique feature of these hairpin structures is that each
would form with mismatched base pairs: the top strand (GT) forming two GT
pairs and the complementary bottom strand (AC) forming two AC pairs.
The sequence shown in figure 4 was chemically synthesized to investigate
hydrogen bonding properties within each strand as well as the duplex
(McMurray et al. 1991). Melting experiments employing the single-stranded
oligonucleotides demonstrated that increases in temperature result in a highly
cooperative increase in absorbance, indicative of the large degree of base
unstacking found in hydrogen-bonded structures upon melting. At neutral
pH, 45 °C was the temperature at the midpoint for transition (tm) of the GT
121
FIGURE 4. Potential secondary structures formed by the human enkephalin
enhancer region. For the duplex structure, the position of
nucleotides relative to the endogenous CAP site are noted. The
shaded boxed sequences represent elements CRE-1 and CRE-2,
which constitute the cAMP-inducible enhancer. For the GT and
AC hairpin structures, the shaded boxes indicate the positions of
the mismatched base pairs.
SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region
of the human enkephalin gene. Copyright 1991 by Cynthia T.
McMurray (Rochester, MN).
strand, 49 °C for the AC strand, and 63 °C for the duplex. In addition, the
hyperchromicity for the melting of each strand was roughly half the value for
the melting of the duplex. Thus, in the absence of their partner strand, the
individual strands are capable of forming stable hairpin structures, despite
the fact that each hairpin forms with two mismatched base pairs.
122
To determine the possibility of induction of structural transitions within the
human enkephalin enhancer, the conformational state of the native enhancer
duplex has been examined under a variety of conditions. A G-50 column
chromatography/nondenaturing PAGE system has served as the means
of analysis of column-purified, radiolabeled oligonucleotides (McMurray et
al. 1991), as shown in figure 5. At a solution pH of 7.0, the double-stranded
enhancer duplex (first lane noted as D) is easily distinguished from the
single-stranded forms (the AC hairpin is sample AC; the GT hairpin is sample
GT) by its greatly reduced mobility. In addition, the single-strand forms are
observed as doublets, with the faster migrating form corresponding to the
hairpin conformation and the slower migrating form corresponding to the
linear strand conformation (C.T. McMurray, unpublished results). In solution,
the duplex form of the 23bp human enkephalin enhancer is able to dissociate
into the AC and GT single-strand forms by altering the pH; lane E represents
radiolabeled enhancer duplex in pH 5.5 buffer analyzed on a pH 7.0
polyacrylamide gel. Under conditions in which the solution pH is raised to
7.0, the oligonucleotides once again migrate as a double-stranded duplex form,
as shown in sample lane D. This pH-dependent conformation/migration pattern
is presumably due to the ability to switch from double-stranded duplex to
single-stranded hairpin conformations, as a nonpalindromic 23mer continues
to migrate as a duplex under pH 5.5 conditions (sample lane C). Thus,
incubation of the enkephalin enhancer oligonucleotide duplex in pH 5.5
buffer results in complete conversion of the duplex form to the single-strand
forms. Furthermore, the process is reversible, as the single-stranded forms
can convert to the duplex form by adjusting the solution pH to 7.0. It is also
noteworthy that the lowest pH condition employed in the study is well above
that where significant base protonation generally occurs and is well within the
pH range where nucleic acid duplexes are stable.
To further examine the nature of this pH effect, the stability of the enhancer
duplex, as well as both of the individual strands that constitute the duplex,
was measured as a function of pH (figure 6) (McMurray et al. 1991). Stability
was determined by observing the midpoint of the thermal transition (tm) for
each conformation. For the native enhancer duplex (open squares), thermal
stability varies only 5 °C over the pH range from 5.5 to 9.0. The duplex exhibits
maximal stability in the pH range from 7.0 to 9.0. The GT hairpin is also
quite stable over the same pH range; from pH 5.0 to 9.0 only a 5 °C change in
tm is observed. In marked contrast, however, the stability of the AC hairpin
displays the opposite pH-dependent stability profile relative to both the duplex
and the GT strand. The AC strand undergoes a 30 °C increase in stability as
the pH decreases from 9.0 to 5.5. At pH 9.0, conditions under which both
the duplex and the GT strand are most stable, the AC strand is essentially
denatured. Between pH 6.0 and 5.5, the stability of the AC hairpin is actually
123
FIGURE 5. PAGE analysis of G-50 column fractions for the GT and AC
strand, a nonpalindromic 23mer duplex, and the human
enkephalin enhancer duplex. In the left three lares are standards
representing the duplex (D), the AC hairpin (AC). and the GT
hairpin (GT). Lane A, GT strand in pH 5.5 buffer analyzed on a
pH 7.0 gel (1x10-8M base pairs); lane B, AC strand in pH 5.5
buffer analyzed on a pH 7.0 gel (1.5x10-8M base pairs); lane C, a
nonpalindromic 23mer duplex in pH 5.5 buffer analyzed on a pH
5.5 gel (1.3x10-8M base pairs); lane D, the human enkephalin
enhancer duplex in pH 7.0 buffer analyzed on a pH 7.0 gel
(1.8x10-8M base pairs); lane E, the human enkephnlin enhancer
duplex in pH 5.5 buffer analyzed on a pH 7.0 gel (1.8x10-8M base
pairs): lane F, the human enkephalin enhancer duplex in pH 5.5
buffer analyzed on a pH 5.5 gel (1.8x10-4M base pairs).
SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region
of the human enkephalin gene. Copyright 1991 by Cynthia T.
McMurray (Rochester, MN).
124
FIGURE 6.
SOURCE:
pH dependence of melting transitions for the human enkephalin
enhancer duplex
the GT strand and the AC strand
T°C represents the temperature at the midpoint for transition.
McMurray et al. 1991. Hairpin formation within the enhancer region
of the human enkephalin gene. Copyright 1991 by Cynthia T.
McMurray (Rochester, MN).
125
greater than that of the double-stranded duplex. Thus, under slightly acidic
conditions, the energy difference between the duplex and both of the hairpin
forms is small, and under low concentration conditions (data not shown), the
hairpin conformation becomes the favored form.
The dramatic pH-dependent increase in stability of the AC strand arises in
part from protonation (figure 7), with additional hydrogen bonding presumably
occurring from protonation of the N1 of adenine within the AC mismatched pair.
From the pH titration curve shown in figure 6, the midpoint for protonation of
the AC strand is near physiological pH of 7.2. The importance of measuring
the pKa is that it allows identification of the relevant range of protonation.
The AC strand is approximately 80 percent protonated at pH 6.6 and 20
percent protonated at pH 7.8. Thus, changes of only 0.5 of a pH unit create
conditions under which the AC hairpin is either largely destabilized or highly
stable. A corresponding pKa shift of this magnitude for protonation of ring
nitrogens could be easily mediated by the presence of a nearby charged
group, such as local interaction of a charged protein containing regions of
acidic or phosphorylated residues. In vivo, the presence of a nearby positive
charge may be the driving force to induce the formation of a cruciform structure
at this specific region of the enkephalin enhancer. Thus, proton transfer and
stabilization of the AC hairpin, at the expense of duplex formation, may be a
biological switch for the formation of a cruciform structure.
Whether this transition has subsequent effects on the binding of additional
trans-acting factors or polymerase subunits that control transcription from the
proenkephalin promoter remains to be determined. There is, however, an
intriguing piece of information that suggests the functional significance of the
ability to form stable cruciform structures. Extensive site-specific mutational
analysis of the human enkephalin enhancer region (Comb et al. 1988) has
identified two mutations within ENKCRE-1 and ENKCRE-2 that stimulate
approximately twofold basal levels of transcription from the promoter, as well
as retaining a high degree of inducibility by CAMP. For one of these mutations,
specific patterns of protein binding have been characterized and appear to be
unaffected. Interestingly, both mutations serve to make perfect base pairs out
of the mismatched pairs. Thus, mutations that potentially stabilize the cruciform
structure, while having no apparent effect on specific DNA-protein interactions,
result in increased expression in vivo.
It is also noteworthy that other cAMP-responsive genes contain CREs within or
near sequences capable of forming imperfect palindromes (figure 8) (McMurray
et al. 1991). Furthermore, many of these regulatory regions have the potential
to form cruciform structures, with AC or GT mismatches representing the major
126
FIGURE 7. Schematic diagram of the hydrogen binding pattern of an AT
base pair and an AC mismatch at pH 7.0 and 5.5. The AC
mismatch pair contains one hydrogen bond at pH 7.0 and binds
an additional proton under acidic conditions. Protonation can
occur at the N1 of adenine, giving rise to an additional hydrogen
bond.
SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region
of the human enkephalin gene. Copyright 1991 by Cynthia T.
McMurray (Rochester, MN)
127
FIGURE 8. Model for cruciform structures that can potentially form at or near
the CREs of several genes. Both linear and potential cruciform
structures are shown for the top strand only. Hatched boxes
represent characterized CREs for each gene. In the cruciform
conformation, the horizontal boxes indicate the position of base
pair mismatches. Henk, human enkephalin gene; VIP, human
vasoactive intestinal peptide gene; CRH, rat corticotropin-
releasing hormone gene; PEPCK, rat phosphoenolpyruvate
carboxykinase gene; TH, rat tyrosine hydroxylase gene.
SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region
of the human enkephalin gene. Copyright 1991 by Cynthia T.
McMurray (Rochester, MN).
128
species of mismatch base pairing. Thus, formation of cruciform structures may
be a general feature of certain classes of CREs. This observation, along with
the unique physical and thermodynamic properties of the enkephalin enhancer
region, suggest a model in which protein-mediated structural changes within the
enhancer region DNA play an active role in regulated expression of the human
proenkephalin gene via the formation of a cruciform structure. The main
challenge for the future will be to determine if such a cruciform structure is
capable of forming in vivo when the enhancer region is placed in the context of
nuclear DNA.
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ACKNOWLEDGMENTS
The studies described were supported in part by National Institute on Drug
Abuse grant DA-04154 (JD) and by postdoctoral fellowship PF-2997 from the
American Cancer Society (CTM).
AUTHORS
Cynthia T. McMurray, Ph.D.
Assistant Professor/Senior Associate Consultant
Departments of Pharmacology and Biochemistry/Molecular Biology
Mayo Foundation
Rochester, MN 55905
Karen Pollock, M.S.
Senior Research Assistant
James Douglass, Ph.D.
Scientist
Vollum Institute-L474
Oregon Health Sciences University
3181 Southwest Sam Jackson Park Road
Portland, OR 97201
131
The Prohormone and Proprotein
Processing Enzymes PC1 and PC2:
Structure, Selective Cleavage of
Mouse POMC and Human Renin at
Pairs of Basic Residues, Cellular
Expression, Tissue Distribution,
and mRNA Regulation
Nabil G. Seidah, Robert Day, Suzanne Benjannet,
Normand Rondeau, Alain Boudreault, Timothy Reudelhuber,
Martin K.-H. Schafer, Stanley J. Watson, and Michel Chrétien
INTRODUCTION
Limited proteolysis of precursors at specific pairs of basic residues and/or
at single basic amino acids is a widespread mechanism by which the cell
expresses a repertoire of biologically active proteins and peptides (Lazure
et al. 1983; Mains et al. 1990). Until recently, the tissue-specific and
developmentally regulated proteinases responsible for such conversions
were not identified at the molecular level. The cloning and cellular expression
of the yeast Kex2 gene product demonstrated that this enzyme belongs to
the Ca2+-dependent subtilisin family of serine proteinases (Julius et al. 1984;
Fuller et al. 1989a) and that it exhibits exquisite selectivity of cleavage at pairs
of basic residues in several yeast (Fuller et al. 1988) and mammalian (Bathurst
et al. 1987; Thomas et al. 1988; Foster et al. 1991) precursors. Furthermore,
the ability of a yeast enzyme to cleave mammalian precursors both in vitro
and in vivo reinforced the hypothesis that Kex2 represents a prototype for a
mammalian subtilisin-like proteinase(s) physiologically responsible for the
cleavage of proproteins at selected pairs of basic residues. The search for
the homologous mammalian convertases led to the recent identification and
molecular cloning of three distinct proteins. These were called furin (Roebroek
et al. 1986; Fuller et al. 1989b; Van den Ouweland et al. 1990), PC1 (Seidah et
al. 1990, 1991a; Smeekens et al. 1991, who also called this enzyme PC3), and
PC2 (Smeekens and Steiner 1990; Seidah et al. 1990).
132
CHROMOSOMAL ASSIGNMENT
The chromosomal localization of the genes coding for the convertases PC1,
PC2, and furin has been achieved by in situ hybridization of both human and
mouse metaphase spreads (Seidah et al. 1991a, 1991b). The data showed
that in both species these three genes are not synthenic. Thus PC1, PC2,
and furin were located on human chromosomes 5q15-21, 20p11.1-11.2, and
15q25-26 and on mouse chromosomes 13 [C1-C3 band], 2 [F3-H2 region],
and 7 [D1-E2 region], respectively. Genetic linkage analysis of each gene
in the mouse demonstrated that PC1, PC2, and furin loci map close to the
G protein Nras-2, homeobox Pax-1, and fes/fps oncogene Fes (Copeland
et al., in press).
STRUCTURAL ANALYSIS
The comparative architectural features of the subtilisin-like proprotein
convertases reported so far and those of the subtilisins are presented in
figure 1. It is apparent that PC1 , PC2, and Kex2 contain disulphide bridges
only within their catalytic domain, whereas furin also exhibits a Cys-rich
segment before the putative transmembrane domain (TMD) and two other Cys
residues within the TMD and the cytosolic C-terminal segment, respectively.
The glycosylation pattern in the mammalian members shows that they each
contain three N-glycosylation sites, and unlike Kex2, none appears within the
putative prosegment. This figure also shows in the unshaded area a segment
(called P-domain) that has been reported to be important for the folding of the
proteinase and the elaboration of the full enzymatic activity of Kex2 (Fuller et
al. 1991). Finally, although the structures of PC1 and PC2 do not predict the
presence of a transmembrane domain, their C-terminal segments code for an
amphipathic structure that could interact with membranes in a pH-dependent
manner, in a similar fashion to the properties of the soluble carboxypeptidase
E (Fricker et al. 1990).
Figure 2 depicts the conservation of sequences around the active sites Asp*,
His*, and Ser* as well as the catalytically important Asn* residues of some
subtilisins and of all the members of the mammalian subtilisin family reported
so far. Aside from the conserved residues in the family (boxed in), certain
residues (inverted triangles) are found only in the mammalian subtilisin family,
and within these, three of them (Arg, Ser, and Ala, shown in bold) are found
only in the Kex2-like convertases and not in the recent structure reported for
the human tripeptidyl peptidase II (hTPP II) (Tomkinson and Jonsson 1991).
Finally, only in PC2 is an Asp* found instead of the usual Asn* residue. This
interesting single point mutation of PC2 (but not PC1 or furin) may have an
important bearing on the catalytic efficiency (kcat/Km) of this proteinase, since
133
FIGURE 1. Schematic diagram depicting the various domains of the Kex2-like
enzymes and their comparison to the primitive subtilisin BPN
enzyme. The number of amino acids (# a.a.) in each primary
translation product is shown on the right. The diagram also points
out the positions of the active sites Asp*(D), His*(H), and Ser*(S)
in each enzyme.
it has been reported that this Asn* residue forms a hydrogen bond with the
developing substrate carbonyl oxygen anion in the transition state complex
(Robertus et al. 1972). This interaction was shown to be important, but not
essential, for the stabilization of this oxyanion hole in subtilisins (Bryan et al.
1986). From these data and the sequence similarity of the catalytic domains in
these various enzymes, it becomes apparent that hTPP II is much closer to
subtilisin BPN’ than to any other member of the Kex2-like proteinases. This
suggests a possible classification of hTPP II as a mammalian degradative
enzyme, rather than a Kex2-like specific proprotein convertase, in agreement
with its observed broad substrate specificity (Bålöw et al. 1986).
134
FIGURE 2. Conservation of the sequences around the Asp*, His*, and Ser* of the active sites found in the subtilisins
(Moehle et al. 1987), Kex2 (Mizuno et al. 1988), furin (Van den Ouweland et al. 1990), mPC1 (Seidah et
al. 1991a; Smeekens et al. 1991), mPC2 (Seidah et al. 1990), and hTPP II (Tomkinson and Jonsson
1991). Boxed amino acids, inverted triangles, and bold residues represent invariant residues, amino
acids found only in the eukaryotic members of the family, and conserved amino acids found only in the
Kex2-like members of the eukaryotic subfamily, respectively.
CLEAVAGE SPECIFICITY OF PC1 AND PC2
To investigate the cleavage specificity of PC1 and PC2 the authors used two
different precursors as substrate: mouse proopiomelanocortin (POMC) and
human renin. Vaccinia virus recombinants of each enzyme were used to
coexpress these proteinases with the prosubstrates in several cell lines.
Following their purification, the intracellular and secreted peptide products
were unambiguously characterized by microsequence.
Vaccinia virus recombinants of the mouse prohormone convertases PC1
and PC2 were coexpressed together with mouse POMC in the constitutively
secreting cells, BSC-40, and in the endocrine-derived cell lines PC12 and
AtT-20, which exhibit regulated secretion. Figure 3 summarizes the cleavage
selectivity of PC1 and PC2 as deduced from the monitoring of the POMC
processing. The data demonstrated the distinct cleavage specificities of
PC1 and PC2, since in the cell lines analyzed it was found that (1) PC1
cleaves POMC into adrenocorticotropic hormone (ACTH) and ß-lipotropin;
(2) PC2 cleaves POMC into biologically active ß-endorphin, an N-terminally
extended ACTH containing the joining peptide, and into either -melanocyte-
stimulating hormone (MSH) or des-acetyl -MSH; and (3) PC2 cleaves POMC
at the five pairs of basic residues analyzed, whereas PC1 cleaves preferentially
two of them, suggesting that PC2 has a broader spectrum of activity than
PC1. These data are consistent with the proposed hypothesis concerning the
physiological role of PC1 and PC2 as distinct proprotein convertases acting
alone or together to produce a set of tissue-specific maturation products both in
the brain and in peripheral tissues (Seidah et al. 1990, 1991a). Interestingly,
since POMC processing can also occur in cells devoid of a regulated pathway
of secretion, these data also show that POMC processing by either PC1 or
PC2 is not dependent on the presence of secretory granules.
Recent data from our laboratory showed that neither PC1 nor PC2 can
process human prorenin in a Chinese Hamster Ovary constitutively secreting
cell line (data not shown). To test the hypothesis that, unlike POMC, human
prorenin processing might require the presence of secretory granules, a
somatomammotroph GH4-C1 cell line, which expresses a stable transfectant
of human prorenin, was infected with PC1 or PC2 vaccinia virus recombinants.
As shown in figure 4, the results of analysis of the secreted renin activity
demonstrated that only PC1 can activate human prorenin in this cell line and
that neither the endogenous furin nor the exogenous PC2 can activate this
zymogen. Pulse chase analysis of the prorenin maturation product confirmed
that PC1 cleaved human prorenin at the expected LysArg-zymogen activation
site, thereby releasing the N-terminal 45 amino acids prosegment (not shown).
These results point out that not all precursors can be processed efficiently in the
136
FIGURE 3. Major end products of POMC processing by PC1 and PC2.
The
arrows represent the cleavage sites following pairs of basic
residues. The dark triangles tally the cleavage sites by each
enzyme. The numbers represent the start and end positions of
the processed peptides based on the mouse POMC sequence.
The N- and O-glycosylation (CHO) sites as well as the pairs of
basic residues are emphasized.
SOURCE: Benjannet et al. 1991
absence of secretory granules and that each case has to be carefully analyzed
before general conclusions can be reached. Furthermore, of the three enzymes
studied, only PC1 is capable of efficient processing of human prorenin. Neither
PC2 nor furin (as shown by work described in this chapter and in Hatsuzawa et
al. 1991) can be shown to activate this zymogen.
BACULOVIRUS EXPRESSION OF PC1 AND PC2
To study the physicochemical and kinetic properties of the convertases
PC1 and PC2 in vitro, we sought to obtain large quantities of each enzyme
using the baculovirus expression system. Recombinant viruses encoding
mouse PC1 and PC2 were obtained using the pJV Nhe I transfer vector
and by a procedure similar to that previously published (Vialard et al. 1990).
Expression of each enzyme was monitored by polyacrylamide electrophoresis
137
FIGURE 4. Human prorenin activation by PC1. A stable transfectant of GH4
cells expressing human prorenin (N.I.=noninfected cells) was
infected with either 5 pfu of the recombinant vaccinia virus
VV:PC1, VV:PC2 or the wild type vaccinia virus VV:WT
(Benjannet et al. 1991). The secreted renin activity was
measured before (active renin) and after (total renin) trypsin
treatment (15 µg for 1 hour at room temperature) by an indirect
assay using human angiotensinogen as a substrate for active
renin and a radioimmunoassay directed against angiotensin I.
The amount of prorenin was calculated from the difference
between the renin activity measured after and before trypsin
treatment.
on SDS/PAGE. Surprisingly, the enzymes produced were not secreted from
the Sf9 cells but, rather, remained intracellular. The proteins were found to
be produced in quantities exceeding 1 mg/L of culture, were associated with
membrane components within the cell, and were recovered by centrifugation
of the cell lysates in the particulate fraction. On SDS/PAGE, PC1 and PC2
migrate with an apparent molecular weight of 87,000 and 74,000 daltons
(not shown). To investigate the enzyme activity of each proteinase, we
selected to affinity label the membrane fraction containing either enzyme
with a pentapeptide chloromethyl ketone 125I-[D-Tyr]GluPheLysArg-COCH2CI,
of which the GluPheLysArg sequence represents the ACTH/ß-lipotropic
hormone junction (Cromlish et al. 1986), which was shown to be cleaved by
either PC1 or PC2 (Benjannet et al. 1991). As shown in figure 5, both PC1
138
FIGURE 5. Labeling of the cellular extract of baculovirus-expressed PC1 and
PC2 with the affinity label [
125I-D-Tyr]GluPheLysArg-COCH2CI.
The labeled proteins migrate exactly at the position of the
Coomassie-colored PC1 (87 kD) and PC2 (67 kD), suggesting
that these proteins are at least partially active proteinases.
This
labeling works both at acidic (pH 5.5) and neutral (pH 7.0) pH
conditions, and no labeling is seen if the enzyme preparation is
first incubated with 1 mM diisopropyl fluorophosphate.
139
and PC2 could be specifically labeled with this pentapeptide, and the labeled
proteins migrate with a similar molecular weight as the unlabeled proteins
stained by Coomassie Blue. However, the specific radioactivity associated
with the labeled proteins were found to be much inferior to that obtained with a
similar amount of active trypsin or plasma kallikrein (N.G. Seidah, unpublished
data). This suggested that either the proteins were synthesized mostly as
inactive enzymes in the Sf9 cells or that the presumed prosegment (see figure
1) was not cleaved in this system. The NH2-terminal sequence analysis of each
protein eluted from the SDS/PAGE gel was found to be KRQFVNEWAAEIPGG
and ERPVFTNHFLVELHK for mPC1 and mPC2, respectively (figure 6). Aside
from confirming the predicted signal peptidase cleavage site in each proteinase
(Seidah et al. 1991a), these data revealed that even though both intracellular
enzymes retained their N-terminal prosegment (figure 6), they retain partial
binding activity toward the pentapeptide chloromethyl ketone, which selectively
labels the His* at the active site of each enzyme (Cromlish et al. 1986).
Efforts are now under way to understand the reason for the absence of
zymogen activation of PC1 and PC2 in the Sf9 cells, which, under similar
conditions, express the active form of the Kex2 proteinase with the concomitant
removal of the prosegment (figure 5) (Germain et al. 1992). A possible
explanation is that, unlike Kex2 and the subtilisins, PC1 and PC2 might not
be capable of autoactivation but, rather, require the presence of another
enzyme to start the zymogen activation process.
PC1 AND PC2 IN THE RAT PITUITARY AND BRAIN
In situ hybridization studies in the mouse central nervous tissues and in
peripheral organs demonstrated that PC1 and PC2 transcripts are mostly
found in endocrine and neuroendocrine tissues and cells (Seidah et al.
1990, 1991a). In contrast, furin was found to be widely distributed both in
endocrine and in nonendocrine tissues and cells (Schalken et al. 1987).
Figure 7 depicts the comparative in situ hybridization of PC1 and PC2 in
the rat pituitary. PC1 transcripts are found in all endocrine cells of the
intermediate lobe and in a majority of pituitary anterior lobe endocrine cells.
PC2 mRNA is highly abundant in the intermediate lobe, with much smaller
amounts found in the anterior lobe. A similar conclusion can also be drawn
from Northern blots (figure 7), which show the relative amounts of PC1 and
PC2 in both the anterior and the neurointermediate lobes of the pituitary. In
the rat, two transcripts are found for both enzymes at about 3 and 5 kb (figure
7), as previously reported in mouse PC1 and PC2 (Seidah et al. 1990, 1991a;
Smeekens et al. 1991) and human PC2 (Smeekens and Steiner 1990).
The difference between these two mRNAs is not yet fully understood, but
preliminary data indicate that the longer forms (5 kb) do not contain an
extended coding region for each enzyme (N.G. Seidah, unpublished data).
140
FIGURE 6. Alignment of the N-terminal segments of the mammalian Kex2-like enzymes. The inverted dark
triangles point to the predicted positions of the signal peptidase cleavage sites, based on von Heijne
(1986) criteria and confirmed by microsequence analysis of the baculovirus-expressed forms of mPC1
and mPC2 (underlined residues). The computer alignment of the prosegments delineates two regions
(shown in bold) where the presumed multibasic cleavage sites exactly align in all cases. By analogy to
the position of the prosegment cleavage site in Kex2 (Wilcox and Fuller 1991), the arrows point to the
probable equivalent site in the other members of the family.
FIGURE 7. (Left) Autoradiography of the rat pituitary distribution of PC1 and
PC2 by in situ hybridization. (Right) Northern gel analysis of PC1
and PC2 mRNA transcripts found in total RNA of anterior (AP;
10 µg) and neurointermediate (NIL; 4 µg) pituitary lobes. X-ray
exposures limes are 4 and 16 hours for PC2 and PC1,
respectively. Markers are in kilobases.
Figure 8 depicts a representative photomicrograph of the distribution of PC1
and PC2 mRNA in a coronal section of the rat brain by in situ hybridization.
It is clear that these enzymes exhibit distinct distribution patterns in the central
nervous system. In general, PC1 has a more restricted distribution, whereas
PC2 is widespread. PC1 mRNA is most highly abundant in the hypothalamic
nuclei such as the supraoptic, paraventricular, and suprachiasmatic nuclei.
142
FIGURE 8. Autoradiography of the rat brain distribution of PC1 and
PC2 by in situ hybridization. CTX=cortex; HC=hippocampus;
PVN=paraventricular nucleus; SON=supraoptic nucleus;
SCN=suprachiasmatic nucleus; Hb=habenula; TH=thalamus;
STR=striatum; Pir=piriform cortex.
143
The habenula and the hippocampus represent extrahypothalamic areas rich in
PC1. Although present throughout the hippocampus, PC1 is expressed with
high abundance in the dentate gyrus. PC1 is also more abundant in the deeper
cortical layers compared with the superficial ones. On the other hand, PC2 is
widely expressed in many brain areas, the most striking example of which is the
thalamus, whose numerous subnuclei all appear to express intermediate to high
levels of PC2. The thalamic distribution of PC1 is much more restricted. PC2 is
also moderately abundant in both the superficial and deeper cortical layers as
well as in the striatum. In the hippocampus, PC2 is found mainly in the CA1,
CA2, and CA3 regions and, to a much lesser extent, in the dentate gyrus.
These unique localization patterns of PC1 and PC2 indicate the differential
roles of these convertases in the brain and may have some significance in
terms of understanding tissue-specific posttranslational processing.
DOPAMINERGIC REGULATION OF PC1 AND PC2 mRNAs IN PITUITARY
PARS INTERMEDIA
PC1 and PC2 are expressed in all pituitary intermediate lobe cells (figure 7).
The rat intermediate lobe melanotrophs, a highly homogeneous cell population,
are highly abundant in POMC and are under inhibitory dopaminergic control.
Figure 9 shows a representative experiment demonstrating the up-regulation
of PC2 in the neurointermediate lobes of rats chronically treated with the
dopaminergic antagonist haloperidol. Also shown is the down-regulation
effect of the dopaminergic agonist bromocryptine on PC2 mRNA in the
neurointermediate lobes of chronically treated rats. Parallel effects were
observed for PC1 mRNA. In the present study, POMC mRNA levels also
increased with haloperidol and decreased with bromocryptine treatments
(data not shown), as previously reported (Chen et al. 1983). Accordingly, in
intermediate lobe melanotrophs, the authors’ data demonstrate the coregulation
of the gene expression of POMC with that of PC1 and PC2.
CONCLUSION
The data presented in this chapter show that PC1 and PC2 are distinct
mammalian subtilisin-like proteinases that originate from two different genes
and that are expressed in neuronal and endocrine tissues in a discrete fashion.
Even though both enzymes cleave POMC at specific pairs of basic residues,
only PC1 can activate human prorenin in cells containing secretory granules.
In the brain, PC2 is generally more abundant and more widely expressed
than PC1. In the pituitary, PC2 mRNA is more abundant than PC1 in the
intermediate lobe, whereas the reverse is true in the anterior lobe. Similar
to POMC, in the intermediate lobe of the pituitary both enzymes are under
144
FIGURE 9. Northern gel analysis of total RNA (2 µg per lane) of
neurointermediate lobes of rats chronically treated with the
dopaminergic antagonist haloperidol (CNT=vehicle; HAL 1=5.0
mg/kg; HAL 2=10 mg/kg) and the dopaminergic agonist
bromoctyptine (CNT=vehicle; BC 1=2.0 mg/kg; BC 2=10 mg/kg).
X-ray exposure times are 4 and 16 hours for PC2 and PC1,
respectively. Markers are in kilobases.
145
negative dopaminergic control. The data from the baculovirus-expressed
enzymes suggest that the zymogen form of PC1 and PC2 is only partially
active and that this form is not secretable. Recent pulse chase experiments
demonstrated that the intracellular activation of PC1 is more efficient than that
of PC2 and that neither enzyme can undergo autocatalytic activation (Benjannet
et al. 1992). Therefore, the zymogen activation of PC1 and PC2 may require
the presence of yet another enzyme, which together with PC1 and PC2 will
fashion in a tissue-specific manner the final molecular products generated from
a given proprotein.
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ACKNOWLEDGMENTS
This chapter was prepared with support from Medical Research Council of
Canada grant PG-2, Mt11268 and from J.A. de Sève Succession.
AUTHORS
Nabil G. Seidah, Ph.D.
Full Professor
Robert Day, Ph.D.
Assistant Professor
Suzanne Benjannet, M.Sc.
Assistant Researcher
Normand Rondeau, M.Sc.
Technician
Alain Boudreault, B.Sc.
Timothy Reudelhuber, Ph.D.
Associate Professor
Michel Chrétien, O.C., M.D.
Scientific Director and Chief Executive Officer
Clinical Research Institute of Montreal
Affiliated to I’Université de Montreal
110 Pine Avenue West
Montreal, Quebec H2W 1R7
CANADA
Martin K.-H. Schafer, M.D.
Assistant Professor
Anatomisches lnstitut
Johannes Gutenberg-Universitat
D-6500 Mainz
GERMANY
149
Stanley J. Watson, M.D., Ph.D.
Professor
Mental Health Research Institute
205 Washtenaw Place
Ann Arbor. MI 48109
150
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CLlNlCAL PRACTICE. Carl G. Leukefeld, D.S.W., and Frank M. Tims, Ph.D.,
eds.
GPO Stock #017-024-01352-8 $7.50 NTIS PB #89-151997/AS $31
87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D., and Bhola N.
Dhawan, M.D., eds.
GPO Stock #017-024-01366-8 $7 NTIS PB #89-158430/AS $45
88 MECHANISMS OF COCAINE ABUSE AND TOXICITY. Doris H. Clouet,
Ph.D.; Khursheed Asghar, Ph.D.; and Roger M. Brown, Ph.D., eds.
GPO Stock #017-024-01359-5 $11 NTIS PB #89-125512/AS $39
89 BIOLOGICAL VULNERABILTY TO DRUG ABUSE. Roy W. Pickens,
Ph.D., and Dace S. Svikis, B.A., eds.
GPO Stock #017-022-01054-2 $5 NTIS PB #89-125520/AS $23
153
90 PROBLEMS OF DRUG DEPENDENCE 1988: PROCEEDINGS OF THE
50TH ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS
OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.
GPO Stock #017-024-01362-5 $17
91
DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION DATA.
Steven W. Gust, Ph.D., and J. Michael Walsh, Ph.D., eds.
GPO Stock #017-024-01384-6 $10 NTIS PB #90-147257/AS $39
92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS. Marian W.
Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.
GPO Stock #017-024-01379-0 $12 NTIS PB #90-148933/AS $45
93 AIDS AND INTRAVENOUS DRUG USE: FUTURE DIRECTIONS FOR
COMMUNITY-BASED PREVENTION RESEARCH. C.G. Leukefeld, D.S.W.;
R.J. Battjes, D.S.W.; and Z. Amsel, D.Sc., eds.
GPO Stock #017-024-01388-9 $10 NTIS PB #90-148941/AS $39
94
PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND
RELATED DESIGNER DRUGS. Khursheed Asghar, Ph.D., and Errol De
Souza, Ph.D., eds.
GPO Stock #017-024-01386-2 $11 NTIS PB #90-148958/AS $39
95 PROBLEMS OF DRUG DEPENDENCE 1989: PROCEEDINGS OF THE
51ST ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS
OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.
GPO Stock #017-024-01399-4 $21 NTIS PB #90-237660/AS $67
96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY,
AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D., eds.
GPO Stock #017-024-01403-6 $8 NTIS PB #90-237678/AS $31
97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY. Lynda
Erinoff, Ph.D., ed.
GPO Stock #017-024-01404-4 $8 NTIS PB #90-237686/AS $31
98 THE COLLECTION AND INTERPRETATION OF DATA FROM HIDDEN
POPULATIONS. Elizabeth Y. Lambert, M.S., ed.
GPO Stock #017-024-01407-9 $4.75 NTIS PB #90-237694/AS $23
99
RESEARCH FINDINGS ON SMOKING OF ABUSED SUBSTANCES.
C. Nora Chiang, Ph.D., and Richard L. Hawks, Ph.D., eds.
GPO Stock #017-024-01412-5 $5 NTIS PB #91-141119 $23
154
100
DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION DATA.
VOL. II. Steven W. Gust, Ph.D.; J. Michael Walsh, Ph.D.; Linda B. Thomas,
B.S.; and Dennis J. Crouch, M.B.A., eds.
GPO Stock #017-024-01458-3 $8
101
RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR. John W.
Spencer, Ph.D., and John J. Boren, Ph.D., eds.
GPO Stock #017-024-01426-7 $6 NTIS PB #91-172858/AS $31
102
ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D., and Lynda Erinoff,
Ph.D., eds.
GPO Stock #017-024-01425-7 $8 NTIS PB #91-172866/AS $31
103 DRUGS AND VIOLENCE: CAUSES, CORRELATES, AND
CONSEQUENCES. Mario De La Rosa, Ph.D.; Elizabeth Y. Lambert, M.S.;
and Bernard Gropper, Ph.D., eds.
GPO Stock #017-024-01427-3 $9 NTIS PB #91-172841/AS $31
104
PSYCHOTHERAPY AND COUNSELING IN THE TREATMENT OF
DRUG ABUSE. Lisa Simon Onken, Ph.D., and Jack D. Blaine, M.D., eds.
GPO Stock #017-024-01429-0 $4 NTIS PB #91-172874/AS $23
105
PROBLEMS OF DRUG DEPENDENCE, 1990: PROCEEDINGS OF THE
52ND ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS
OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.
GPO Stock #017-024-01435-4 $22
106
IMPROVING DRUG ABUSE TREATMENT. Roy W. Pickens, Ph.D.; Carl
G. Leukefeld, D.S.W.; and Charles R. Schuster, Ph.D., eds.
GPO Stock #017-024-01439-7 $12 NTIS PB #92-105873
Paperback $50 Microfiche $19
107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:
METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and William J.
Bukoski, Ph.D., eds.
GPO Stock #017-024-01446-0 $9 NTIS PB #92-160985
Paperback $35 Microfiche $17
108 CARDIOVASCULAR TOXICITY OF COCAINE: UNDERLYING
MECHANISMS. Pushpa V. Thadani, Ph.D., ed.
GPO Stock #017-024-01446-0 $7 NTIS PB #92-106608
Paperback $35 Microfiche $17
155
109
LONGITUDINAL STUDIES OF HIV INFECTION IN INTRAVENOUS DRUG
USERS: METHODOLOGICAL ISSUES IN NATURAL HISTORY RESEARCH.
Peter Hartsock, Dr.P.H., and Sander G. Genser, M.D., M.P.H., eds.
GPO Stock #017-024-01445-1 $4.50 NTIS PB #92-106616
Paperback $26 Microfiche $12.50
110
THE EPIDEMIOLOGY OF COCAINE USE AND ABUSE. Susan Schober,
Ph.D., and Charles Schade, M.D., M.P.H., eds.
GPO Stock #017-024-01456-7 $11 NTIS PB #92-14624-0
Paperback $43 Microfiche $17
111
MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH VOLUME I:
RECEPTOR CLONING, NEUROTRANSMITTER EXPRESSION, AND
MOLECULAR GENETICS. Theresa N.H. Lee, Ph.D., ed.
Not for sale at GPO NTIS PB #92-135743
Paperback $35 Microfiche $17
112
EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN DRUG
ABUSE RESEARCH. Rao S. Rapaka, Ph.D.; Alexandros Makriyannis, Ph.D.;
and Michael J. Kuhar, Ph.D., eds.
GPO Stock #017-024-01455-9 $11
113 ECONOMIC COSTS, COST-EFFECTIVENESS, FINANCING, AND
COMMUNITY-BASED DRUG TREATMENT. William S. Cartwright, Ph.D.,
and James M. Kaple, Ph.D., eds.
Not for sale at GPO NTIS PB #92-155795
Paperback $35 Microfiche $17
114
METHODOLOGICAL ISSUES IN CONTROLLED STUDIES ON EFFECTS
OF PRENATAL EXPOSURE TO DRUG ABUSE. M. Marlyne Kilbey, Ph.D., and
Khursheed Asghar, Ph.D., eds.
GPO Stock #017-024-01459-1 $12 NTIS PB #92-146216
Paperback $43 Microfiche $17
115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES AND
IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H.. and Nicholas J. Kozel, M.S.,
eds.
GPO Stock #017-024-01460-5 $4
116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE
RESEARCH. Richard A. Glennon, Ph.D.; Torbjörn U.C. Järbe, Ph.D.; and
Jerry Frankenheim, Ph.D., eds.
GPO Stock #017-024-01470-2 $13
156
117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGICAL, PREVENTION,
AND TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN AND THEIR
CHILDREN. M. Marlyne Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.
GPO Stock #017-024-01472-9 $12
118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. Carl G.
Leukefeld, D.S.W., and Frank M. Tims, Ph.D., eds.
GPO Stock #017-024-01473-7 $16
119 PROBLEMS OF DRUG DEPENDENCE 1991: 53RD ANNUAL
SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG
DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.
GPO Stock #017-024-01474-5 $22
120
BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE BLOOD-
BRAIN BARRIER. Jerry Frankenheim, Ph.D., and Roger M. Brown, Ph.D., eds.
121
BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID
DEPENDENCE. Jack D. Blaine, Ph.D., ed.
122
RESEARCH METHODS IN WORKPLACE SETTINGS. Helen Axel, M.A.,
and Dennis J. Crouch, M.B.A., eds.
123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF
TREATMENT. Heinz Sorer, Ph.D., ed.
124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR
INTERACTION. Roger M. Brown, Ph.D., and Joseph Frascella, Ph.D., eds.
125 ACTIVATION OF IMMEDIATE EARLY GENES OF ABUSE. Reinhard
Grzanna, Ph.D., and Roger M. Brown, Ph.D., eds.
157
DHHS Publication No. (ADM) 92-1945
Alcohol, Drug Abuse, and Mental Health Administration
Printed 1992
