Alternative drug discovery approaches for orphan GPCRs.

Abstract

G protein-coupled receptors (GPCRs) are well-known drug targets. However, a question mark remains for the more than 100 orphan GPCRs as current deorphanisation strategies failed to identify specific ligands for these receptors. Recent advances have shown that orphan GPCRs may have important functions that are ligand-independent. Orphan GPCRs can modulate the function of well-defined drug targets such as GPCRs with identified ligands and neurotransmitter transporters though physical association with those molecules. Thus, compounds that bind to orphan GPCRs and allosterically regulate the function of the interacting partner or even disrupt the interaction with the latter could become new drugs.

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REVIEWS
Drug Discovery Today?Volume 13,Numbers 1/2?January 2008
Alternative drug discovery approaches
for orphan GPCRs
Ange ´lique Levoye1,2,3and Ralf Jockers1,2
1Institut Cochin, Universite ´ Paris Descartes, CNRS (UMR 8104), Department of Cell Biology, Paris, France
2Inserm, U567, Paris, France
Gprotein-coupledreceptors(GPCRs)arewell-knowndrugtargets.However,aquestionmarkremainsfor
the more than 100 orphan GPCRs as current deorphanisation strategies failed to identify specific
ligands for these receptors. Recent advances have shown that orphan GPCRs may have important
functions that are ligand-independent. Orphan GPCRs can modulate the function of well-defined drug
targets such as GPCRs with identified ligands and neurotransmitter transporters though physical
association with those molecules. Thus, compounds that bind to orphan GPCRs and allosterically
regulate the function of the interacting partner or even disrupt the interaction with the latter could
become new drugs.
Current deorphanisation strategies
GPCRs constitute the largest family of membrane receptors. They
are involved in virtually every biological process. They are typi-
cally expressed at the cell surface and possess well-defined orthos-
teric and allosteric ligand-binding pockets that can be targeted by
small molecular weight compounds. Not surprising, many top-
selling drugs target GPCRs. Based on the completed sequence of
the human genome, approximately 400 non-odorant GPCRs can
be predicted. During the last 20 years most of them were matched
with their respective ligands using several different strategies.
However, a considerable number of GPCRs, more than 100, are
still orphans without identified ligand. In this review we will
update recent progress on new ligand-independent functions of
orphan GPCRs and discuss the potential of these orphan GPCRs as
drug targets.
From the early days of GPCR cDNA cloning in the late 1980s
until now, a strategy termed ‘reverse pharmacology’ (see Glossary)
was used for the deorphanisation (see Glossary) of GPCRs [1]. This
approach is based on the exogenous expression of the orphan
GPCR in a suitable cell system. Activation of the receptor by
exposure to a variety of potential ligands is monitored by changes
in intracellular second messenger (see Glossary) levels involving
heterotrimeric G proteins (Figure 1). As the number of identified
natural ligands was rapidly outnumbered bythe GPCRs discovered
by DNA cloning, a modified version called ‘orphan receptor strat-
egy’ emerged in the mid-1990s [2]. This strategy is also referred to
as the ‘tissue-extract based approach’ where orphan GPCRs are
exposed to tissue extracts instead of purified ligands. Activation of
orphan receptors is again measured by changes in second mes-
senger responses. Positive extracts are fractionated until the active
component is isolated and characterised. Despite difficulties of
assay specificity because of the complex composition of tissue
extracts, the orphan receptor strategy proved to be useful for
the identification of several natural ligands for orphan GPCRs
[3–6].
More recently,approaches based onthecellular translocationof
proteins that participate in the GPCR desensitisation/internalisa-
tion cycle, that is b-arrestin, have been added to the toolbox. This
system commercialised as ‘Transfluor technology’ monitors the
change in distribution of a b-arrestin-GFP construct from homo-
genous and diffuse intracellular localisation to aggregated distri-
bution as a consequenceof ligand-dependent internalisation [7,8].
Other strategies exploit the acidic nature of the endosomal recy-
cling compartments and the pH-sensitive cyanine dye CypHer 5,
which is non-fluorescent at pH 7.4 and fluoresces brightly in an
Reviews?POST SCREEN
Corresponding author: Jockers, R. (jockers@cochin.inserm.fr)
3Present address: Institut Pasteur, laboratoire de pathoge ´nie virale mole ´-
culaire, Inserm U819, De ´partement de Virologie, 28 rue du Dr Roux, 75724
Paris, France.
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acidicenvironment[9].Surfaceexposedreceptorsarelabelledwith
CypHer 5-labeled antibody and ligand-dependent internalisation
is monitored by the occurrence of intense intracellular fluores-
cence. The expression pattern of receptors may provide another
clue for ligand-receptor pairing. The nicotinic acid receptor
GPR109B was identified based on its restricted expression pattern
in adipose tissue and the spleen, the pharmacological sites of
nicotinic acid action [10,11]. Generation of knock out animals
has also been used to increase our understanding of the biological
functionoforphanGPCRsintheabsenceofknowledgeabouttheir
endogenous ligand [12,13].
Limitation of current deorphanisation strategies
A recent update of the number of deorphanised GPCRs clearly
shows that the deorphanisation rate slowed down considerably
since 2004, with 4–6 deorphanisations per year (Figure 2). What
mightbethepotentiallimitationsofcurrentstrategies?Functional
screening assays for orphan GPCRs traditionally focus on the
monitoring of intracellular second messengers of G protein-
dependent pathways. However, some receptors may not rely on
the activation of heterotrimeric G proteins but rather on the
activation of G protein-independent pathways [14]. This could
be the case for GPCR C5L2 (also known as GPR77), which binds to
C5a anaphylotoxin in binding assays, but which is completely
inactive in functional G protein-dependent assays [15,16].
Whether this receptor is only a ‘silent’ (non-signalling) C5a ana-
phylotoxin scavenger or whether G protein-independent path-
ways are activated remains to be clarified [17]. Similarly, some
chemokine receptors such as D6 receptors are known to bind to
chemokines without inducing cell migration or G protein-depen-
dent signalling [18].
The absence of specific GPCR-associated proteins in a given
cell system may also represent a major obstacle for successful
expression of functional receptors. Many GPCRs have indeed
been reported to interact with several scaffolding (b-arrestin,
homer, spinophilin) and cytoskeletal proteins (filamin A, cofilin)
or so-called receptor activity modulating proteins (RAMPs). For
some GPCRs, coexpression of these accessory proteins is obliga-
tory to promote efficient surface expression, ligand binding and/
or signal transduction. A well-known example is the calcitonin
receptor-like receptor (CRLR) that requires RAMP for efficient
transport to the cell surface and ligand binding [19]. Other
GPCRs require coexpression with another GPCR. Accumulating
structural, functional and pharmacological evidences suggest
that GPCRs form dimers or higher order oligomers [20–22].
The best characterised example is the metabotropic g-aminobu-
tyric acid B (GABAB) receptor heterodimer, where the coexpres-
sion of the GABAB2subunit is obligatory for the GABAB1subunit
to reach the cell surface [23]. Similarly, coexpression of the b2-
adrenergic receptor appears to promote a1D-adrenergic receptor
surface expression and functional activity [24]. Moreover, coex-
pression of the different taste receptor subunits is necessary for
the formation of functional receptors. Whereas T1R2/T1R3het-
erodimers respond rather to sweet stimuli such as aspartame, the
T1R1/T1R3heterodimer responds to the umami taste of L-gluta-
mate [25,26].
Taken together, these examples clearly indicate that new assays
have to be designed to deorphanise the remaining orphan recep-
tors. This includes the enlargement of functional readouts towards
Gprotein-independent (i.e. b-arrestin-dependent)signalling path-
ways and the coexpression of appropriate interacting partners,
which promote either cell surface expression or ligand binding. In
addition, recent evidence suggests that orphan GPCRs could also
have ligand-independent functions. Such a concept would offer
additional options for future drug screening strategies as discussed
below.
Ligand-independent functions of orphan 7TM proteins
Recent advances suggest that at least some of the remaining
orphan receptors may have ligand-independent functions [27].
Considering that the term ‘orphan GPCR’ may be to restrictive, as
some of them may be ‘real’ orphans and others are orphans with
known ligand-independent functions for which ligand binding
cannot be completely excluded, we will use the term orphan
seven-transmembrane (7TM) protein in this review. Two types
of ligand-independent functions have emerged so far, constitutive
activity (see Glossary) of orphan 7TM proteins (see Glossary) and
the modulation of the function of other proteins (i.e. non-orphan
GPCRs or transporters) by hetero-oligomerisation with orphan
7TM proteins.
Constitutively active orphan 7TM proteins
The importance of agonist-independent constitutive activity of
GPCRs in native tissues has been questioned for a long time,
however, there is now significant evidence that this is physiolo-
gically relevant [28,29]. Some of the most convincing evidence
comes from orphan 7TM proteins displaying a substantial degree
of constitutive activity. Several virus-related (virus-encoded or
virus-induced) orphan receptors are constitutively active. The
human cytomegalovirus (HCMV) encodes three constitutively
active GPCRs that exploit the cytokine system of the host cell
to survive. Among these three receptors, UL33 is an orphan 7TM
protein [30,31]. The Epstein Barr virus-induced receptor 2 (EBI2)
Drug Discovery Today?Volume 13,Numbers 1/2?January 2008REVIEWS
GLOSSARY
Allosteric binding site : a binding site on the receptor that is
topographically distinct from (does not exhibit any overlap
with) the orthosteric site and which influences the functional
effects of ligand binding to the orthosteric site.
Constitutive activity significant activity of GPCR in absence
of ligand.
Deorphanisation : a process, which consists of the
identification of a natural ligand acting on an orphan
receptor.
Inverseagonist:molecule,whichbindstothesamereceptor
binding site as the endogenous agonist but exerts the
opposite pharmacological effect.
Orphan 7TM protein : a seven-transmembrane protein
withoutanassignedligand,definedbyanovelsequencewith
a certain degree of homology to other proteins with similar
topology.
Orthosteric binding site : the primary binding site on the
receptor that is recognized by the endogenous agonist.
Reverse pharmacology : strategy aiming to identify the
endogenous ligand of a novel receptor.
Second messenger : cytosolic molecules such as cAMP or
calcium, which are modulated upon activation of GPCRs.
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[32], originally identified as the most upregulated gene in EBV-
infected cells, is an orphan 7TM protein, which constitutively
signals through the inhibitory (Gi) subunit of G proteins [33].
Constitutive activity is also observed for endogenous, non-virus-
related orphan GPCRs. GPR3, GPR6 and GPR12 constitute a sub-
family with more than 60% homology that promote constitutive
activation of the adenylyl cyclase pathway [34–36]. Similar obser-
vations have been made for the two homologous receptors GPR26
and GPR78 [37]. However, it should be mentioned that the pos-
sibility always remains that the apparent constitutive activity
might actually reflect the presence of an endogenous ligand that
is difficult to remove. This is illustrated by a recent study on
GPR40, whose apparent constitutive activity can be explained
by the occupation of receptor bindings sites with endogenous
fatty acid ligands [38]. Screening programs for inverse agonists
(see Glossary) should take into account such potential complica-
tions. Conversely, binding of endogenous inverse agonists may
also exist and mask the ligand-binding site.
Association of orphan 7TM proteins with non-orphan GPCR
In line with the general notion that GPCRs can form dimers or
higher order oligomers, some of these reported oligomeric com-
plexes are composed of GPCRs with known ligands and orphan
7TM proteins [39]. This has been most convincingly demon-
stratedforseveralclassCGPCRs.TheGABABreceptoriscomposed
of two functionally distinct subunits, GABAB1 and GABAB2.
Whereas the GABAB1subunit binds the natural ligand, GABA,
the GABAB2subunit couples to the heterotrimeric G protein.
Importantly, the GABAB2subunit can be considered as an orphan
7TM protein without functional GABA binding site, a conclusion
that is supported by an evolutionary trace analysis of the putative
binding pocket [40]. An additional function of GABAB2 is to
promote cell surface expression of GABAB1. A similar scenario
has been reported for the Drosophila odorant receptor 83b
(DOR83b), a 7TM protein with no apparent affinity for odorants
that heterodimerise with classical odorant receptors such as
DOR22a and DOR43a. Expression of DOR83b is required for
REVIEWSDrug Discovery Today?Volume 13,Numbers 1/2?January 2008
FIGURE 1
Schematicrepresentationofcurrentdeorphanisationapproaches.OrphanreceptorsareexpressedincellsandGprotein-dependentsecondmessengerresponses
aremonitored.(a)Reversepharmacologyapproach.Orphanreceptor-expressingcellsareincubatedinthepresenceofsyntheticornatural(purified)compounds.
(b) Orphan receptor strategy. Orphanreceptor-expressing cells are incubated in the presenceof tissue extracts, which are fractionated until an active component
is isolated.
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functional membrane expression of DOR22a and DOR43a in
olfactory sensory neurons [41,42]. The receptors for sweet and
umami tastes are also composed of two receptor subunits, firstly
the ligand-binding subunits T1R1or T1R2, and secondly their
orphan obligatory heterodimerisation partner T1R3, which has
no apparent ligand-binding capacity [43].
Tworecentexamplesoftheassociationoforphan7TMproteins
with non-orphan GPCRs show that orphans can also modify the
signalling of their interacting partners. The Mas-related gene
(Mrg) family members MrgD and MrgE have been shown to form
heterodimers. ß-alanine binding to MrgD, when expressed alone,
activates the ERK pathway and receptor internalisation. Coex-
pression of MrgD with the orphan MrgE potentiates ß-alanine-
inducedMrgDsignallingandinhibitsMrgDinternalisationinthe
MrgD/MrgE heterodimer [44]. In the case of the heterodimer
between the MT1melatonin receptor and the orphan GPR50
7TM protein, the orphan drastically inhibits ligand-promoted
signalling of MT1. Indeed, engagement of MT1with GPR50 inhi-
bits high-affinity agonist binding, heterotrimeric G protein cou-
pling and ß-arrestin binding in cells expressing both proteins
heterologously and endogenously [39]. Consequently, GPR50
can be considered as a natural negative modulator of MT1. Taken
together, these examples show that orphan 7TM proteins may be
important regulators of the function of GPCRs with known
ligands by modulating their trafficking, signalling and pharma-
cological properties.
Association of orphan 7TM proteins with other membrane
proteins
Orphan 7TM proteins can form heteromeric complexes contain-
ing proteins other than non-orphan GPCRs. The orphan GPR37,
which is associated with Parkinson’s disease has recently been
reported to associate with the dopamine transporter DAT and to
modulate its activity. GPR37-null mice show enhanced dopamine
uptake in striatal membranes associated with increased DAT cell
surface expression [45]. A further example concerns Smoothened
(Smo), a 7TM protein that mediates the effects of Hedgehog (Hh)
in embryonic development. Interestingly, Hh does not bind
directly to Smo but rather to the 12TM receptor Patched (Ptch),
which inhibits the constitutive activity of Smo. Binding of Hh to
Ptch relieves this repression of Smo. Consequently, the latter can
be considered as orphan 7TM protein whose activity is regulated
by its binding partner Ptch [46].
Fast progress in the field of functional proteomics is likely to
reveal further, unexpected, protein complexes that contain
orphan 7TM proteins. One possibility is the complex formation
betweenorphan7TMproteinsandion channelsasalreadyshown
for non-orphan GPCRs. Complexes containing the ß2-adrenergic
receptor and the calcium activated potassium channel [47] or the
L type calcium channel Cav1.2 [48], complexes between the
dopamine D5 receptor and the GABAA-ligand-gated channel
and complexes between the nociceptin (ORL1) receptor and
the voltage-gated calcium channel Cav2.2 [49] have been docu-
mented. It is important to note that the majority of these com-
plexes are formed constitutively and do not require ligand
activation of the receptor. For example, coexpression of ORL1
and Cav2.2 results in a constitutive voltage-dependent inhibition
ofthechannel,aphenomenonthatcanbeobservedindorsalroot
ganglion neurons.
Implications for drug design
Inverse agonists of orphan GPCRs with constitutive activity
Numerous GPCRs exhibit constitutive activity and the potential
for identifying novel inverse agonists at such GPCRs has been
recognised for drug discovery (Figure 3A) [28,50]. The only iden-
tified naturally occurring potent inverse agonist for GPCRs is the
agouti-related peptide, which acts on the MC4 receptor to parti-
cipate in body weight regulation [51,52]. Several potent synthetic
inverse agonists have been developed for constitutively active
GPCRs [50]. US28, a GPCR with constitutive activity that is
encoded by the HCMV has also attracted attention. Recently,
the first small nonpeptidergic inverse agonist for US28 has been
described [53]. So far, no inverse agonist has been described for
orphan 7TM proteins. As an initial step towards drug discovery,
the identification of the biological function and signalling path-
ways used by these orphans will be of vital importance. The recent
development of transcription-based reporter gene high through-
put screening assays intended to identify the signalling pathways
of orphan 7TM proteins such as GPR3, GPR12 and GPR26 should
speed up this process [54].
Heterodimer-selective ligands
Development of selective ligands for dimeric (oligomeric) com-
plexes between orphan 7TM proteins and non-orphan GPCRs
constitutes a potential drug design strategy, which, however, still
needs to prove its efficiency. Even the development of ligands that
selectively target heterodimers of non-orphan GPCRs are still in its
infancy. Two strategies are under current investigation for GPCR
heterodimers: first, the design of small compounds that target
receptor heterodimers, as proposed for 60-GNTI, a compound that
shows some selectivity for k/d opioid receptor heterodimers [55]
and second bivalent ligands such as KDN21 that contains a d and a
k opioid antagonist pharmacophore linked through a 21-atom
Drug Discovery Today?Volume 13,Numbers 1/2?January 2008 REVIEWS
FIGURE 2
Success rate of deorphanisation between 2000 and 2006. Adapted from [67].
MrgD and MrgX3, Mas related gene D and X3; GPR, G Protein Coupled
Receptor; CCR6, Chemokine CC motif Receptor 6; RDC1 or chemokine orphan
receptor 1 (CMKOR1); GPRC6A, G-Protein-coupled Receptor family C 6 A;
ChemR23, Chemerin Receptor 23.
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spacer. The first strategy could be applied to complexes between
orphan 7TM proteins and non-orphan GPCRs.
Allosteric modulators
The therapeutical potential of allosteric modulators of GPCRs is
gaining increasing acceptance. Two comprehensive state-of-the-
art reviews have been published recently [56,57]. Allosteric mod-
ulators act per definition outside of the orthosteric binding site
(see Glossary) of endogenous ligands (Figure 3B). Several examples
illustrate the great potential of such modulators for orphan 7TM
proteins. As described earlier, the GABABreceptor is composed of a
ligand-binding competent GABAB1and the G protein coupling-
competent GABAB2subunit. Although GABAB2has no functional
orthosteric binding site, this subunit can be targeted by the
CGP7930 compound, which allosterically enhances GABA bind-
ing to the heterodimer [58]. It is important to note that the GABAB
receptor is a member of the class C GPCR subfamily that is
characterised by a large bilobate extracellular orthosteric ligand-
binding domain that binds GABA. Interestingly, CGP7930 and
allosteric modulators of the structurally similar metabotropic
glutamate receptors, preferentially target the 7TM domain in a
mannersimilar toorthostericligands ofclass AGPCRs[59].Similar
observations have been made for taste receptors belonging to the
class C subfamily. Lactisole and cyclamate have been proposed to
REVIEWSDrug Discovery Today?Volume 13,Numbers 1/2?January 2008
FIGURE 3
Design of ‘drugs’ for orphan 7TM proteins. (A) Inverse agonistes mayinhibit the activity of constitutively active orphan 7TM proteins. (B) The interaction between
orphan 7TM proteins and non-orphan GPCRs, neurotransmitter transporters or ion channels may be regulated by allosteric modulators that bind to the orphan
7TM protein to allosterically regulate the function of the interacting partner or may be inhibited by compounds that disrupt this interaction.
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bind to the 7TM domain of the orphan T1R3subunit and thus
allosterically regulate ligand binding to the bilobate extracellular
orthosteric sites of T1R1and T1R2in their respective heterodimers.
Collectively, this indicates that ligand binding competent GPCRs
can be subject to allosteric regulation by orphan 7TM proteins in
oligomeric complexes.
Targeting of protein–protein interfaces
An emerging class of compounds target the interacting interface
between proteins. This interesting new class of molecules may
become relevant for orphan 7TM proteins that modulate the
function of other proteins by direct interaction (Figure 3B). The
design and optimisation of such protein–protein interaction inhi-
bitors requires detailed structural knowledge to identify relevant
regions of the interface. Unfortunately, structural information, in
particular of the 7TM domain, is still very poor and limited to the
structure of the rhodopsin GPCR. Despite this general lack of
knowledge, two specific examples illustrate the great potential
of such compounds in the GPCR field.
The well-known antithrombotic compound clopidogrel and its
active metabolite Act-Met have been shown to disrupt P2Y12
receptor oligomer complexes in platelets and to redistribute the
receptors out of lipid rafts [60]. As raft-associated P2Y12 oligomers
represent the functional form of the receptor, clopidogrel acts as
an inhibitor of P2Y12 function. Although the precise mechanism
of action of clopidogrel is currently unknown, modification of
cysteine 97 within the first extracellular loop of P2Y12 by the free
thiol of the Act-Met metabolite appears to be involved.
The pharmacological phenotype of the class B CRLR can be
modulated through interaction with three distinct RAMPs [61].
Development of selective, high-affinity CGRP1 receptor (the
heterodimer of CRLR and RAMP1) antagonists has been intensi-
fied in recent years because of mounting evidence that CGRP
receptors are involved in the pathophysiology of migraine head-
ache. Although RAMPs are not orphan 7TM proteins but rather
singleTMaccessoryproteins,thenatureofCGRPreceptorantago-
nists sheds light on possible mechanisms of action of compounds
targeting interaction interfaces involving orphan 7TM proteins.
Antagonists such as BIBN4096BS act in fact at the interface
between CRLR and RAMP. Key sites for this interaction are tryp-
tophane 74 of RAMP1 and the amino terminus of CRLR. These
datasuggestthattheinterfacebetweenaGPCRandotherproteins
can also constitute allosteric binding pockets [62,63]. Clearly,
more precise structural information is needed to exploit the full
therapeuticpotentialofthisnewclassofcompoundsasshownfor
inhibitors of the p53 and Mdm2 interaction [64], iNOS dimerisa-
tion [65] and TNF-alpha trimerisation [66].
Concluding remarks and perspectives
Toovercometheincreasingresistanceofthemorethan100remain-
ing orphan 7TM proteins to current deorphanisation strategies,
alternative approaches and concepts are needed. Whereas some
of these orphans must be matched with their endogenous ligands,
others may be true orphans for which no natural ligand does exist.
However, even true orphans could become interesting drug targets
as detailed in the present review. Elucidating the function of these
orphans continues to be a priority for the understanding of their
biological role and for drug discovery. Whereas the complete
sequence of the human genome provided us with a fairly precise
idea about the total number of orphan 7TM proteins, current
progress in systems biology and proteomics will provide us with
valuableinsightsintheorphan7TMprotein-containingcomplexes,
toultimatelyelucidatetheinvivofunctionoftheseproteins.Clearly,
more than 100 orphan 7TM proteins will keep us busy for several
more years.
Acknowledgements
This work was supported by grants from SERVIER, the Fondation
Recherche Me ´dicale (‘Equipe FRM’), FRC Neurodon, Association
pour la Recherche contre le Cancer (ARC, N83970), Institut
National de la Sante ´ et de la Recherche Me ´dicale (INSERM), Centre
National de la Recherche Scientifique (CNRS). We thank J. Dam, P.
Maurice, P. Chen and J.L. Guillaume (Institut Cochin, Paris) for
comments on the manuscript.
References
1 Civelli, O. (2005) GPCR deorphanizations: the novel, the known and the
unexpected transmitters. Trends Pharmacol. Sci. 26, 15–19
2 Civelli, O. et al. (2001) Novel neurotransmitters as natural ligands of orphan
G-protein-coupled receptors. Trends Neurosci. 24, 230–237
3 Reinscheid, R.K. et al. (1995) Orphanin FQ: a neuropeptide that activates an
opioidlike G protein-coupled receptor. Science 270, 792–794
4 Civelli, O. et al. (1998) Reverse physiology: discovery of the novel neuropeptide,
orphanin FQ/nociceptin. Crit. Rev. Neurobiol. 12, 163–176
5 Hinuma, S. et al. (1998) A prolactin-releasing peptide in the brain. Nature 393,
272–276
6 Kojima, M. et al. (1999) Ghrelin is a growth-hormone-releasing acylated peptide
from stomach. Nature 402, 656–660
7 Fergusson, S.S. and Caron, M.G. (2004) Green fluorescent protein-tagged beta-
arrestin translocation as a measure of G protein-coupled receptor activation.
Methods Mol. Biol. 237, 121–126
8 Oakley, R.H. et al. (2006) The ligand-independent translocation assay: an enabling
technology for screening orphan G protein-coupled receptors by arrestin
recruitment. Methods Enzymol. 414, 50–63
9 Adie,E.J.etal.(2003)CypHer5:agenericapproachformeasuringtheactivationand
trafficking of G protein-coupled receptors in live cells. Assay Drug Dev. Technol. 1,
251–259
10 Tunaru, S. et al. (2003) PUMA-G and HM74 are receptors for nicotinic acid and
mediate its anti-lipolytic effect. Nat. Med. 9, 352–355
11 Wise, A. et al. (2003) Molecular identification of high and low affinity receptors for
nicotinic acid. J. Biol. Chem. 278, 9869–9874
12 Mendive, F. et al. (2006) Defective postnatal development of the male reproductive
tract in LGR4 knockout mice. Dev. Biol. 290, 421–434
13 Morita, H. et al. (2004) Neonatal lethality of LGR5 null mice is associated with
ankyloglossia and gastrointestinal distension. Mol. Cell. Biol. 24, 9736–9743
14 Lefkowitz, R.J. and Shenoy, S.K. (2005) Transduction of receptor signals by beta-
arrestins. Science 308, 512–517
15 Cain, S.A. and Monk, P.N. (2002) The orphan receptor C5L2 has high affinity
bindingsitesforcomplementfragmentsC5aandC5ades-Arg(74).J.Biol.Chem.277,
7165–7169
16 Kalant, D. et al. (2003) The chemoattractant receptor-like protein C5L2 binds the
C3a des-Arg77/acylation-stimulating protein. J. Biol. Chem. 278, 11123–11129
17 Chen, N.J. et al. (2007) C5L2 is critical for the biological activities of the
anaphylatoxins C5a and C3a. Nature 446, 203–207
18 Mantovani, A. et al. (2001) Decoy receptors: a strategy to regulate inflammatory
cytokines and chemokines. Trends Immunol. 22, 328–336
19 McLatchie, L.M. et al. (1998) RAMPs regulate the transport and ligand specificity of
the calcitonin-receptor-like receptor. Nature 393, 333–339
Drug Discovery Today?Volume 13,Numbers 1/2?January 2008 REVIEWS
www.drugdiscoverytoday.com
57
Reviews?POST SCREEN

Page 7

20 Bouvier, M. (2001) Oligomerization of G-protein-coupled transmitter receptors.
Nat. Rev. Neurosci. 2, 274–286
21 Milligan, G. (2006) G-protein-coupled receptor heterodimers: pharmacology,
function and relevance to drug discovery. Drug Discov. Today 11, 541–549
22 Prinster, S.C. et al. (2005) Heterodimerization of g protein-coupled receptors:
specificity and functional significance. Pharmacol. Rev. 57, 289–298
23 White, J.H. et al. (1998) Heterodimerization is required for the formation of a
functional GABA(B) receptor. Nature 396, 679–682
24 Uberti, M.A. et al. (2005) Heterodimerization with beta2-adrenergic receptors
promotes surface expression and functional activity of alpha1D-adrenergic
receptors. J. Pharmacol. Exp. Ther. 313, 16–23
25 Nelson, G. et al. (2001) Mammalian sweet taste receptors. Cell 106, 381–390
26 Nelson, G. et al. (2002) An amino-acid taste receptor. Nature 416, 199–202
27 Levoye, A. et al. (2006) Do orphan G-protein-coupled receptors have ligand-
independent functions? New insights from receptor heterodimers. EMBO Rep 7,
1094–1098
28 Bond, R.A. and Ijzerman, A.P. (2006) Recent developments in constitutive receptor
activity and inverse agonism, and their potential for GPCR drug discovery. Trends
Pharmacol. Sci. 27, 92–96
29 Milligan, G. (2003) Constitutive activity and inverse agonists of G protein-coupled
receptors: a current perspective. Mol. Pharmacol. 64, 1271–1276
30 Waldhoer, M. et al. (2002) Murine cytomegalovirus (CMV) M33 and human CMV
US28receptorsexhibitsimilarconstitutivesignalingactivities.J.Virol.76,8161–8168
31 Vischer, H.F. et al. (2006) HCMV-encoded G-protein-coupled receptors as
constitutively active modulators of cellular signaling networks. Trends Pharmacol.
Sci. 27, 56–63
32 Davis, D.J. et al. (1992) Insulin Inhibits beta-Adrenergic Responses in Fetal Rabbit
Lung in Explant Culture. Am. J. Physiol. 263 (5 Pt. 1), L562–L567
33 Rosenkilde, M.M. et al. (2006) Molecular pharmacological phenotyping of EBI2. An
orphanseven-transmembranereceptorwithconstitutiveactivity.J.Biol. Chem.281,
13199–13208
34 Tanaka, S. et al. (2007) Neural expression of G protein-coupled receptors GPR3,
GPR6, and GPR12 upregulates cyclic AMP levels and promotes neurite outgrowth.
J. Biol. Chem. 282, 10506–10515
35 Ledent, C. et al. (2005) Premature ovarian aging in mice deficient for Gpr3. Proc.
Natl. Acad. Sci. U.S.A. 102, 8922–8926
36 Mehlmann, L.M. et al. (2004) The Gs-linked receptor GPR3 maintains meiotic arrest
in mammalian oocytes. Science 306, 1947–1950
37 Jones, P.G. et al. (2007) Tissue distribution and functional analyses of the
constitutively active orphan G protein coupled receptors, GPR26 and GPR78.
Biochim. Biophys. Acta 1770, 890–901
38 Stoddart, L.A. et al. (2007) Uncovering the pharmacology of the G protein-coupled
receptor GPR40: high apparent constitutive activity in guanosine 50-O-(3-
[35S]thio)triphosphate binding studies reflects binding of an endogenous agonist.
Mol. Pharmacol. 71, 994–1005
39 Levoye, A. et al. (2006) The orphan GPR50 receptor specifically inhibits MT(1)
melatonin receptor function through heterodimerization. EMBO J. 25, 3012–3023
40 Kniazeff, J. et al. (2002) No ligand binding in the GB2 subunit of the GABA(B)
receptor is required for activation and allosteric interaction between the subunits.
J. Neurosci. 22, 7352–7361
41 Neuhaus, E.M. et al. (2005) Odorant receptor heterodimerization in the olfactory
system of Drosophila melanogaster. Nat. Neurosci. 8, 15–17
42 Benton, R. et al. (2006) Atypical membrane topology and heteromeric function of
Drosophila odorant receptors in vivo. PLoS Biol. 4, e20
43 Xu, H. et al. (2004) Different functional roles of T1R subunits in the heteromeric
taste receptors. Proc. Natl. Acad. Sci. U.S.A. 101, 14258–14263
44 Milasta, S. et al. (2006) Interactions between the Mas-related receptors MrgD and
MrgE alter signalling and trafficking of MrgD. Mol. Pharmacol. 69, 479–491
45 Marazziti, D. et al. (2007) GPR37 associates with the dopamine transporter to
modulate dopamine uptake and behavioral responses to dopaminergic drugs. Proc.
Natl. Acad. Sci. U.S.A. 104, 9846–9851
46 Riobo, N.A. et al. (2006) Hedgehog signal transduction: signal integration and cross
talk in development and cancer. Cell Cycle 5, 1612–1615
47 Liu, G. et al. (2004) Assembly of a Ca2+-dependent BK channel signaling complex by
binding to beta2 adrenergic receptor. EMBO J. 23, 2196–2205
48 Davare, M.A. et al. (2001) A beta2 adrenergic receptor signaling complex assembled
with the Ca2+channel Cav1.2. Science 293, 98–101
49 Beedle, A.M. et al. (2004) Agonist-independent modulation of N-type calcium
channels by ORL1 receptors. Nat. Neurosci. 7, 118–125
50 Lange, J.H. and Kruse, C.G. (2005) Keynote review: Medicinal chemistry strategies
to CB1 cannabinoid receptor antagonists. Drug Discov. Today 10, 693–702
51 Nijenhuis, W.A. et al. (2001) AgRP(83-132) acts as an inverse agonist on the human-
melanocortin-4 receptor. Mol. Endocrinol. 15, 164–171
52 Adan, R.A. and Kas, M.J. (2003) Inverse agonism gains weight. Trends Pharmacol.Sci.
24, 315–321
53 Casarosa,P.etal.(2003)Identificationof thefirstnonpeptidergicinverseagonistfor
a constitutively active viral-encoded G protein-coupled receptor. J. Biol. Chem. 278,
5172–5178
54 Bresnick, J.N. et al. (2003) Identification of signal transduction pathways used by
orphan g protein-coupled receptors. Assay Drug Dev. Technol. 1, 239–249
55 Waldhoer,M.etal.(2005)Aheterodimer-selectiveagonistshowsinvivorelevanceof
G protein-coupled receptor dimmers. Proc. Natl. Acad. Sci. U.S.A. 102, 9050–9055
56 May, L.T. et al. (2007) Allosteric modulation of G protein-coupled receptors. Annu.
Rev. Pharmacol. Toxicol. 47, 1–51
57 Smit, M.J. et al. (2007) Pharmacogenomic and structural analysis of constitutive g
protein-coupled receptor activity. Annu. Rev. Pharmacol. Toxicol. 47, 53–87
58 Binet, V. et al. (2004) The heptahelical domain of GABA(B2) is activated directly by
CGP7930, a positive allosteric modulator of the GABA(B) receptor. J. Biol. Chem.
279, 29085–29091
59 Pin, J.P. et al. (2005) Allosteric functioning of dimeric class C G-protein-coupled
receptors. FEBS J. 272, 2947–2955
60 Savi, P. et al. (2006) The active metabolite of Clopidogrel disrupts P2Y12 receptor
oligomers and partitions them out of lipid rafts. Proc. Natl. Acad. Sci. U.S.A. 103,
11069–11074
61 Hay, D.L. et al. (2006) GPCR modulation by RAMPs. Pharmacol. Ther. 109,
173–197
62 Mallee,J.J.etal.(2002)Receptoractivity-modifyingprotein1determinesthespecies
selectivity of non-peptide CGRP receptor antagonists. J. Biol. Chem. 277, 14294–
14298
63 Salvatore, C.A. et al. (2006) Identification and pharmacological characterization of
domains involved in binding of CGRP receptor antagonists to the calcitonin-like
receptor. Biochemistry 45, 1881–1887
64 Grasberger, B.L. et al. (2005) Discovery and cocrystal structure of
benzodiazepinedione HDM2antagonists that activatep53 in cells. J.Med. Chem. 48,
909–912
65 McMillan, K. et al. (2000) Allosteric inhibitors of inducible nitric oxide synthase
dimerization discoveredviacombinatorialchemistry.Proc.Natl.Acad. Sci.U.S.A.97,
1506–1511
66 He, M.M. et al. (2005) Small-molecule inhibition of TNF-alpha. Science 310,
1022–1025
67 Oh da, Y. et al. (2006) Cellular and molecular biology of orphan G protein-coupled
receptors. Int. Rev. Cytol. 252, 163–218
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