GOLF?Mediates Dopamine D1Receptor Signaling
Xiaoxi Zhuang, Leonardo Belluscio, and Rene Hen
Center for Neurobiology and Behavior, Columbia University, New York, New York 10032
It is generally assumed that the coupling of dopamine D1
receptors to adenylyl cyclase is mediated by the stimulatory
GTP-binding protein Gs. However, the striatum contains little
Gs?subunit, whereas it expresses high levels of Golf?, a close
relative of Gs?that is also expressed in olfactory receptor
neurons. We used Golf?knockout mice to examine the func-
tional coupling of D1receptors. We found that these mice
showed no hyperlocomotor response to either the D1agonist
SKF-81297 or the psychostimulant cocaine. Moreover, Golf?
knockout mice did not display cocaine-induced c-fos expres-
sion in the striatum. Finally, in the absence of Golf?, striatal D1
receptors have a decreased affinity for dopamine. Thus cou-
pling to Golf?appears to mediate D1signaling in the striatum.
Key words: dopamine; D1 receptor; Golf; Gs; striatum;
The dopamine D1receptor is the most abundant and widespread
of the five known dopamine receptor subtypes. It is highly ex-
pressed in the striatum, nucleus accumbens, and olfactory tuber-
cle and is moderately expressed in the cortex, amygdala, hypo-
thalamus, and thalamus (Gingrich and Caron, 1993; Jaber et al.,
1996; Missale et al., 1998). The D1receptor is also found on the
terminals of striatal neurons in the substantia nigra pars reticulata
(Altar and Hauser, 1987). D1receptors stimulate the formation of
cAMP in response to agonists both in intact preparations (Hess et
al., 1987; Watts et al., 1993) and in a number of D1-transfected
cell lines (Dearry et al., 1990; Monsma et al., 1990; Zhou et al.,
1990). It is thus generally assumed that the coupling of dopamine
D1receptors to adenylyl cyclase is mediated by Gs. However,
several studies have shown that the striatum, despite its high D1
receptor level, has very little Gs?subunit, whereas it does express
high levels of another G-protein subunit, Golf?(Drinnan et al.,
1991; Herve et al., 1995; Belluscio et al., 1998), which was found
originally to mediate olfactory receptor signaling (Jones and
Reed, 1989). Golf?and Gs?share 88% homology in amino acid
composition, both stimulate adenylyl cyclase (Jones and Reed,
1989), and both are substrates for covalent ADP-ribosylation
catalyzed by cholera toxin (Jones et al., 1990). In addition, there
are no specific Golf?or Gs?inhibitors.
Recently, a mouse line deficient in Golf?has been generated
(Golf?knockout). These mice are anosmic because of the lack of
olfactory receptor signaling (Belluscio et al., 1998). They also
provide the means to test the role of Golf?in D1signaling. We
have found that Golf?knockout mice are deficient in striatal
dopamine D1receptor-mediated behavioral and biochemical ef-
fects, consistent with the hypothesis that Golf?mediates D1sig-
naling in the striatum.
MATERALS AND METHODS
Animals. The generation of Golf?knockout mice has been detailed
previously (Belluscio et al., 1998). Wild-type and knockout littermates
have a mixed (129/Sv X C57BL/6) background and were backcrossed at
least three times into the C57BL/6 strain. Only male mice were used in
all of the studies. All mice were kept on a 6 A.M.-6 P.M. light cycle. All
animal procedures were approved by the Institutional Animal Care and
In situ hybridization. In situ hybridization was performed on 20 ?m
fresh frozen sections with
encoding Golf?(Jones and Reed, 1989) and Gs?(Sullivan et al., 1986)
were obtained by RT-PCR. Under the in situ hybridization conditions
that were used, the Golf?and Gs?probes did not cross-hybridize (Bel-
luscio et al., 1998).
Immunohistochemistry. Animals (n ? 3 for both genotypes) were
deeply anesthetized with ketamine and transcardially perfused with 4%
paraformaldehyde. Sections (45 ?m) were cut on a freezing-sliding
microtome. The primary antibody was directed against the c-fos
N-peptide (AB-2; Oncogene Sciences, Mineola, NY) and used at 1:500
dilution. Fos immunoreactivity was visualized with the avidin–biotin–
peroxidase method (Vectostatin Elite ABC; Vector, Burlingame, CA).
The peroxidase reaction was developed in diaminobenzidine and H2O2.
Locomotor activity. All mice (n ? 5 for both genotypes) were kept on
a 6 A.M.-6 P.M. light cycle. Male mice between 3 and 5 months old were
used and tested during the light period. Animals were placed in square
open chambers (40 cm long ? 40 cm wide ? 37 cm high). They were
monitored throughout the test session by a video tracking system
equipped with infrared beams (PolyTrack, San Diego Instruments) that
records the animal’s location and path (horizontal activity) as well as the
number of rearings (vertical activity). Before each test, the open fields
were cleaned to maintain constant olfactory cues.
Drug treatment. Drugs were dissolved in saline and administered in-
traperitoneally. Animals’ locomotor activities were monitored right after
the injection (n ? 5 for each genotypes). (?)-SKF-81297 hydrobromide
was obtained from RBI (Natick, MA), and cocaine was obtained from
Sigma (St. Louis, MO).
Autoradiography. Coronal fresh frozen sections were cut at 20 ?m and
thaw-mounted onto slides. Wild-type and mutant brain (n ? 4 for each
genotype) sections of comparable brain regions were mounted on the
same slides. For dopamine D1binding, sections were dried at room
temperature, preincubated for 30 min in 50 mM Tris buffer containing (in
mM): 120 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 0.001% ascorbic acid, pH
7.4, and then incubated for 90 min in the same buffer supplemented with
2 nM n-methyl-3H-SCH23390 (85.0 Ci/mmol; Amersham, Arlington
33P-UTP-labeled riboprobes. cDNA clones
Received April 10, 2000; revised June 1, 2000; accepted June 2, 2000.
This work was supported in part by a Bristol-Myers Squibb Neuroscience Award
(R.H.). We thank Richard Axel for providing the Golf knockout mice. We thank
Suhan Kassir for help with autoradiography study and Kimberly Scearce-Levie for
setting up the open field.
Correspondence should be addressed to Rene Hen, Center for Neurobiology and
Behavior, Columbia University, 722 West 168th Street, PI Annex, New York, NY
10032. E-mail: email@example.com.
Dr. Belluscio’s present address: Department of Neurobiology, Duke University
Medical Center, Durham, NC 27710.
Copyright © 2000 Society for Neuroscience 0270-6474/00/200001-05$15.00/0
This article is published in The Journal of Neuroscience, Rapid
Communications Section, which publishes brief, peer-reviewed
papers online, not in print. Rapid Communications are posted
online approximately one month earlier than they would appear
if printed. They are listed in the Table of Contents of the next
open issue of JNeurosci. Cite this article as: JNeuro-
sci, 2000, 20:RC91 (1–5). The publication date is the date of
posting online at www.jneurosci.org.
The Journal of Neuroscience, 2000, Vol. 20 RC91 1 of 5
Heights, IL) and 50 nM ketanserin (to block 5-HT2 receptor binding).
Nonspecific binding was determined in the presence of 10 ?M flu-
penthixol. For displacement studies, alternate slides were incubated with
various concentrations of dopamine (1, 4, 10, 25, 50, 100, 200, 400, 800,
and 1600 ?M) (Altar and Marien, 1987; Richfield et al., 1989).
Data analysis. Data were analyzed using StatView 4.5 (Abacus Con-
cepts Inc.). Unpaired two-tailed Student’s t test was used when genotype
was the only grouping variable. ANOVA was used when genotype was
not the only grouping variable and when data were collected in a single
trial of a single session. Repeated measure ANOVA was used when data
were collected in multiple trials of a single session. Nested repeated
measure ANOVA was used when data were collected in multiple trials in
more than one session.
Golf?but not Gs?is highly expressed in the striatum
The distribution of Golf?mRNA and Gs?mRNA in wild-type
mice was studied by in situ hybridization with Golf?and Gs?RNA
probes, respectively. Golf?mRNA is highly expressed in the
caudate-putamen, nucleus accumbens, olfactory tubercle, piri-
form cortex, dentate gyrus, CA3 region of the hippocampus (Fig.
1a,c), and Purkinje cells of the cerebellum (data not shown).
There are low levels of Golf?expression in the thalamus, hypo-
thalamus, lateral septum, bed nucleus of the stria terminalis,
preoptic area (Fig. 1a,c), and substantia nigra (data not shown).
In contrast, Gs?mRNA is widely expressed except in the caudate-
putamen, nucleus accumbens, and olfactory tubercle, where it is
barely detectable (Fig. 1b,d). In Golf?knockout mice, Golf?
mRNA is undetectable, whereas the level of Gs?is unchanged
(Belluscio et al., 1998).
Golf?knockout mice do not display D1receptor-
dependent locomotor responses
Because striatal D1receptor activation leads to behavioral stim-
ulation, we examined the locomotor and rearing activities of Golf?
knockout mice both in baseline conditions and in response to
direct and indirect D1agonists. Both locomotor (Fig. 2a,b) and
rearing activities (data not shown) of knockout mice were signif-
icantly higher in three 1 hr open field daily sessions compared
with wild-type mice. Nevertheless, knockout mice showed normal
As previously reported, the D1-selective agonist SKF-81297 (8
mg/kg) evoked increases in locomotor activity (Fig. 2c,d) and
rearing (data not shown) in wild-type mice. In contrast, SKF-
81297 did not stimulate locomotor activity (Fig. 2c,d) or rearing
in Golf?knockout mice. Similarly, cocaine (20 mg/kg) dramati-
cally increased locomotor activity (Fig. 2c,d) and rearing (data
not shown) in wild-type mice but had no effect in Golf?knockout
mice (Fig. 2c,d). Cocaine also caused mild stereotypy in wild-type
mice but not in knockout mice (data not shown).
Golf?knockout mice do not display cocaine-induced
c-fos expression in the striatum
The expression of the immediate-early gene c-fos is markedly
induced in the striatum in response to psychostimulants such as
cocaine (Graybiel et al., 1990; Lucas et al., 1997). Mice were
injected with 20 mg/kg body weight cocaine or saline, killed 2 hr
later, and studied for c-fos expression by immunohistochemistry.
In saline-injected wild-type or Golf?knockout mice, virtually no
c-fos expression was detected in the brain (data not shown). In
response to cocaine, wild-type but not Golf?knockout mice dis-
played c-fos expression in the striatum (Fig. 3). In the cingulate
cortex, lateral septum (Fig. 3), and piriform cortex (data not
shown), c-fos was markedly induced in both genotypes. There
were also low levels of cocaine-induced c-fos expression in the
thalamus, hypothalamus, parietal cortex, and perirhinal cortex in
both genotypes (data not shown).
Decreased affinity of D1receptors for dopamine in the
striatum of Golf?knockout mice
G-protein coupling is usually necessary for high affinity agonist
binding (Adham et al., 1998; Zhao et al., 1998). To compare the
state of D1receptor coupling in wild-type and Golf?knockout
mice, we performed in vitro autoradiography on brain sections.
We examined the effects of increasing concentrations of the
agonist dopamine on binding of the D1antagonist ligand
SCH23390. As shown in Figure 4, dopamine is less efficient at
displacing the radioligand in the striatum of Golf?knockout mice
as compared with wild-type mice. The IC50is significantly higher
in Golf?knockout mice than in wild-type mice (t(6)? 2.6; p ?
0.04), consistent with a decreased affinity of striatal D1receptors
for dopamine. No genotype difference in IC50was found in the
nucleus accumbens, olfactory tubercle, or substantia nigra. Spe-
cific radioligand binding was the same for both genotypes in all
Dopamine D1receptors are coupled to Golf?in
The present study provides the first functional evidence that the
dopamine D1receptor in the striatum is coupled to Golf?. Spe-
cifically, we show in three ways that striatal D1receptors are not
functional in mice lacking Golf?. First, Golf?knockout mice do
not display locomotor responses to the D1-selective agonist SKF-
81297. Second, both the locomotor and the c-fos-inducing effects
of cocaine are absent in Golf?knockout mice. Although SKF-
81297 directly activates D1receptors, cocaine increases extracel-
lular dopamine concentration by blocking dopamine reuptake
and acts therefore as an indirect D1agonist. It has been shown
using D1antagonists (Cabib et al., 1991; Young et al., 1991;
Ushijima et al., 1995) and D1receptor knockout mice (Xu et al.,
1994a,b; Drago et al., 1996; Moratalla et al., 1996) that cocaine-
mRNA (a, c) and Gs?mRNA (b, d) in wild-type mice was studied by in
situ hybridization with Golf?and Gs?RNA probes, respectively. Golf?
mRNA is highly expressed in the CPu, NAc, OT, Pir, DG, and CA3 (a, c).
There is a low level of Golf?expression in the Th, HyT, LS, BST, and POA.
Gs?mRNA is highly expressed everywhere except in CPu, NAc, and OT,
where it is barely detectable. ac, Anterior commissure; AO, anterior
olfactory nuclei; BST, bed nucleus stria terminalis; CA1, CA1 field of
hippocampus; CA2, CA2 field of hippocampus; CA3, CA3 field of hip-
pocampus; cc, corpus callosum; CPu, caudate-putamen (striatum); Cx,
cortex; DG, dentate gyrus; fi, fimbria hippocampus; HyT, hypothalamus;
LPA, lateral preoptic area; LS, lateral septum; MCPO, magnocellular
preoptic nucleus; MPA, medial preoptic area; MS, medial septum; NAc,
nucleus accumbens; OT, olfactory tubercle; ox, optic chiasm; Pir, piriform
cortex; POA, preoptic area; Th, thalamus; VP, ventral pallidum.
The distribution of Golf?and Gs?. The distribution of Golf?
2 of 5 J. Neurosci., 2000, Vol. 20Zhuang et al. • Dopamine D1Receptor Couples to Golf
induced locomotion and striatal c-fos expression are D1depen-
dent. The lack of these responses in the Golf?knockout mice is
therefore consistent with an inactivity of striatal D1receptors.
Cocaine-induced c-fos expression in other brain regions of the
Golf?knockout mice such as the cingulate cortex and the lateral
septum could be attributable either to intact D1-signaling in these
structures or to D1-independent mechanisms. The latter alterna-
tive is suggested by observations that cocaine induces c-fos ex-
pression in the cingulate cortex and lateral septum of D1knock-
out mice (Moratalla et al., 1996).
A third line of evidence suggesting that striatal D1receptors
are inactive in Golf?knockout mice is the decrease in the affinity
of these receptors for dopamine. A decrease in the affinity of a
G-protein-coupled receptor for agonists is often associated with
G-protein uncoupling (Adham et al., 1998; Zhao et al., 1998). In
the absence of Golf?, striatal D1receptors may not be coupled to
a G-protein. The other two regions with high levels of Golf?but
not Gs?, namely the nucleus accumbens and olfactory tubercle,
did not show decreased affinity for dopamine, suggesting that D1
receptors in these regions may be coupled to a different
Although Golf knockout and wild-type mice are on a mixed
genetic background (C57BL/6 X 129/Sv; see Materials and Meth-
ods), it is highly unlikely that the lack of responsiveness to cocaine
and dopamine agonist of Golf knockout mice results from differ-
ences between two parental strains, for two reasons. First, the
experimental animals are littermates and therefore contain sim-
ilar proportions of both strains. Second, both 129/Sv and C57BL/6
parental strains are similarly responsive to cocaine and dopamine
agonists (our unpublished results).
mg/kg cocaine and killed 2 hr later for immunohistochemical studies.
There was a dramatic increase in c-fos immunoreactive nuclear staining in
the striatum, cingulate cortex, and lateral septum in the wild-type mice in
contrast to saline-injected controls. In Golf?knockout mice, cocaine-
induced c-fos expression was seen in the cingulate cortex and lateral
septum but not in the striatum (genotype difference in the striatum: t(4)?
3.1, p ? 0.035). CPu, Caudate-putamen (striatum); LS, lateral septum; V,
Cocaine-induced c-fos expression. Mice were injected with 20
path length) was monitored for 1 hr (each point represents 5 min in a and c; the average activity during the hour is shown in b and d) for 3 consecutive
days. Locomotor activity of knockout mice is significantly higher than that of wild-type mice on all 3 d (a, b, F(1,8)? 26.0; p ? 0.001). The D1-selec-
tive agonist SKF-81297 (8 mg/kg) and cocaine (20 mg/kg) elicited a significant increase in locomotor activity in wild-type mice, whereas they had no
effect in Golf?knockout mice (c, d). There is a significant genotype ? treatment interaction (F(2,16)? 24.5; p ? 0.0001).
Basal and drug-induced locomotor activity in the open field. Naive animals were exposed to the open field, and their horizontal activity (total
Zhuang et al. • Dopamine D1Receptor Couples to Golf
J. Neurosci., 2000, Vol. 20 3 of 5
Two distinct D1signaling pathways
The midbrain dopamine system has three major projections: the
nigrostriatal pathway, which is involved in motor function; the
mesolimbic pathway, which is involved in reward; and the meso-
cortical pathway, which is involved in cognitive functions (Ging-
rich and Caron, 1993; Jaber et al., 1996; Missale et al., 1998).
Although D1receptors are found in all three projection areas,
there appears to be a clear segregation of their downstream
pathways. Golf?is highly expressed in the striatum (nigrostriatal
pathway), whereas Gs?is barely detectable there but highly ex-
pressed in the cortex (mesocortical pathway). The mesolimbic
pathway, on the other hand, has both kinds of stimulatory
G-proteins, with Golf?in the nucleus accumbens and the olfactory
tubercle, Gs?in the septum, and both Golf?and Gs?in the
Segregation in adenylyl cyclase distribution has also been re-
ported. Specifically, adenylyl cyclase type V (AC5) is found to be
restricted to the striatum, nucleus accumbens, and olfactory tu-
bercle, whereas adenylyl cyclase type I (AC1) is barely detectable
in these three regions but is widely distributed in other brain
regions (Mons et al., 1995; Matsuoka et al., 1997; Shishido et al.,
1997). This AC1 versus AC5 segregation matches well with the
Gs?versus Golf?segregation. It is therefore likely that D1signal-
ing in the nigrostriatal pathway is primarily mediated by D1–
Golf?–AC5 coupling, whereas D1signaling in the mesocortical
pathway is mediated by D1–Gs?–AC1 coupling.
The distinction between these two D1signaling pathways is
also seen within the striatum during development. The striatum
expresses Gs?and AC1 but not Golf?or AC5 before the first
postnatal week (Rius et al., 1994; Matsuoka et al., 1997), and
there is a progressive switch from D1–Gs?–AC1 to D1–Golf?–
AC5 during the first 3 postnatal weeks. It will be interesting to
investigate whether these two signaling pathways have different
functional properties and how such differences may impact on the
development and function of the nigrostriatal pathway.
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