From the
Coupling between D-1 dopamine receptors and G proteins in cell
lines expressing human D-1 receptors and different G proteins was
examined. Pertussis toxin (PTX) treatment of rat pituitary
GH
Dopamine, a key neurotransmitter in both the central and
peripheral nervous systems, exerts its effects via at least five
genetically distinct receptor subtypes: D-1, D-2, D-3, D-4, and D-5
(Gingrich and Caron, 1993; O'Dowd, 1993). In addition, a sixth
receptor, D
G proteins are
To identify which G proteins can
couple to rat D-1 dopamine receptors, we reconstituted soluble rat
striatal D-1 receptors with exogenous sources of soluble G proteins,
after first inactivating endogenous G proteins by prior treatment of
striatal membranes with the sulfhydryl-modifying reagent, N-ethylmaleimide (Sidhu et al., 1991). In this
cell-free reconstituted system, we have demonstrated that rat D-1
receptors couple to not only G
In the present study, we
have explored human D-1 receptor-G protein couplings in membranes from
two different cellular systems: rat pituitary GH
SK-N-MC neuroblastoma cells were grown in 175-cm
The harvested tissue
culture cells from above were suspended in ice-cold 10 mM Tris-HCl, pH 7.4, containing 1 mM each EDTA and EGTA, and
homogenized in an all glass Dounce homogenizer. The suspension was
centrifuged (5 min at 1,500
Western blot
analyses was undertaken by SDS-polyacrylamide gel electrophoresis (10%
polyacrylamide) using 10 µg each of membranes from
GH
D-1 receptor binding activity in the supernatant
fraction was measured by prior removal of the detergent with SM-2
BioBeads and simultaneous incorporation of the receptors into
phospholipid vesicles (Sidhu et al., 1994). After 1 h of
treatment with SM-2 BioBeads, the supernatant was removed and used
immediately in binding assays as described below.
Cells treated with both CTX and PTX were tested for
their ability to accumulate cAMP, after stimulation with increasing
concentrations of dopamine (Fig. 1C). In control,
untreated cells, K
NEI-805 was able to coimmunoprecipitate solubilized
D-1 dopamine receptors into the pellet fraction; 24 ± 4.3% (n = 3) of the total receptor activity was associated
with the pellet fraction (Fig. 4A). There was a
corresponding loss of receptors in the supernatant fraction where 69.8
± 8.8% (n = 4) of the total receptor activity
was detected (Fig. 4B).
The D-1
dopamine receptor is associated with G
Using different cell lines which express D-1 dopamine
receptors and different PTX-sensitive G proteins, the results of this
study indicate that D-1 dopamine receptors couple to the
That
D-1-like dopamine receptors are able to couple to PTX-sensitive G
proteins had been suggested earlier (Bertorello and Aperia, 1988).
However, the inhibition of
Na
In this
study we not only confirm that human D-1 receptors couple to
PTX-sensitive G proteins, but also show that such couplings exist in
membranes. Thus, a reduction in the proportion of D-1 receptors in the
high affinity state was observed upon treatment of
GH
The most important
finding in our studies is the ability of D-1 dopamine receptors to
couple to the PTX-sensitive G
An unexpected
result of our studies is the failure of D-1 dopamine receptors to be
associated with G
There is growing
consensus that receptors affect multiple signaling systems by
differential coupling to G proteins. For instance, the
We thank Dr. David Manning for his generous gift of
the different antisera.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C
cells significantly reduced, but did not
abolish, agonist high affinity binding sites of the D-1 dopamine
receptor; in SK-N-MC neuroblastoma cells, PTX failed to have any effect
on D-1 high affinity sites. Cholera toxin (CTX) treatment of
GH
C
cells reduced but did not abolish the high
affinity sites of D-1 receptors, while in SK-N-MC cells, treatment with
CTX abolished all the high affinity sites. Western blot analyses with
specific antisera indicated that G
,
G
, G
, and G
were
expressed in both cell lines, while G
and
G
were expressed in GH
C
but
not SK-N-MC cells. Antisera NEI-805 (anti-G
) and 9072
(anti-G
) immunoprecipitated 24 ± 4.3 and 34.4
± 6.9%, respectively, of G protein-associated D-1 dopamine
receptors. Antisera 3646 (anti-G
), 1521
(anti-G
), 1518 (anti-G
), and 0941
(anti-G
) failed to coimmunoprecipitate appreciable
levels of soluble receptors. These data indicate that D-1 dopamine
receptors are coupled to both G
and G
but not to G
.
, has been recently isolated from Xenopus
laevis (Sugamori et al., 1994).(
)
D-1-like dopamine receptors are encoded by intronless genes, are
able to stimulate adenylyl cyclase, and share similar pharmacological
properties. D-1-like dopamine receptors are also coupled to other
signaling systems: stimulation of phospholipase C (Felder et
al., 1989; Undie et al., 1994) and translocation of
protein kinase C (McMillan et al., 1992), stimulation of
inositol phosphate production and Ca
mobilization in Xenopus oocytes (Mahan et al., 1990), inhibition of
Na
/K
-ATPase activity (Bertorello and
Aperia, 1989), activation of the arachidonic acid cascade system
(Piomelli et al., 1991), regulation of
Na
/H
-antiport activity (Felder et
al., 1990), and stimulation of K
efflux
(Laitinen, 1993). There are also other reports which indicate that D-1
receptors have either no effect on PI metabolism (Kelly et
al., 1988) or may actually inhibit PI metabolism (Wallace and
Claro, 1990; Rubinstein and Hitzemann, 1990). The mechanism(s) by which
D-1-like dopamine receptors are coupled to such diverse signal
transducing pathways remains to be established. Some of these effects
are likely to occur through selective activation of specific members of
the D-1 family of receptors and through differential coupling of
specific receptors to different G proteins.
(
)
heterotrimers, and
members of this family include the PTX-sensitive (G
and
G
) and CTX-sensitive (G
) G proteins; these
toxins cause ADP-ribosylation of the
-subunits of the respective G
proteins (Birnbaumer, 1990; Hepler and Gilman, 1992; Neer, 1994).
G
mediates the activation of adenylyl cyclase and may
regulate the stimulation of dihydropyridine-sensitive voltage-gated
Ca
channels (Mattera et al., 1989) and
inhibition of Na
channels (Schubert et al.,
1989). The three
-subunits of G
, G
,
G
, and G
oppose the effects of G
and cause inhibition of the adenylyl cyclase system, in addition
to stimulation of K
channels (Yatani et al.,
1988) and inhibition of Ca
channels (Hille, 1994).
G
has been shown to be widely distributed in neuronal
tissues and brain, where it comprises 1-2% of membrane protein
(Huff et al., 1985), and its role in mediating the inhibition
of Ca
currents is becoming increasingly apparent
(Schultz and Hescheler, 1993; Hille, 1994). Antibodies to
G
reduced the voltage-gated C
current inhibition caused by norepinephrine in superior cervical
ganglion neurons (Caulfield et al., 1994) and mediated by
µ-opioid receptors in dorsal root ganglion (Moises et al.,
1994). G
, which is both PTX and CTX insensitive, is known
to mediate the stimulation of phospholipase C
-isoforms (Waldo et al., 1991; Rhee and Choi, 1992; Hepler and Gilman, 1992).
Thus, from the physiological properties associated with D-1-like
dopamine receptors, the G proteins most likely coupled to D-1 receptors
appear to be G
and the PTX-insensitive G
.
However, coupling of the D-1-like receptors to these G proteins does
not adequately explain the signaling responses regulated by these
receptors. Many of the observed physiological functions of D-1-like
receptors occur independent of adenylyl cyclase stimulation. Also, the
inhibition of Na
/K
-ATPase by D-1-like
dopamine sites has been shown to occur via a PTX-sensitive G protein
(Bertorello and Aperia, 1988), suggesting that either G
or
G
may be involved.
, but also to a PTX-sensitive
G protein (either G
or G
), and that this
coupling to G
/G
occurs in the simultaneous
presence of G
(Sidhu et al., 1991). Although we
documented the ability of rat D-1 sites to couple to multiple G
proteins in reconstituted lipid vesicles, it was unclear if such
couplings also existed in membranes, where interaction between
receptors and G proteins are likely to be restricted. In addition, we
were not able to define the specific identity of the PTX-sensitive G
protein coupled to these rat D-1 sites.
C
cells stably transfected with the human D-1 receptor cDNA (Kimura et al., 1995) and human SK-N-MC neuroblastoma cells
endogenously expressing D-1 dopamine receptors
(
)(Sidhu and Fishman, 1990; Zhou et al.,
1991). These two cell lines were chosen because a large proportion of
the D-1 receptors in these cells exist in the high affinity state (60.7
± 2.5% in GH
C
and 43.3 ± 15.3% in
SK-N-MC), so that any perturbations in receptor/G protein couplings can
be reflected by changes in the high affinity sites. We report here that
D-1 receptors interact with PTX-sensitive and CTX-sensitive G proteins
in the membrane-bound state. Through a combination of Western blot and
immunoprecipitation analyses, we show that D-1 receptors are coupled to
both G
and to the PTX-sensitive G
.
From both physiological and immunoprecipitation studies, we were unable
to detect any coupling between D-1 dopamine receptors and
G
.
Materials
All drugs used in this study were
obtained from Research Biochemicals Inc. (Natick, MA); the
D-1-selective radioligand,
8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol, I-SCH 23982 (Sidhu, 1990), was purchased from DuPont NEN.
Guanyl-5`-yl imidodiphosphate (Gpp(NH)p), was from Boehringer Mannheim.
PTX and CTX were from Calbiochem (San Diego, CA). All other materials
were from sources previously described and are of the highest purity
commercially available (Sidhu, 1990).
Tissue Culture and Preparation of Membranes
Rat
somatomammotrophic GHC
cells were stably
transfected with human D-1 receptor cDNA as described previously
(Kimura et al., 1995). The transfected cells were grown in
175-cm
tissue culture flasks containing Ham's F-10
medium (Mediatech, Washington D. C.), supplemented with 10%
heat-inactivated fetal bovine serum (Hyclone, Logan, UT). Cells were
detached from the culture flasks with Versene (Life Technologies, Inc.)
and collected by centrifugation (5 min at 800
g).
flasks
containing Eagle's minimum essential medium (from Mediatech)
supplemented with 10% Nu-Serum (Collaborative Biomedical Products,
Bedford, MA). Cells were detached with phosphate-buffered saline
containing 2 mM each of EGTA and EDTA and collected by
centrifugation as described above. Except where indicated,
GH
C
and SK-N-MC cells were treated with PTX
(10-50 ng/ml of growth media) or with CTX (1 µg/ml of growth
media) for 24 h prior to harvesting the cells.
g) to remove nuclei, and
the supernatant was centrifuged (20 min at 31,000
g)
to yield a crude plasma membrane pellet. The latter was resuspended in
the same homogenizing buffer and recentrifuged as before. The washed
pellet was suspended at 0.06-0.1 mg/ml in buffer A (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2
mM CaCl
and 1 mM MgCl
) and
used immediately in either binding assays or for solubilization.
Alternately, the membranes were stored frozen at -80 °C in
0.25 M sucrose, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl
at a protein concentration of 0.3-0.5 mg/ml
of protein (Sidhu, 1990).
Solubilization of D-1 Dopamine Receptors
D-1
dopamine receptors from GHC
cells were
solubilized using methods we have described elsewhere for extraction of
D-1 receptors (Sidhu and Kimura, 1994). Briefly, membranes from
GH
C
cells were suspended in solubilization
buffer (50 mM Tris-HCl, pH 7.4, 1 M NaCl, 5 mM KCl, 2 mM CaCl
, 1 mM
MgCl
, 250 mM sucrose, 1 mM DTT, 1 mM EDTA, 5 µg each of leupeptin and pepstatin, and 1 mM
phenylmethylsulfonyl fluoride) at a protein concentration of
1-1.5 mg/ml. Sonicated phospholipids (Type VII, Sigma) in 10
mM Tris-HCl, pH 7.4, containing 10 µg/ml butylated
hydroxytoluene and 1% sodium cholate was added to the membrane solution
to a final concentration of 1.2 mg/ml. After 5 min on ice, sodium
cholate (20% in water, w/v) was added to a final concentration of 1%.
The mixture was shaken gently on ice for 30 min. The sample was then
centrifuged at 45,000
g for 45 min at 4 °C. The
supernatant was removed and diluted 1:3 with buffer A containing the
protease inhibitors described above. The concentration of protein in
the soluble extract was 0.2-0.5 mg/ml and 200-400 µl of
sample was used for each immunoprecipitation reaction.
G Protein Antisera and Immunoblotting
The G
protein antisera used were all obtained after injection of synthetic
peptides corresponding to specific regions of G. Antisera NEI-801
(anti-G
/G
) and NEI-805
(anti-G
) was purchased from DuPont NEN; antisera 0941
(anti-G
/G
), 1518
(anti-G
), 1521 (anti-G
), 3646
(anti-G
), and 9072 (anti-G
) were a
kind gift of Dr. David Manning (Department of Pharmacology, University
of Pennsylvania). These antisera and their specificities are
extensively described elsewhere (Law et al., 1991; Okuma and
Reisine, 1992; Lounsbury et al., 1993).
C
and SK-N-MC cells; as a control, 10 µg
of rat striatal membranes were coelectrophoresed. Following transfer to
nitrocellulose, the filters were probed with 1:1000 dilutions of the
various antisera. The proteins were visualized using the alkaline
phosphatase-conjugated biotin-avidin system, with p-nitroblue
tetrazolium and 4-bromo-4-chloro-3-indolyl phosphate as substrate
(Bio-Rad).
Immunoprecipitation of D-1 Dopamine Receptor
To immunoprecipitate D-1 dopamine
receptorG
Protein Complexes
G protein complexes, essentially the same procedure was
used as described previously for similar studies with somatostatin (Law et al., 1991) and
-adrenergic receptors
(Okuma and Reisine, 1992), with minor modifications. A sample of
solubilized D-1 dopamine receptors (200-400 µl) from
GH
C
membranes was incubated with constant
agitation for 16 h at 4 °C with 1:50 dilutions of the various
antisera; preliminary titration studies indicated that at 1:50
dilutions of antisera, the D-1 receptors were maximally coprecipitated.
An aliquot (100 µl) of protein A-Sepharose beads (CL-4B, Sigma)
washed three times and diluted to 50% (w/v) in buffer A was added. The
samples were incubated for an additional 90 min and centrifuged at
16,000
g for 5 min in a microfuge; the supernatant was
removed and saved as the ``supernatant fraction.'' The
pellets were washed once in buffer A containing protease inhibitors
(0.5 mM phenylmethylsulfonyl fluoride and 5 µg each of
leupeptin and pepstatin) and resuspended in 200-400 µl of
binding assay buffer. Radioligand binding assays on the pellets were
conducted immediately following the wash, using 1 nM of
I-SCH 23982 in the radioligand binding assay procedure
described below.
Radioligand Binding Assays
The standard binding
assay for membranes was performed as described previously by this
laboratory (Kimura and Sidhu, 1994; Sidhu et al., 1994).
Briefly, 50 µl of membranes (0.1 mg/ml) were incubated with
0.5-1 nM (final concentration) of I-SCH
23982, in the absence or presence of competing drugs in a total volume
of 150 µl. All drug dilutions were conducted using buffer A with
protease inhibitors described above. After incubation at room
temperature for 60 min, the reactions were terminated by filtration
onto glass fiber filters under reduced pressure. The filters were
washed as described elsewhere (Kimura and Sidhu, 1994) and counted in a
Beckman 4000 gamma-counter (80% efficiency). Specific binding was
obtained by subtraction of nonspecific binding (determined in the
presence of 1 µM SCH 23390) from total binding (performed
in the presence of buffer A alone). In a typical experiment with
GH
C
membranes and 0.5 nM
I-SCH 23982, total binding obtained was 4,950 cpm,
while nonspecific binding was 142 cpm (<3%). For assays in which
Gpp(NH)p was used, the nonhydrolyzable guanyl nucleotide analog was
added to the binding assay to a final concentration of 100
µM. D-1 dopamine receptors in both the pellet fraction and
the supernatant fraction (after reconstitution into phospholipid
vesicles) were assayed using the same binding assay procedures and
buffers, described above for membrane-bound receptors.
Other Procedures and Data Analysis
Proteins were
detected by the method of Lowry et al.(1951). PI turnover
assays in GHC
cells were conducted essentially
as described before by us (Kimura et al., 1995). Accumulation
of cAMP was determined by radioimmunoassay as described previously
(Kimura et al., 1995). Analysis of binding data was performed
with the curve fitting program LIGAND (Munson and Rodbard, 1980). In
each case, a two-site model was considered to be a better fit according
to the F test at p < 0.05. Statistical
significance (p < 0.05) between two different groups was
analyzed by the Student's t test. All values represent
means ± S.D. from separate, independent experiments where n equals the number of experiments.
Coupling of D-1 Receptors to G Proteins in
GH
Since agonist high affinity
binding sites of receptors represents coupling between receptors and G
proteins, agonist competition curves were analyzed and the affinity
values and proportion of receptors existing in the high affinity state
were determined in membranes prepared from GHC
Cells
C
cells. Competition curves with dopamine were extremely shallow (Fig. 1A), and the agonist bound to two binding sites
with high and low affinity binding values of 25.7 ± 3.9 and
3,200 ± 1,200 nM, respectively (n = 6).
Approximately 60.7 ± 2.5% (n = 6; p < 0.05) of the total receptor population was in the high
affinity state. These high affinity sites were sensitive to modulation
by guanine nucleotide analogs: they were abolished by 100 µM Gpp(NH)p (Fig. 1A) and converted to a single low
affinity state (K
= 2,600 ±
500 nM, n = 3, p < 0.001).
Treatment of cells with N-ethylmaleimide similarly abolished
D-1 receptor high affinity sites (Fig. 1B), with
conversion to a single low affinity state (K
= 3, 250 ± 850, n = 5, p < 0.001). These results indicate that the observed high
affinity binding sites were due to coupling of D-1 receptors to G
proteins.
Figure 1:
Effect of CTX and PTX on D-1 dopamine
receptors expressed in GHC
cells.
GH
C
cells were treated with 1 µg/ml CTX (A) or 10 ng/ml PTX (B) for 24 h. For conducting the
cAMP accumulation studies (C), GH
C
cells were treated with the same concentrations of toxins for 1
h. Cells were washed, membranes prepared, and competition binding
studies with
I-SCH 23982 were conducted as described
under ``Experimental Procedures.'' Data shown are from a
representative experiment conducted in triplicate. Additional separate
experiments (n = 3 for CTX and n = 5
for PTX) gave similar results. For assaying cAMP accumulation, the
studies were conducted as described under ``Experimental
Procedures,'' and each value was corrected by subtracting the
basal values. For control and PTX-treated cells, basal values were
0.112 ± 0.019 and 0.116 ± 0.019 nmol/mg protein/20 min,
respectively.
Competition studies (Fig. 1A) were
conducted using membranes prepared from GHC
cells treated with CTX, which significantly (p <
0.01) reduced, but did not abolish, the percent of D-1 receptors
present in the high affinity state (32 ± 1.6%, n = 3), with K
and K
values of 85.0 ± 40.4 and 12,800
± 5,400 nM, respectively (n = 3, p < 0.01). When GH
C
cells were treated
with PTX (Fig. 1B), which causes ADP-ribosylation of the
-subunits of G
and G
, the proportion of
D-1 receptors in the high affinity state was significantly (p < 0.05) reduced to 45.8 ± 6.3% (n = 5),
without any alteration in either K
or K
of binding (34.8 ± 11.9 and
5,800 ± 2, 900 nM, respectively, n =
5). These combined data suggest that the D-1 sites in
GH
C
cells couple to not only G
, but
also to PTX-sensitive G proteins such as G
or
G
. Moreover, similar to cell-free systems (Sidhu et
al., 1991), D-1 dopamine receptors couple to G
/G
in the membrane-bound state, in the simultaneous presence of
G
.
for cAMP accumulation was
15.3 ± 1.4 nM and maximal accumulation was 2.1 ±
0.3 nmol cAMP/mg protein/20 min (n = 8). In PTX-treated
cells, neither the K
(13.0 ± 0.7
nM, n = 3) nor the maximal accumulation (2.1
± 0.1 nmol cAMP/mg protein/20 min, n = 4) were
significantly different than control cells, indicating that
differential coupling of D-1 sites to PTX-sensitive G proteins did not
affect the G
-mediated ability of these receptors to
stimulate adenylyl cyclase. In CTX-treated cells, the basal levels were
high (3.33 ± 0.36 nmol cAMP/mg protein/20 min, n = 4); when cells were challenged with dopamine and the
results plotted after subtraction of basal values, dopamine tended to
suppress the cAMP accumulation in a dose-dependent manner (Fig. 1C). Thus, coupling of D-1 receptors to
PTX-sensitive G proteins may cause suppression of adenylyl cyclase, but
only in the absence of functional receptor/G
couplings.
Coupling of D-1 Dopamine Receptors to G Proteins in
SK-N-MC Cells
Since coupling of dopamine receptors to signal
transducing systems is highly dependent on the cellular milieu in which
these receptors are expressed (Vallar et al., 1990), we
assessed the ability of D-1 receptors to couple to PTX-sensitive G
proteins in another cell line, SK-N-MC neuroblastoma cells. In control,
untreated SK-N-MC cells, dopamine bound to D-1 receptors with K and K
values of 0.66 ± 0.3 and 1,600 ± 78
nM, respectively; 35 ± 3% of the receptors were in the
high affinity state (Fig. 2A). When cells were treated
with PTX, there was no loss in the high affinity sites, and dopamine
continued to bind to two sites on the D-1 receptors with K
and K
values of 0.71 ± 0.14 and 843 ± 275
nM, respectively (n = 4), with 46 ± 6% (n = 4) of the receptors in the high affinity state (Fig. 2A). When SK-N-MC cells were treated with CTX, the
agonist high affinity sites were abolished and the receptors were
converted to a single low affinity state (K
= 400 ± 76 nM, n =
6).
Figure 2:
Effect of CTX and PTX on D-1 dopamine
receptors expressed in SK-N-MC cells. SK-N-MC cells were treated with 1
µg/ml CTX or 50 ng/ml PTX for 24 h. Cells were washed, membranes
prepared (1 mg/ml), and radioligand binding assays were conducted as
described in the legend to Fig. 1, using increasing concentrations of
dopamine (A) or SKF R-38393 (B). In a typical
experiment with 1 nM of I-SCH 23982, total and
nonspecific binding obtained was 6,900 and 2,000 cpm, respectively. The
data are from a representative experiment conducted in triplicate.
Additional separate experiments gave similar
results.
The absence of PTX couplings in SK-N-MC was puzzling, and so
these studies were repeated using a D-1-selective agonist, SKF R-38393.
In membranes from control, untreated SK-N-MC cells, SKF R-38393 bound
to the receptor with K and K
values of 12.2 ± 1.4 and 670
± 400 nM, respectively (n = 3); 33.6
± 5% of the receptors were in the high affinity state. Treatment
of cells with PTX failed to cause any changes in either the proportion
of the receptors in the high affinity state (34.3 ± 4.4%) or the
affinity value of these high affinity sites (K
= 900 ± 240 nM, n =
3), while treatment of cells with CTX caused all the receptors to be
converted to the low affinity state (K
= 810 ± 500 nM, n =
3). These data confirm the results obtained with dopamine and indicate
that in SK-N-MC cells, D-1 receptors are exclusively coupled to
G
but not to PTX-sensitive G proteins.
Immunodetection of the G Proteins Expressed in
GH
Since different
cells express different G proteins, we examined the expression of
PTX-sensitive G proteins in GHC
and SK-N-MC Cells
C
and SK-N-MC
cells, which could account for the inability of D-1 sites to display
PTX-sensitive couplings in SK-N-MC cells. Aliquots of membranes from
GH
C
and SK-N-MC cells were subject to
SDS-polyacrylamide gel electrophoresis followed by Western blot
analysis. Membranes from rat striatum were coanalyzed as a positive
control. As expected, the presence of G
was detected
in rat striata, GH
C
and SK-N-MC membranes by
antiserum NEI-805, which recognized a major 44 kDa band, along with a
minor band at approximately 47 kDa (Fig. 3A). Antiserum
3646, specific for G
subunit, detected a single
41-kDa polypeptide in rat striatal membranes (Fig. 3B, lanes 1 and 4). Antiserum 3646 also recognized a
41-kDa subunit in GH
C
and SK-N-MC membranes,
but this protein was present at much lower amounts, suggesting lower
levels of expression of G
in these cells relative to
rat striata.
Figure 3:
Immunochemical localization of G proteins
in membranes from rat striata, GHC
, and SK-N-MC
cells. Membranes (10 µg/lane) were prepared from rat striata (lanes 1 and 4), GH
C
(lane 2), or SK-N-MC (lane 3) cells and
subjected to SDS-polyacrylamide gel electrophoresis on 10% gels. The
membrane proteins were transferred to nitrocellulose membrane, and the
-subunit of each G protein was detected by using specific
antiserum diluted to 1:1000. A, antiserum NEI-805; B,
antiserum 3646; C, antiserum 1521; D, antiserum 1518; E, antiserum 9072.
Antiserum 1521 recognized a 39 kDa band corresponding
to G in both rat striata (Fig. 3C, lanes 1 and 4) and in GH
C
(lane 2) membranes, but failed to similarly detect this
protein in membranes from SK-N-MC cells (lane 3), suggesting
that these cells do not express G
. Antiserum 1518,
which is specific for G
, recognized a 40-kDa subunit
in rat striata, GH
C
, and SK-N-MC membranes (Fig. 3D), indicating that G
is
present in all three cell types. Antiserum 9072, which is specific for
G
, recognized a 39-kDa subunit in both rat striata (Fig. 3E, lanes 1 and 4) and in
GH
C
(lane 2), but not in SK-N-MC (lane 3) membranes. Thus, from these Western blot studies, the
absence of PTX-sensitive D-1 receptor-G protein couplings in SK-N-MC
cells may be related to the lack of expression of either
G
or G
in these cells.
Immunoprecipitation of D-1 Dopamine Receptor
Using the sodium cholate method we established
for solubilization of D-1 dopamine receptors (Sidhu, 1990; Sidhu et
al., 1994), GHG
Protein Complexes
C
membranes were
solubilized. Approximately 42% of membrane-bound receptors were
extracted by these procedures (B
= 283
± 5 fmol/mg protein, n = 3) with K
values of binding to
I-SCH 23982 of 0.85 ± 0.04 nM (n = 3), similiar to K
values of
membrane-bound receptors (0.6 ± 0.3 nM). In a typical
experiment using 1 nM of
I-SCH 23982, total
binding obtained was 11,400 ± 2,300 cpm, while nonspecific
binding was 4,800 ± 1,300 cpm. Aliquots of the soluble extracts
were incubated with aliquots of the different antisera, used at a final
dilution of 1:50 since this concentration was estimated to be optimal
from preliminary titration studies. Control studies were simultaneously
conducted, whereby soluble D-1 receptors from GH
C
cells were incubated at 4 °C for 16 h in the absence of
antisera, but treated with protein A-Sepharose. There was no receptor
binding activity detected in the subsequent protein A-Sepharose pellet
fraction, and all the activity was recovered in the supernatant
fraction (specific binding = 6,000 ± 1, 400 cpm, n = 5), suggesting that these incubation procedures did not
result in receptor inactivation or nonspecific adsorption to the
Sepharose beads.
Figure 4:
Immunoprecipitation of D-1 receptor/G
protein complexes by anti-G antisera. D-1 dopamine receptors were
solubilized with sodium cholate from GH
C
cells,
as described under ``Experimental Procedures.'' Each
antiserum against the
subunits of the various G proteins was
incubated with solubilized preparations and then subjected to
immunoprecipitation with protein A-Sepharose. The binding of
I-SCH 23982 to the washed immunoprecipitate pellet (A), or the supernatant obtained after immunoprecipitation (B) was determined. The numbers on the x axis denote
antisera used: 1, NEI-805; 2, 9072; 3, 3646; 4, 1521; 5, 1518; 6, 0941; 7,
NEI-801. Data represent the mean ± S.D. for the values obtained
from separate experiments.
To determine which
-subunits of G
were associated with D-1 dopamine
receptors, similar studies were conducted with different
G
antisera to coimmunoprecipitate D-1
receptor
G
complexes. Antisera 3646 and 1518
failed to coimmunoprecipitate D-1 dopamine receptors and only 4
± 1 and 8.1 ± 4.2% (n = 3), respectively,
of the D-1 receptor activity was detected in the pellet fraction (Fig. 4A). In addition, there was no appreciable loss of
receptor binding activity in the corresponding supernatant fraction (Fig. 4B), indicating lack of association of D-1 sites
with either G
or G
. Using antiserum
1521, 8.9 ± 3.6% (n = 3) of the D-1 activity was
detected in the pellet fraction (Fig. 4A) and 89.5
± 5.5% (n = 3) of the receptor binding activity
was recovered in the soluble fraction (Fig. 4B). To
eliminate the possibility that the antiserum 1521 was not strongly
immunogenic, in some studies the concentration of the antiserum was
increased to 1:20, while in other studies, a different antiserum,
NEI-801, which recognizes both G
and
G
, was used. Under either of these conditions, only
insignificant amounts of D-1 dopamine receptors were
coimmunoprecipitated in the pellet (2-6%), indicating that the
receptor was not associated with G
.
. When antiserum
9072 was used in these immunoprecipitation studies, 34.4 ± 6.9% (n = 4) of the D-1 receptor binding activity was
detected in the pellet fraction (Fig. 4A). This was also
accompanied by a corresponding decrease of D-1 receptors in the
supernatant fraction, and only 56.3 ± 9.5% (n =
4) of the total receptor activity remained in the supernatant (Fig. 4B). These data indicate that the PTX-sensitive G
protein coupling to D-1 dopamine receptors is G
.
D-1 Dopamine Receptors Do Not Appear to be Coupled to
G
Since there are many contradictory reports in
the literature regarding the effects of D-1 dopamine receptors on PI
metabolism, we also examined D-1 receptor/G couplings
in this study. Western blots studies with antiserum 0941 indicated that
G
was expressed in both GH
C
and SK-N-MC cells (Fig. 5A). When
GH
C
cells were challenged with dopamine (1
µM), D-1 dopamine receptors failed to stimulate PI
hydrolysis, and the amount of IP
detected in the stimulated
cells was identical to the amount of IP
detected in
control, unstimulated cells (Fig. 5B). Raising the
levels of dopamine to 100 µM failed to elicit any IP
production. That these transfected GH
C
cells are able to mediate PI hydrolysis under identical
experimental conditions was confirmed by testing the ability of the
thyrotropin-releasing hormone to stimulate PI hydrolysis, which caused
a 2.3-fold increase in IP
levels (Fig. 5B).
In SK-N-MC cells, D-1 dopamine receptors failed to similarly mediate PI
turnover (not shown). When immunoprecipitation studies were conducted
with antiserum 0941 (Fig. 4A), there was no D-1 dopamine
receptor binding activity detected in the pellet fraction (1.5 ±
1.5%, n = 4) and virtually all the receptor binding
activity (116 ± 11%, n = 4) remained in the
reconstituted supernatant fraction (Fig. 4B).
Figure 5:
Coupling of D-1 receptors to
G. A, Western blots using antiserum 0941 were
conducted as described in the legend to Fig. 3. B,
phosphoinositide turnover studies were conducted in
GH
C
cells. Cells were labeled with
[
H]myoinositol and stimulated with dopamine or
thyrotropin releasing hormone for 10 min at 37 °C, and IP
released was measured, as described under ``Experimental
Procedures.'' Unstimulated values of control cells were 1,421
± 189 cpm, and the data represent the mean ± S.E. from
three to five separate experiments.
-subunits
of both G
and G
. That the D-1 dopamine receptor
is coupled to G
is well known, since a definitive
function of this receptor is the stimulation of adenylyl cyclase
activity (Gingrich and Caron, 1993; O'Dowd, 1993). We confirmed
the ability of D-1 receptors to associate with G
in
the immunoprecipitation studies presented in this report, which also
validates these techniques for analyzing other existing associations
between D-1 sites and
-subunits of different G proteins.
/K
-ATPase activity seen in these
studies required the simultaneous stimulation of both D-1-like and D-2
dopamine sites, and these studies could not distinguish if the observed
PTX sensitivity was in fact due to a G protein which was coupled to
D-2, rather than to D-1-like receptors. Our own studies in cell-free
systems indicated that rat D-1 dopamine receptors were coupled to
PTX-sensitive G proteins (Sidhu et al., 1991).
C
cells with PTX. The absence of similar
PTX-sensitive couplings in SK-N-MC was concomitant with the absence of
expression of two PTX-sensitive G proteins, G
and
G
. Since both these PTX-sensitive G proteins were
expressed in GH
C
cells, we concluded that D-1
receptors in GH
C
cells must be coupling to
these G proteins. However, the failure of antiserum 1521 to
coimmunoprecipitate D-1 dopamine receptors in the pellet fraction,
coupled with detection of 95% of starting binding activity in the
supernatant fraction, eliminated the possibility that D-1 receptors
were associated with G
. Whereas G
and G
were expressed in both GH
C
and SK-N-MC cells, only the former, but not the latter, displayed
PTX-sensitive couplings, eliminating the possibility that D-1 sites
were coupled to either of these G proteins. Further, antisera against
these subunits did not coimmunoprecipitate appreciable levels of D-1
receptors, indicating absence of coupling between D-1 sites and
G
and G
.
. Thus, the absence of
PTX-sensitive D-1 receptor-G protein couplings paralleled the lack of
expression of G
in SK-N-MC. Moreover, the results
obtained using antiserum 9072 were quite dramatic, whereby 34% of
receptor binding activity was recovered in the pellet fraction,
indicating association between G
and D-1 dopamine
receptors. From physiological studies, there is increasing evidence
indicating that D-1-like dopamine receptors may couple to signal
transducing systems which regulate ion channel function, via
G
. Indeed, dopamine depresses and slows a
voltage-dependent Ca
current in embryonic chick
sympathetic neuron, through a PTX-sensitive G protein, which may or may
not be G
(Hille, 1994). It has also been reported that
rat D-1 receptor activation in Xenopus oocytes induced
Ca
mobilization in a cAMP-independent manner (Mahan et al., 1990). Moreover, in the resting state of medium-size
spiny neuron, activation of D-1 dopamine receptors may inactivate a
slow K
current (Kitai and Surmeier, 1993). Thus, it is
plausible that D-1 dopamine receptors modulate ion channel function
through coupling to G
. The suppression of cAMP accumulation
by dopamine upon disruption of functional D-1/G
couplings seen in this study is unlikely to be due to a direct effect
of G
on adenylyl cyclase, but it is possible that
G
modulates cyclase activity by alteration of
[K
]
or
[Ca
]
.
. Since G
is not
subject to ADP-ribosylation by either PTX or CTX (Hepler and Gilman,
1992), the coupling studies using membranes from cell pretreated with
these toxins would not be able to detect D-1
receptor/G
couplings. However, from physiological
studies, the effects of D-1-like dopamine receptors on the PI pathway
may be mediated through G
since G
has
been shown to be the G protein mediating the stimulation of
phospholipase C (Waldo et al., 1991; Hepler and Gilman, 1992;
Rhee and Choi, 1992). That G
is expressed in both
GH
C
and SK-N-MC cells is evident from the
Western blot analyses. However, the absence of D-1 receptor-mediated
stimulation of PI hydrolysis, coupled with the failure of antiserum
0941 to coimmunoprecipitate D-1 dopamine receptors, suggests that these
receptors are not associated with G
. It remains to be
established whether the ability of D-1 sites to activate PI metabolism
occurs via direct coupling to G
in certain cellular
milieu or whether this an indirect effect as a result of activation of
other, as yet unknown, factors. In this regard, functional linkage of
cloned D-1-like receptors to signaling systems other than adenylyl
cyclase activation has never been demonstrated in transfected cells
lines for either rat (Dearry et al., 1990), Xenopus (Sugamori et al., 1994), or human (Kimura et
al., 1995) receptors. Further, there are several reports in the
literature documenting the lack of effect of D-1 stimulation on PI
metabolism (Kelley et al., 1988), in addition to reports which
indicate that D-1 receptors may actually inhibit PI metabolism (Wallace
and Claro, 1990; Rubinstein and Hitzemann, 1990).
-adrenergic receptor is known to couple to all three
-subunits of G
and to G
(Okuma and
Reisine, 1992), while the SSTR2 subtype of somatostatin receptors was
found to couple to G
and G
(Law et al., 1993). In hepatocytes, cloned endothelin B receptors
coupled to the Ca
pump and phospholipase C through
G
and G
, respectively (Jouneaux et al., 1994). In human thyroid membranes, the thyrotropin
receptor was similarly shown to be coupled to G
and
G
(Allgeier et al., 1994). Coupling of human
parathyroid hormone to multiple G proteins, G
and
G
, was dependent on a core region of the receptor
comprising the first, second and third intracellular loops (Schneider et al., 1994). In this regard, D-1-like receptors show
significant sequence divergence in the third intracellular loop, in
addition to the carboxyl-terminal tails (Sugamori et al.,
1994). Thus, other D-1-like receptors, such as D-5 and D
may similarly be able to differentially couple to G proteins in
addition to G
, enabling the activation of multiple and
diverse signaling pathways. Indeed, our recent studies indicate that
D-5 dopamine receptors are not coupled to G
/G
,
but can couple to G
and to a pertussis toxin-insensitive G
protein (Kimura et al., 1995).
, stimulatory
regulator of adenylyl cyclase; G
, inhibitory regulator of
adenylyl cyclase; G
, the pertussis toxin-sensitive G
protein isolated from brain; G
, stimulatory regulator of
phosphoinositide metabolism; G
, the
-subunit of
G
; G
, the
-subunit of G
;
G
, the
-subunit of G
; PTX, pertussis
toxin; CTX, cholera toxin; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; PI,
phosphatidyl inositol; cpm, counts/min.
) and D-5
(D
) denote the human receptors, while D
and
D
represent the rat homologues, respectively. D
is a novel receptor found in X. laevis (Sugamori et al., 1994). The term D-1-like is used as a
catch-all phrase to describe the adenylyl cyclase stimulatory family of
dopamine receptors, irrespective of species origin (D-1/D
,
D-5/D
, and D
).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.