©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Coupling of Human D-1 Dopamine Receptors to Different Guanine Nucleotide Binding Proteins
EVIDENCE THAT D-1 DOPAMINE RECEPTORS CAN COUPLE TO BOTH G AND G(*)

Kazuhiro Kimura , Beatrix H. White , Anita Sidhu (§)

From the (1)Department of Pediatrics, Georgetown University Medical Center, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 GHC 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 GHC 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 GHC 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.


INTRODUCTION

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, 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.()

G proteins are 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.

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, 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.

In the present study, we have explored human D-1 receptor-G protein couplings in membranes from two different cellular systems: rat pituitary GHC 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 GHC 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.


EXPERIMENTAL PROCEDURES

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).

SK-N-MC neuroblastoma cells were grown in 175-cm 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, GHC 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.

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 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 GHC 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).

Western blot analyses was undertaken by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) using 10 µg each of membranes from GHC 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 ReceptorG Protein Complexes

To immunoprecipitate D-1 dopamine receptorG 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 GHC 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.

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.

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 GHC membranes and 0.5 nMI-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.


RESULTS

Coupling of D-1 Receptors to G Proteins in GHC Cells

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. 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. GHC 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), GHC 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 GHC 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 GHC 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.

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 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 Kvalues 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 GHC and SK-N-MC Cells

Since different cells express different G proteins, we examined the expression of PTX-sensitive G proteins in GHC 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 GHC 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, GHC 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 GHC 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), GHC (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 GHC (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, GHC, 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 GHC (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 ReceptorG Protein Complexes

Using the sodium cholate method we established for solubilization of D-1 dopamine receptors (Sidhu, 1990; Sidhu et al., 1994), GHC 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 GHC 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.

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).


Figure 4: Immunoprecipitation of D-1 receptor/G protein complexes by anti-G antisera. D-1 dopamine receptors were solubilized with sodium cholate from GHC 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 receptorG 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.

The D-1 dopamine receptor is 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 GHC and SK-N-MC cells (Fig. 5A). When GHC 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 GHC 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 GHC 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.




DISCUSSION

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 -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.

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/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).

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 GHC 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 GHC cells, we concluded that D-1 receptors in GHC 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 GHC 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.

The most important finding in our studies is the ability of D-1 dopamine receptors to couple to the PTX-sensitive 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].

An unexpected result of our studies is the failure of D-1 dopamine receptors to be associated with G. 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 GHC 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).

There is growing consensus that receptors affect multiple signaling systems by differential coupling to G proteins. For instance, the -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).


FOOTNOTES

*
This work was supported by research grants from the National Institute of Neurological Disorders and Stroke and National Institutes of Health Grants NS-29685 and NS-30912. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Georgetown University Medical Center, Box 17, 3900 Reservoir Rd., NW, Washington D. C. 20007. Tel.: 202-687-0273; Fax: 202-687-0279.

The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; G, 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.

The nomenclature scheme used in this paper is in line with that originally suggested by Gingrich and Caron, 1993: D-1 (or D) 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).

We have confirmed the genetic identity of the receptor expressed in SK-N-MC cells to be the D-1 subtype from reverse transcriptase-polymerase chain reaction and sequencing studies (A. Sidhu, manuscript in preparation).


ACKNOWLEDGEMENTS

We thank Dr. David Manning for his generous gift of the different antisera.


REFERENCES
  1. Allgeier, A., Offermanns, S., Van Sande, J., Spicher, K., Schultz, G., and Dumont, J. E.(1994) J. Biol. Chem.269, 13733-13735 [Abstract/Free Full Text]
  2. Bertorello, A., and Aperia, A.(1988) Acta Physiol. Scand.132, 441-443 [Medline] [Order article via Infotrieve]
  3. Bertorello, A., and Aperia, A.(1989) Am. J. Physiol.256, F9-14
  4. Birnbaumer, L.(1990) FASEB J.4, 3068-3078 [Abstract]
  5. Caulfield, M. P., Jones, S., Vallis, Y., Buckley, N. J., Kim, G., Milligan, G., and Brown, D. A.(1994) J. Physiol.477, 415-422 [Abstract]
  6. Dearry, A., Gingrich, J. A., Falardeau, P., Fremeau, R. T., Bates, M. D., and Caron, M. G.(1990) Nature347, 72-76 [CrossRef][Medline] [Order article via Infotrieve]
  7. Felder, C. C., Jose, P. A., and Axelrod, J.(1989) J. Pharmacol. Expt. Ther.248, 171-175 [Abstract]
  8. Felder, C. C., Campbel, T., Albrecht, F., and Jose, P. A.(1990) Am. J. Physiol.259, F297-F303
  9. Gingrich, J. A., and Caron, M. G.(1993) Annu. Rev. Neurosci.16, 299-321 [CrossRef][Medline] [Order article via Infotrieve]
  10. Hepler, J. R., and Gilman, A. G.(1992) Trends Biochem. Sci.17, 383-387 [CrossRef][Medline] [Order article via Infotrieve]
  11. Hille, B.(1994) Trends Neurosci.17, 531-536 [CrossRef][Medline] [Order article via Infotrieve]
  12. Huff, R. M., Axton, J. M., and Neer, E. J.(1985) J. Biol. Chem.260, 10864-10871 [Abstract/Free Full Text]
  13. Jouneaux, C., Mallat, A., Serradeil-Le Gal, C., Goldsmith, P., Hanoune, J., and Lotersztajn, S.(1994) J. Biol. Chem.269, 1845-1851 [Abstract/Free Full Text]
  14. Kelly, E., Batty, I., and Nahorski, S. R.(1988) J. Neurochem.51, 918-924 [Medline] [Order article via Infotrieve]
  15. Kimura, K., and Sidhu, A.(1994) J. Neurochem.63, 2093-2098 [Medline] [Order article via Infotrieve]
  16. Kimura, K., Sela, S., Bouvier, C., Grandy, D. K., and Sidhu, A. (1995) J. Neurochem.64, 2118-2124 [Medline] [Order article via Infotrieve]
  17. Kitai, S. T., and Surmeier, D. J.(1993) Adv. Neurol.60, 40-52 [Medline] [Order article via Infotrieve]
  18. Latinen, J. T.(1993) J. Neurochem.61, 1461-1469 [Medline] [Order article via Infotrieve]
  19. Law, S. F., Manning, D. R., and Reisine, T.(1991) J. Biol. Chem.266, 17885-17897 [Abstract/Free Full Text]
  20. Law, S. F., Yasuda, K., Bell, G. I., and Reisine, T.(1993) J. Biol. Chem.268, 10721-10727 [Abstract/Free Full Text]
  21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.(1951) J. Biol. Chem.193, 265-275 [Free Full Text]
  22. Lounsbury, K. M., Schlegel, B., Poncz, P., Brass, L. F., and Manning, D. R.(1993) J. Biol. Chem.268, 3494-3498 [Abstract/Free Full Text]
  23. Mahan, L. C., Burch, R. M., Monsma, F. J., and Sibley, D. R.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 2196-2200 [Abstract]
  24. Mattera, R., Graziano, M. P., Yatani, A., Zhou, Z., Graf, R., Codina, J., Birnbaumer, L., Gilman, A. G., and Brown, A. M.(1989) Science243, 804-807 [Medline] [Order article via Infotrieve]
  25. McMillian, M. K., He, X. P., Hong, J. S., and Pennypacker, K. R. (1992) J. Neurochem.58, 1308-1312 [Medline] [Order article via Infotrieve]
  26. Moises, H. C., Rusin, K. I., and Macdonald, R. L.(1994) J. Neurosci.14, 3842-3851 [Abstract]
  27. Munson, P., and Rodbard, D.(1980) Anal. Biochem.107, 220-239 [Medline] [Order article via Infotrieve]
  28. Neer, E. J.(1994) Protein Sci.3, 3-14 [Abstract/Free Full Text]
  29. O'Dowd, B. F.(1993) J. Neurochem.60, 804-816 [Medline] [Order article via Infotrieve]
  30. Okuma, Y., and Reisine, T.(1992) J. Biol. Chem.267, 14826-14831 [Abstract/Free Full Text]
  31. Piomelli, D., Pilon, C., Giros, B., Sokoloff, P., Martes, M-P., and Schwartz, J.-C.(1991) Nature353, 164-167 [CrossRef][Medline] [Order article via Infotrieve]
  32. Rhee, S. G., and Choi, K. D.(1992) J. Biol. Chem.267, 12393-12396 [Free Full Text]
  33. Rubinstein, J. E., and Hitzemann, R. J.(1990) Biochem. Pharmacol.39, 1965-1970 [Medline] [Order article via Infotrieve]
  34. Schneider, H., Feyen, J. H., and Seuwen, K.(1994) FEBS Lett.351, 281-285 [CrossRef][Medline] [Order article via Infotrieve]
  35. Schubert, B., Van Dongen, A. M. J., Kirsch, G. E., and Brown, A. M. (1989) Science245, 516-519 [Medline] [Order article via Infotrieve]
  36. Schultz, G., and Heschler, J.(1993) Arzneim.-Forsch./Drug Res.43, 229-232
  37. Sidhu, A.(1990) J. Biol. Chem.265, 10065-10072 [Abstract/Free Full Text]
  38. Sidhu, A., and Fishman, P.(1990) Biochem. Biophys. Res. Commun.166, 1849-1856
  39. Sidhu, A., and Kimura, K.(1994) J. Neurochem.63, 201-206 [Medline] [Order article via Infotrieve]
  40. Sidhu, A., Sullivan, M., Kohout, T., Balen, P., and Fishman, P. (1991) J. Neurochem.57, 1445-1451 [Medline] [Order article via Infotrieve]
  41. Sidhu, A., Kimura, K., and Vachvanichsanong, P.(1994) Biochemistry33, 11246-11253 [Medline] [Order article via Infotrieve]
  42. Sugamori, K. S., Demchyshyn, L. L., Chung, M., and Niznik, H. B.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 10536-10540 [Abstract/Free Full Text]
  43. Undie, A. S., Weinstaock, J., Sarua, H. M., and Friedman, E.(1994) J. Neurochem.62, 2045-2048 [Medline] [Order article via Infotrieve]
  44. Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J., and Civelli, O.(1990) J. Biol. Chem.265, 10320-10326 [Abstract/Free Full Text]
  45. Waldo, G. L., Boyer, J. L., Morris, A. J., and Harden, T. K.(1992) J. Biol. Chem.267, 25798-25802 [Abstract/Free Full Text]
  46. Wallace, M. A., and Claro, E.(1990) Neurosci. Lett.110, 155-161 [Medline] [Order article via Infotrieve]
  47. Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnbaumer, L.(1988) Nature336, 680-682 [CrossRef][Medline] [Order article via Infotrieve]
  48. Zhou, X.-M., Sidhu, A., and Fishman, P. H.(1991) Mol. Cell. Neurosci.2, 464-472

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.