Adenylyl Cyclase Interaction with the D2 Dopamine Receptor Family; Differential Coupling to Gi, Gz, and Gs

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1 Cellular and Molecular Neurobiology, Vol. 19, No. 5, 1999 Adenylyl Cyclase Interaction with the D2 Dopamine Receptor Family; Differential Coupling to Gi, Gz, and Gs Joseph Obadiah, 1 Tomer Avidor-Reiss, 2 C. Simone Fishburn, 1,3 Shari Carmon, 1 Michael Bayewitch, 2 Zvi Vogel, 2 Sara Fuchs, 1,4 and Berta Levavi-Sivan 1 Recived September 8, 1998; accepted October 29, 1998 SUMMARY 1. The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D 2S,D 2L,D 3S,D 3L, and D 4 ) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity. 2. COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the subunit of Gz. PTX treatment was employed to inactivate endogenous i, and coupling to Gi and Gz was estimated by measuring the inhibition of camp accumulation induced by quinpirole, in forskolin-stimulated cells. 3. D 2S or D 2L receptors can couple to the same extent to Gi and to Gz. The D 4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of camp accumulation. The D 3S and D 3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of camp accumulation, respectively. 4. We have demonstrated for the first time that the two D 3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX. 5. Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function. KEY WORDS: GTP-binding protein Gz; GTP-binding protein Gs; GTP-binding protein Gi;D2(D 2,D 3,D 4 ) dopamine receptor; adenylyl cyclase. INTRODUCTION Dopamine is a major neurotransmitter in the CNS that is involved in the control of locomotor activity, emotion, affect, and neuroendocrine secretion. On the basis of their structure, pharmacology, and signaling properties, the dopamine receptors are divided into two subgroups: D1- and D2-like receptors (Vallar and Meldolesi, 1 Department of Immunology. 2 Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel. 3 Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California To whom correspondence should be addressed. Fax: lifuchs@wiccmail; weizmann.ac.il /99/ $16.00/ Plenum Publishing Corporation

2 654 Obadiah et al. 1989). The D2 dopamine receptor family represents the primary target of drugs employed in the treatment of number of neurological disorders such as Parkinson s disease and schizophrenia (Seeman et al., 1993). In addition, dopaminergic systems of the D2 receptors have recently been implicated in experimental cocaine selfadministration as well as in alcoholism (Caine and Koob, 1993; Sander et al., 1995). The actions of dopamine are mediated by a family of receptors that are part of the large superfamily of GTP-binding (G) protein-coupled receptors. A multiplicity of dopaminergic receptors have been revealed by cloning studies. Each receptor arises from a different gene but still adheres to the original D1/D2 classification. The D2 family consists, of the D 2,D 3, and D 4 subtypes, and the D 2 and D 3 receptor subtypes have, furthermore, been found to be alternatively spliced in the coding region, yielding functional short and long receptor isoforms [D 2S,D 2L,D 3S,D 3L (Monsma et al., 1989; Fishburn et al., 1993)]. The D 2,D 3, and D 4 receptors share a typical structure of G protein-coupled receptors that couple to Gi and mediate the inhibition of adenylyl cyclase (AC). Those receptors have a large third cytoplasmic loop and a short carboxy-terminal tail, ending at the putatively palmitoylated cysteine residue (reviewed by Sibley et al., 1993). Based on their structures, the D 3 and D 4 receptors were thought to inhibit AC activity as the D 2 receptors do. Studies of the functional properties of these novel receptors required their expression in transfected cell lines. The D 4 was found to inhibit AC activity to a smaller extent than the D 2 (Chio et al., 1994a), while the D 3 receptor was found, in some cases, not to inhibit AC (Sokoloff et al., 1990; Mackenzie et al., 1994) or to inhibit it to a small extent (Chio et al., 1994a; Potenza et al., 1994; Cox et al., 1995). These results raised the possibility that the transfected cells employed may not express the same cellular components (e.g., G-protein subunits) as the native brain tissue (Kenakin, 1996). The complexity of G protein-mediated signal transduction has increased with the isolation of a large number of signaling molecules exhibiting similar functions (e.g., i, o, and z). Although the mechanism by which z inhibits camp accumulation is unknown, comparison of -chain amino acid sequences indicates that z resembles the i subfamily (which includes also o and t) more closely than it resembles other chains such as q and s (Simon et al., 1991). This resemblance probably indicates a close evolutionary relationship between z and i proteins, which may account for the overlap in their signaling function. Because z lacks the cysteine residue that is ADP-ribosylated by PTX in i and o, ligands for receptors that activate z should inhibit camp accumulation in a PTX-insensitive fashion. An increasing number of Gi-coupled receptors have been shown to share the ability to interact with the PTXinsensitive G protein, Gz. Thus far, this group includes the 2 -adrenergic, dopamine D 2, adenosine A1, N-formylmethionyl-leucyl-phenylalanine peptide, C5a complement, melatonin, and the three types of opioid receptors (Wong et al., 1992; Tsu et al., 1995a c; Chan et al., 1995; Lai et al., 1995; Yung et al., 1995). In the present study we report that the D 2,D 3 and D 4 dopamine receptors can couple to Gz, varying in the extent of their coupling. In addition, we demonstrate that the D 3 receptor can also interact with the stimulatory G protein Gs.

3 Differential Coupling of D2 Dopamine Receptors to Gi, Gz, and Gs 655 MATERIALS AND METHODS Materials The plasmid (pmv-7) encoding rat Gz was a gift from Prof. H. Bourne (San Francisco, CA). The cdna clones encoding murine D 2S,D 2L,D 3S,D 3L, and D 4, respectively (Fishburn et al., 1993, 1995a,b), were subcloned into the eukaryotic expression vector pcdnai/neo (Stratagene, La Jolla, CA) or pcdna-ampi, for stable or transient transfections, respectively. The expression constructs contained identical sizes of coding and noncoding sequences for the various cdnas, except for the alternatively spliced part in the putative third cytoplasmic loop [87 or 63 base pairs (bp) for D 2 or D 3, respectively]. [ 3 H]Adenine was from DuPont New England Nuclear (Boston, MA); [ 3 H]7- OH-DPAT, and [ 125 I]sulpride were from Amersham (Arlington Heights, IL). Quinpirole was obtained from RBI (Natick, MA); forskolin, 1-methyl-3-isobutylxantine (IBMX), pertussis toxin (PTX), and cholera toxin (CTX) were from Sigma (St. Louis, MO); and RO was from Calbiochem (La Jolla, CA). Culture media and serum were from Life Technologies, Inc. (Grand Island, NY). Methods Transient Cell Transfection. Twenty-four hours before transfection, COS-7 cells were trypsinized and split into five 10-cm plates in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. The cells were kept in a humidified atmosphere consisting of 5% CO 2 at 37 C. The cells were transfected, using the DEAE dextran chloroquine method (Keown et al., 1990), with 2 g/plate of either one of the murine dopamine receptors cdnas and 3 g/plate of Gz cdna or pxmd1-gal (for mock DNA transfection). Twenty-four hours later, the cells were trypsinized and recultured in 24-well plates, in the absence or presence of PTX (130 ng/ml) or CTX (20 g/ml), and after an additional 24hr the cells were assayed for AC activity as described below. Transfection efficiencies were normally in the range of 40 80%, as determined by staining for -galactosidase ( -Gal) activity (Lim and Chae, 1989). The specificity of the inhibitory response was shown by the failure of cells transfected with -Gal or vector alone to inhibit forskolin-stimulated camp accumulation. The expression of the dopamine receptors was confirmed by metabolic labeling with 35 S-methionine followed by immunoprecipitation with specific antibodies as described (David and Fuchs, 1991). Receptor expression was also monitored by saturation binding as described previously (Fishburn et al., 1993; Avidor-Reiss et al., 1995), with [ 125 I]sulpride as a ligand and butaclamol, 7-OH- DPAT, or clozapine as the displacement drug for D 2,D 3,orD 4, respectively, and it was always in the range of 1 4 pmol/mg protein. CHO Cell Transfection and Culture. Stably transfected CHO cells were prepared as described previously (Fishburn et al., 1993, 1995b). Briefly, CHO cells were transfected with 20 g ofd 2L,D 3L,orD 3S receptor DNA, in pcdnai/neu, using electroporation (250 V, 500 mf). Stable transfectants were selected in the

4 656 Obadiah et al. presence of 500 g/ml G418 (Life Technologies, Inc.) and were subcloned by limiting dilution. Positive receptor-expressing cell lines were identified by Northern blot analysis and by binding experiments, using [ 125 I]sulpride for the D 2L and [ 3 H] 7-OH-DPAT for the D 3S and D 3L receptors. Comparative analyses of D 2L,D 3S, and D 3L receptors were performed on cell lines expressing similar levels of receptor (approximately 400 fmol/mg protein). Cells were cultured in Ham s F-12 medium containing the same concentrations of antibiotics as described above. AC Activity. The assay was performed in triplicate as previously described (Avidor-Reiss et al., 1995). Briefly, cells cultured in 24-well plates were incubated for 2 hr with 0.25 ml/well of fresh DMEM containing 5 Ci/ml [ 3 H]adenine. The medium was replaced with 0.5 ml/well of DMEM containing 20 mm Hepes (ph 7.4), 0.1 mg/ml bovine serum albumin, and the phosphodiesterase inhibitors IBMX (0.5 mm) and RO (0.5 mm). AC activity was stimulated in the presence of forskolin (20 M) in the absence or presence of quinpirole for 20 min at 37 C. The medium was then removed, and the reaction was terminated by the addition of perchloric acid containing 0.1 mm unlabeled camp, followed by neutralization with KOH. The amount of [ 3 H]cAMP was determined by two-step column separation procedure according to Salomon (1991). Data Analysis. camp levels in the presence of forskolin and quinpirole are expressed as a percentage of the camp accumulation or inhibition observed following stimulation with 20 M forskolin alone. Results of individual experiments were analyzed by one-way ANOVA, followed by Fisher PLSD test. Significance was imparted at the P 0.05 level. Data are presented as the mean SE. RESULTS The ability of the D 2L dopamine receptor to inhibit camp accumulation was tested in COS-7 cells transiently transfected with the murine D 2L receptor. Forskolin stimulation elevated intracellular camp levels by nine fold above the basal level (Fig. 1). In the presence of the dopamine agonist quinpirole, the forskolin-stimulated camp accumulation was reduced by approximately 50%, an inhibition that was antagonized by the dopamine antagonist spiperone. When the cells were preincubated with PTX, to inactivate the endogenous Gi, quinpirole did not inhibit the forskolin-stimulated camp accumulation (Fig. 1). We then investigated the ability of D 2L and D 2S receptors to couple to Gz, which is also a member of the Gi family. Gz lacks the cysteine residue that is ADPribosylated by PTX in i and o (Matsuoka et al., 1988; Casey et al., 1990), thus activation of z should inhibit camp accumulation in a PTX-insensitive manner. In COS-7 cells cotransfected with z and the D 2L receptor, quinpirole-mediated inhibition of forskolin-stimulated camp accumulation became insensitive to PTX. However, in the absence of Gz, PTX inactivated the endogenous Gi and totally abolished the inhibitory response to quinpirole (Fig. 2). The same maximal inhibition (of around 40%) was observed in the presence or absence of Gz. Interestingly, this level of inhibition was similar to the previously reported inhibition of hcg-stimulated camp accumulation in HEK-293 cells cotransfected with the rat D 2L receptor,

5 Differential Coupling of D2 Dopamine Receptors to Gi, Gz, and Gs 657 Fig. 1. Quinpirole-mediated inhibition of camp accumulation in the presence or absence of PTX. COS-7 cells were transfected with D 2L in the presence or absence of pertussis toxin (PTX; 130 ng/ml, 24hr), as described under Materials and Methods. The camp accumulation in the presence of forskolin (FSK; 20 M) or FSK and quinpirole (Quin; 100 M), in the presence or absence of spiperone (Spip; 10 M), is expressed. Data represent the mean SE of at least three experiments performed in triplicate. Fig. 2. Coupling of D 2 and D 4 dopamine receptors to Gz. COS- 7 cells were contransfected with D 2S,D 2L,orD 4 receptors as indicated, with or without z, as described under Materials and Methods. The camp accumulation in the presence of 20 M forskolin was normalized to 100% and the quinpirole-mediated inhibition was calculated. Data represent the mean SE of at least three experiments performed in triplicate.

6 658 Obadiah et al. the LH receptor, and z (Wong et al., 1992). In COS-7 cells contransfected with z and the D 2S receptor, quinpirole-mediated and forskolin-inhibited camp accumulation also became insensitive to PTX, although the maximal levels of inhibition of AC accumulation were lower (20%). Thus, D 2L and D 2S dopamine receptors can couple to both Gi and Gz. A PTX-sensitive inhibition of quinpirole-mediated forskolin-stimulated camp accumulation was also observed in cells transfected with the D 4 dopamine receptor (Fig. 2). However, cotransfection with D 4 and Gz led to a strong inhibition, about twice that observed in the absence of Gz, of the forskolin-stimulated camp accumulation. This inhibition was obtained in both the absence and the presence of PTX treatment ( and %, respectively), thus demonstrating a preferential efficiency of coupling between D 4 and z compared with that of D 2 receptors. Forskolin-stimulated accumulation of camp in COS-7 cells transfected with D 2L or D 4 in the presence or absence of z was dose dependent with respect to the Fig. 3. The effect of Gz on the dose response for quinpirole on camp accumulation in COS- 7 cells expressing D 2L or D 4 receptors. Cells were transfected with D 2L or D 4, with ( ) or without ( ) z, as described under Materials and Methods. The camp accumulation was calculated as described in the legend to Fig. 1. Data represent the mean SE of at least three experiments performed in triplicate.

7 Differential Coupling of D2 Dopamine Receptors to Gi, Gz, and Gs 659 dopamine agonist quipirole. Cotransfection with the z subunit mediated a shift in camp accumulation in D 4 -contransfected cells but not in D 2L -contransfected cells (Fig. 3). This supports the notion that D 2L couples equally to Gz and Gi and that D 4 shows a preferential coupling to Gz. Cotransfection of COS-7 cells with D 3S or D 3L and z led to a PTX-resistant inhibition of quinpirole-mediated forskolin-stimulated camp accumulation ( and %, respectively; Fig. 4), demonstrating the ability of both D 3 isoforms to couple to z. In COS-7 cells transfected with D 3L or D 3S alone, quinpirole resulted in only a marginal inhibition of forskolin-stimulated camp accumulation. Moreover, the addition of PTX in the absence of Gz led to an increase in camp accumulation for both of the D 3 isoforms ( % for D 3L and % for D 3S ). This result points to a coupling between the D 3 isoforms and the stimulatory G protein (Gs) endogenously present in COS-7 cells. In order to investigate further the coupling of the D 3 isoforms to G s and G i, we used CHO cells stably transfected with either D 3S (CHO-D 3S ), D 3L (CHO-D 3L ), or D 2L (CHO- D 2L ; as a control). These cells were preincubated with either CTX or PTX, to ablate selectively receptor Gs or receptor Gi coupling, respectively. By ADP-ribosylating target G-protein subunits, CTX constitutively activates s, while PTX inactivates i/ o. Pretreatment with CTX is known to activate the G protein Gs, causing a maximal elevation of camp levels and uncoupling the receptors from Gs. CTX incubation was performed at a concentration of 20 g/ml, which was shown to yield maximal ADP-ribosylation of G s in CHO cells (Eason et al., 1992). We found that PTX pretreatment yielded a stimulation of camp accumulation at all doses of quinpirole, in both CHO-D 3L and CHO-D 3S cells but not in CHO-D 2L (Fig. 5). On the other hand, CTX caused a dose-dependent inhibition of camp accumulation due to the abolishment of the coupling of D 3L (and to a lesser extent of D 3S )togs Fig. 4. Quinpirole-mediated effect on camp accumulation in COS-7 cells expressing the D 3S or D 3L dopamine receptors. COS- 7 cells were cotransfected with D 3S or D 3L with or without z as described under Materials and Methods. The camp accumulation was calculated as described in the legend to Fig. 2. Data represent the mean SE of at least three experiments performed in triplicate.

8 660 Obadiah et al. Fig. 5. The effect of PTX and CTX on camp accumulation in CHO cells stably transfected with D 3L, D 3S,orD 2L. CHO cells were transfected as described under Materials and Methods. Cells were either untreated ( ) or treated with pertussis toxin (130 ng/ ml; ) or with cholera toxin (20 g/ml; ) for 24hr before AC assay. Data represent the mean SEm of at least three experiments performed in triplicate. (Fig. 5). However, no such inhibition was observed with the CHO-D 2L in the presence of CTX. These data reveal that in CHO cells, both isoforms of the D 3 dopamine receptors couple to both Gi and Gs and that the observed agonist-mediated modulation of AC in the untreated D 3 transfected cells is a combination of coupling of two or more G proteins, which could have opposing effects. DISCUSSION Gz is a member of the family of inhibitory guanine nucleotide binding regulatory proteins. It does not serve as a substrate for ADP-ribosylation by PTX and is thus classified as a PTX-insensitive G protein. It has been shown to interact and trigger effector pathways in response to hormone receptors that routinely interact with PTX-sensitive Gi proteins (Wong et al., 1992; Casey et al., 1990). Expressing dopamine receptor subtypes ectopically in a cell line creates a good model for studying the interactions of these receptors with G proteins and their effectors naturally expressed in that cell line. In the present study we showed that

9 Differential Coupling of D2 Dopamine Receptors to Gi, Gz, and Gs 661 COS-7 cells cotransfected with D2 dopamine receptors and z and stimulated with forskolin serve as an appropriate tool to test the ability of Gz to couple to the different dopamine receptor subtypes. It is well documented that the D 2 dopamine receptor interacts with Gi (reviewed by Vallar and Meldolesi, 1989). Recent studies also demonstrated the interaction of D 2L receptor with Gz (Wong et al., 1992). We demonstrated that both D 2L and D 2S, expressed in COS-7 cells, couple to Gi, as well as to the PTX-insensitive Gz. The interaction between these isoforms of D 2 and the two types of G proteins was approximately equivalent. In previous studies on D 4 receptor-mediated signaling, inhibition of forskolin-stimulated camp accumulation was demonstrated (Chio et al., 1994a). A stably transfected CHO cell line expressing D 4 showed more than 50% inhibition of the forskolin-stimulated camp accumulation. This inhibition was abolished following PTX pretreatment, indicating that D 4 couples to Gi (Chio et al., 1994a). We have also observed an interaction between D 4 and Gi in COS-7 cells; moreover, we have demonstrated that the D 4 dopamine receptor interacts strongly and preferably with Gz. COS-7 cells transfected with either of the two D 3 isoforms failed to demonstrate appreciable inhibition of camp accumulation (Fig. 5). However, quinpirole-mediated inhibition of forskolin-stimulated camp accumulation in cells cotransfected with Gz and either one of the D 3 isoforms yielded an inhibition (32% for the D 3L and 15% for the D 3S ), demonstrating a greater degree of coupling to Gz than to endogenous Gi. Observations on the coupling to the D 3 receptor in expression systems to specific signal transduction cascades have varied. In some systems, a G shift in D 3 binding was observed but alterations in second messengers such as camp, phosphoinositides, and arachidonic acid were not detected (MacKenzie et al., 1994); other groups observed a variety of D 3 -initiated signaling events including inhibition of AC, mitogenesis, increased extracellular acidification, and alteration in Ca 2 concentration; all of these signaling events were blocked by PTX, suggesting coupling to Gi (Chio et al., 1994b; Potenza et al., 1994; Cox et al., 1995). It is interesting to note that C6 glioma cells, from which Gz has been cloned (Matsuoka et al., 1988), transiently transfected with D 3, yielded inhibition of AC activity (by more than 50%) in cells treated with dopamine (Cox et al., 1995). This can be explained not only by coupling to Gi, as the authors have implicated, but also by functional coupling of the D 3 receptors to the endogenous Gz present in those cells. We have demonstrated that quinpirole yielded a rise in forskolin-stimulated camp accumulation in D 3 -transfected and PTX-pretreated COS-7 or CHO cells. On the other hand, in the presence of CTX, used to ablate selectively receptor-gs coupling, there was an inhibition of camp accumulation. This result suggests that the D 3 receptor isoforms can couple with Gs proteins. It has been reported that an elevation of intracellular camp levels in CHO cells inhibits mitogenesis (LeCam et al., 1980). In addition, D 3 -mediated mitogenesis was abolished in PTX-treated cells, a fact that can be explained by an elevation of intracellular camp levels by D 3 through activation of Gs (Chio et al., 1994b). It has been reported recently that other receptors known to be coupled to Gi or Gq may also couple to Gs. CHO cells expressing 2 -adrenergic receptors were shown to mediate both the inhibition and the activation of AC (Eason et al., 1992). Similar patterns of G protein coupling

10 662 Obadiah et al. have been observed for the m 2 -muscarinic receptor (Buford et al., 1995). Here we show for the first time that such a coupling to Gs also occurs for the D 3 isoforms. It is still not known whether the coupling between D 2,D 3,orD 4 and Gz demonstrated here in vitro also takes place in vivo. Interestingly, there are several reports which indicate that Gz is present in neuronal regions where D2 receptors are found. High levels of the D 2 subtype are found in the striatum and sabstantia nigra, as well as in the pituitary. The latter indicates that this subtype is involved in the dopaminergic control of prolactin secretion (Mansour et al., 1990). The D 3 receptor is expressed predominantly in limbic areas, including the nucleus accumbens and hypothalamus and in dopaminergic neurons of the substantia nigra (Sokoloff et al., 1990). The D 4 receptor has been shown to be localized to the cortex, the hippocampus, and the substantia nigra as well as to other structures (Mauger et al., 1998). Moreover both D 2 and D 4 dopamine receptors were found in the retina (Dearry et al., 1991; Yamaguchi et al., 1997). Most of these neural regions, such as the pituitary lactotrophs, retina, and regions in and around the limbic areas including the hippocampus, striatum, substanial nigra, and hypothalamus, have been linked by various techniques to the expression of Gz (Fields et al., 1997). In addition, a GTPase-activating protein specific for G z was shown to be present in the cerebral cortex and also in the retina (Wang et al., 1997). The coupling between the D2 dopamine receptors and the G protein Gz, along with their colocalization to certain limbic regions, may suggest a functional connection. Despite the fact that the physiological significance of results obtained with transfected cell lines is often difficult to interpret due to the overexpression of transfected proteins, the demonstration of the differential coupling of the D2 dopamine receptors to different G proteins may add another aspect to the diversity of their function. Moreover, the robust second-messenger coupling of the various D2 dopamine receptor subtypes in a heterologous system with defined components should facilitate the development and characterization of new receptor ligands. ACKNOWLEDGMENTS This work was supported by grants from the Irwin Green Research Fund in Neurosciences, the United States Israel Binational Science Foundation (B.S.F.), and the Leo and Julia Forchheimer Center for Molecular Genetics at The Weizmann Institute of Science. REFERENCES Avidor-Reiss, T., Bayewitch, M., Levy, R., Matus-Leibovitch, N., Nevo, I., and Vogel, Z. (1995). Adenylyl cyclase supersensitization in -opioid receptor-transfected Chinese Hamster ovary cells following chronic opioid treatment. J. Biol. Chem. 270: Burford, N. T., Tobin, A. B., and Nahorski, S. R. (1995). Differential coupling of m1, m2 and m3 muscarinic receptor subtypes to inositol 1,4,5-trisphosphate and adenosine 3,5 -cyclic monophosphate accumulation in Chinese Hamster ovary cells. J. Pharmacol. Exp. Ther. 274: Caine, S. B., and Koob, G. F. (1993). Effects of mesolimbic dopamine depletion on responding maintained by cocaine and food. Science 260:

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