D2 Dopamine Receptors Modulate G -Subunit Coupling of the CB1 Cannabinoid Receptor

CB1 cannabinoid (CB1) and D2 dopamine (D2) receptors are known to couple to the G protein G i/o. It has been reported that concurrent activation of D2 receptors and CB1 receptors, in primary striatal neuronal culture, promotes functional CB1 receptor coupling to G s resulting in elevations in intracellular cyclic AMP levels. We now report that in the absence of D2 receptors, acute activation of CB1 receptors inhibits cyclic AMP accumulation, whereas the presence of D2 receptors promotes CB1-stimulated cAMP accumulation, presumably through G s. This G s subunit switching was not prevented by pertussis toxin treatment and occurred in the presence and absence of D2 receptor activation. Thus, coexpression of the D2 receptor with the CB1 receptor was sufficient to switch the coupling of the CB1 receptors from G i/o to G s. Persistent activation of D2 receptors resulted in heterologous sensitization of adenylate cyclase to subsequent stimulation by forskolin, whereas the persistent activation of CB1 receptors did not. Additional studies in human embryonic kidney cells cotransfected with D2 and CB1 receptors revealed that persistent activation (18 h) of D2 receptors induced a switch of CB1 receptor coupling from G s to G i/o. This D2 receptor-induced effect allowed for CB1 receptor-mediated inhibition of cyclic AMP accumulation. The present studies suggest D2 receptors may have a significant modulatory role in determining the G protein coupling specificity of CB1 receptors. The CB1 cannabinoid (CB1) receptor is expressed primarily in the central nervous system, especially in the basal ganglia and cortex (Herkenham et al., 1990; Matsuda et al., 1993; Tsou et al., 1998), with some expression occurring in peripheral tissues such as the uterus, testes, and ileum (Pacheco et al., 1991; Pertwee et al., 1992; Das et al., 1995). This distribution pattern in the brain suggests that the cannabinoid system and the ascending dopamine pathways may interact with one another. Indeed, functional links between dopaminergic and cannabinoid systems have been reported (Mailleux and Vanderhaeghen, 1993; Glass and Felder, 1997; Giuffrida et al., 1999). Microdialysis experiments have demonstrated that activation of D2 dopamine receptors (D2) promotes elevations in extracellular concentrations of the endocannabinoid anandamide (Giuffrida et al., 1999). Chronic treatment with D2 receptor antagonists causes upregulation of CB1 mRNA in striatum (Giuffrida et al., 1999). When expressed individually, activation of either the D2 or CB1 receptor inhibits cAMP accumulation. Several studies have shown that the CB1 receptor inhibits adenylate cyclase activity through coupling with a pertussis toxin-sensitive G i/o-protein (for review, see Howlett, 1995). The D2 receptor also inhibits adenylate cyclase via pertussis toxin-sensitive G i/o-proteins (Sibley and Monsma, 1992). Our interest in dopamine-cannabinoid interactions stems from reports that concurrent activation of D2 and CB1 receptors alters CB1 receptor coupling to signal transduction mechanisms. Specifically, in primary striatal cultured neurons, D2 receptor activation shifts CB1 receptor coupling from an inhibitory effect on the formation of the signaling molecule cAMP to a stimulatory effect on cAMP formation (Glass and Felder, 1997). Glass and Felder (1997) have suggested this increase in cAMP is brought about by the CB1 receptor switching from G i/o to G s linkage. Additional studies demonstrating the G s linkage of CB1 receptors were carried out in Chinese This work was supported by a National Association for Research in Schizophrenia and Depression Young Investigator Award (to E.L.B) and MH60397 (to V.J.W.). A preliminary report of these findings was made at the 2001 meeting of the Society for Neuroscience and the 2003 meeting of the Federation of American Societies for Experimental Biology. 1 Current address: Department of Pharmaceutical Sciences, Butler University College of Pharmacy and Health Sciences, 4600 Sunset Ave., Indianapolis, IN 46208. E-mail: [email protected] Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.057620. ABBREVIATIONS: CB1, cannabinoid receptor; CHO, Chinese hamster ovary cell line; D2, type 2 dopamine receptor; HEK, human embryonic kidney; CP55,940, ( )-cis-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; SR141716A, N-(piperidin-1-yl)-5-(4chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide. 0022-3565/04/3083-880–886$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 57620/1128129 JPET 308:880–886, 2004 Printed in U.S.A. 880 at A PE T Jornals on A uust 1, 2017 jpet.asjournals.org D ow nladed from hamster ovary (CHO) cells stably expressing only the human CB1 receptors (Glass and Felder, 1997; Bonhaus et al., 1998; Felder et al., 1998). In the CB1 receptor/CHO cells augmentation of forskolin-stimulated cAMP accumulation upon CB1 receptor activation was observed only after pertussis toxin pretreatment. Together, the studies described above indicate that CB1 receptors can couple to multiple G proteins (i.e., G s and G i/o) after acute activation. Acute activation of G s-coupled receptors enhances adenylate cyclase activity, which increases cAMP levels, whereas acute activation of G i/o-coupled receptors decreases adenylate cyclase activity, which results in decreased cAMP levels. However, long-term activation of G i/o-coupled receptors enhances subsequent stimulation of adenylate cyclase, a pharmacological phenomena known as heterologous sensitization (for review, see Watts, 2002). Although the exact mechanisms are not yet fully elucidated, persistent activation of a G i/o-coupled receptor induces heterologous sensitization via a pertussis toxin-sensitive G protein (Watts, 2002). Chronic activation of G i/o-coupled receptors such as D2 and CB1 receptors has been reported to potentiate adenylate cyclase responsiveness upon subsequent drug-stimulated cAMP accumulation (Watts and Neve, 1996; Rhee et al., 2000). Because cultured cells have a unique composition of G proteins and adenylate cyclase isoforms, our initial experiments have focused on asking the question how generalized is the D2 receptor effect on CB1 receptor coupling? Do the D2 receptors promote the CB1 receptors to switch from G i/o to G s in transfected systems? Moreover, does the influence of the D2 receptor also affect the ability of the CB1 receptor to sensitize adenylate cyclase and vice versa? What are the effects of persistent activation of the D2 and CB1 receptor on adenylate cyclase? To address these questions, we investigated D2 and CB1 receptor signaling in HEK-293 cells. Our studies showed that the D2 receptor dramatically influences CB1 receptor coupling to G subunits. We determined that coexpression of the two receptors induces the CB1 receptor to switch to G s coupling, although activation of the D2 receptor is not necessary. Furthermore, overexpression of G i1 or persistent activation of the D2 receptor seemed to facilitate the reestablishment of G i/o coupling for the CB1 receptor. We asked questions seeking to further refine the relationship between D2 and CB1 receptor signaling. In the present study, we provide data that demonstrate the D2 receptor’s ability to regulate the G protein coupling specificity of CB1 receptors. Materials and Methods Materials. [H]Cyclic AMP (32 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Forskolin, pertussis toxin, and ( )-quinpirole were purchased from Sigma/RBI (Natick, MA). CB1 receptor cDNA was a gift from Dr. Tom Bonner (National Institute of Mental Health, Bethesda, MD). The G protein subunit cDNAs were purchased from the Guthrie Research Institute (Sayre, PA). CP55,940 was a generous gift of the Research Triangle Institute (Research Triangle Park, NC). All other drugs and chemicals were of the highest grade possible and were purchased from standard com-