The D 1 dopamine receptor (D 1 R) has been proposed to form a hetero-oligomer with the D 2 dopamine receptor (D 2 R), which in turn results in a complex that couples to phospholipase Cmediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose-response curves assaying intracellular calcium mobilization in cells heterologously expressing the D 1 and D 2 subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D 1 -D 2 heteromer-selective ligand, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol (SKF83959), found no stimulation of calcium release, but it did find a broad range of cross-reactivity with other G protein-coupled receptors. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Overexpression of G qa with the D 1 and D 2 dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G qa with only the D 1 R. Inactivation of G i or G s with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D 1 R and D 2 R cotransfected cells. Moreover, sequestration of G bg subunits through overexpression of G protein receptor kinase 2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggest that the mechanism of D 1 R/D 2 R-mediated calcium signaling involves more than receptor-mediated G q protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition, SKF83959 may not exhibit selective activation of D 1 -D 2 heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D 1 receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the ␣ isoform of GRK4; the ␤, ␥, and ␦ isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4␣ is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D 1 receptor by GRK4␣ results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number-all of which are abolished in a D 1 receptor mutant containing T428V and S431A. The increase in internalized D 1 receptors induced by GRK4␣ phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D 1 receptor by GRK4␣ does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4␣. These observations shift the attenuation of D 1 receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization. Dopamine signaling in mammals is mediated by five G protein-coupled receptor (GPCR) proteins divided into two groups based upon sequence homology, G protein coupling, signaling pathways, pharmacological profiles, and desensitization kinetics (Sibley and Monsma, 1992;Missale et al., 1998 Upon agonist activation, GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response under continued agonist stimulation (Ferguson et al., 1996;Gainetdinov et al., 2004). Desensitization involves phosphorylation of the receptor by GRKs and/or second messenger-activated kinases (cAMP-dependent protein kinase or protein kinase C). Homologous desensitization of GPCRs involves only activated receptors and is primarily mediated by GRKs. GRKs are serine/threonine-directed protein kinases composed of seven isoforms divided into three families (Penela et al., 2003). GRK1 and GRK7 compose the rhodopsin kinase/visual family, are expressed exclusively in retina, and participate in desensitization of opsins in rods and cones (Somers and Klein, 1984;Hisatomi et al., 1998;Weiss et al., 1998). GRK2 (␤ARK1) and GRK3 (␤ARK2) were originally identified as regulating the ␤-adrenergic receptor and com- ABBREVIATIONS: GPCR, G protein-coupled receptor; ␤ARK, ␤-adrenergic ...