The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC 50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization.Phosphorylation is an important mechanism involved in the regulation of numerous cellular responses, notably the responsiveness of G protein-coupled receptors (1). This phosphorylation process is believed to be the triggering mechanism that leads to receptor desensitization. The cellular responses elicited upon activation of G protein-coupled receptors are regulated in a dynamic fashion by the action of two classes of serine/threonine kinases. The first class consists of the second messenger-dependent kinases such as protein kinase A and protein kinase C (1). The second class consists of receptorspecific kinases that phosphorylate the agonist-occupied or activated form of the G protein-coupled receptors (1-3). These receptor kinases were originally described for rhodopsin (rhodopsin kinase) and the ␤ 2 -adrenergic receptor (␤-adrenergic receptor kinase) and are referred to as the G protein-coupled receptor kinases or GRKs 1 (1-3). This large family of kinases includes six members (GRK1 to GRK6) whose activities are regulated by phospholipids, posttranslational modifications, or G protein ␤␥ subunits (2-6). The GRKs are widely distributed in brain and periphery, suggesting an important role in the regulation of responsiveness of various G protein-coupled receptors (2, 7). Moreover, Arriza et al. (7) have shown that ␤-adrenergic receptor kinase 1 (GRK2) and ␤-adrenergic receptor kinase 2 (GRK3) are found in presynaptic and postsynaptic localizations in various brain r...

Multiple DI dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the DI dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned DI receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no DI dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called DIB1The actions of dopamine were originally thought to be mediated by an interaction with two distinct receptor subtypes: D1 receptors, which were coupled to the stimulation of adenylyl cyclase, and D2 receptors, which were either linked or not linked to the inhibition of adenylyl cyclase (1). Recently, molecular biological approaches have supported and extended this pharmacological classification. At least three different receptor genes code for dopamine receptor subtypes-namely, D1, D2, and D3 (2-9). These receptors belong to the large family of receptors coupled to guanine nucleotide-binding regulatory protein (G protein) and are believed to contain seven membrane-spanning domains (10).Traditionally, the central actions of dopaminergic compounds have been attributed to their interactions with D2 dopamine receptors (11). However, recent studies have indicated that D1 dopamine receptors have important functions in the central nervous system (12). The distribution of D1 receptors in brain has been studied by using the selective ligand SCH 23390. Although this ligand binds to a single class ofreceptor sites, recent evidence has suggested the existence of multiple D1 receptor subtypes (13). For instance, it has been demonstrated (14) that injection of rat striatal mRNA into Xenopus oocytes directs the expression of a D1 dopamine receptor coupled to activation of phospholipase C and phosphatidylinositol phosphate metabolism. Furthermore, dopamine does not stimulate adenylyl cyclase in the amygdala, a tissue known to contain specific binding sites for the radiolabeled Dl-selective antagonist SCT{ 23390 (13). In the periphery, D1 receptors have been shown to stimulate adenyly...

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