The D1 dopamine receptor is constitutively phosphorylated by G protein-coupled receptor kinase 4

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 alpha isoform of GRK4; the beta, gamma, and delta isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4 alpha 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 alpha 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 alpha 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 alpha 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 alpha. 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.