Abstract

Exposure of D₁ dopamine receptors to agonists results in rapid desensitization of the receptor-stimulated accumulation of cAMP. It is believed that agonist-induced phosphorylation of the receptor plays a critical role in the processes that underlie this phenomenon. To investigate the role of agonist-induced receptor phosphorylation, a FLAG epitope was added to the amino terminus of the rat D₁dopamine receptor and this construct was stably expressed in C6 glioma cells. It was found that the D₁ receptor was stoichiometrically phosphorylated under basal conditions and that its phosphorylation state was increased by 2- to 3-fold upon exposure of the cells to dopamine for 10 min. The dopamine-induced receptor phosphorylation could be blocked by D₁-selective antagonists but was unaffected by inhibitors of either protein kinase A or protein kinase C. The incorporation of phosphate into the receptor was rapid but transient, despite the continued presence of dopamine. A comparison of the rates of receptor phosphorylation (t_1/2 < 1 min) and dopamine-induced desensitization (t_1/2 ∼7 min) revealed that receptor phosphorylation was not the rate limiting step for receptor desensitization. Upon removal of dopamine, the receptor was rapidly dephosphorylated (t_1/2 ∼10 min) and this was not blocked by agents (i.e., concanavalin A or hypertonic sucrose) that inhibit D₁receptor internalization. Using specific inhibitors, the phosphatase involved in D₁ receptor dephosphorylation was shown not to correlate with the recently identified “G protein-coupled receptor phosphatase” (Proc Natl Acad Sci USA 92:8343–8347, 1995). These results suggest that the phosphorylated D₁ receptor is processed through a novel recovery pathway and that internalization is not required for receptor dephosphorylation.