The role of phosphorylation/dephosphorylation in agonist-induced desensitization of D-1 dopamine receptor function: Evidence for a novel pathway for receptor dephosphorylation

Exposure of D-1 dopamine receptors to agonists results in rapid desensitiza tion of the receptor-stimulated accumulation of cAMP. It is believed that a gonist-induced phosphorylation of the receptor plays a critical role in the processes that underlie this phenomenon. To investigate the role of agonis t-induced receptor phosphorylation, a FLAG epitope was added to the amino t erminus of the rat D-1 dopamine receptor and this construct was stably expr essed in C6 glioma cells. It was found that the D-1 receptor was stoichiome trically 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 D-1-selective antagonists but was unaffected by inhibitors of either pr otein kinase A or protein kinase C. The incorporation of phosphate into the receptor was rapid but transient, despite the continued presence of dopami ne. A comparison of the rates of receptor phosphorylation (t(1/2), < 1 min) and dopamine-induced desensitization (t(1/2) <similar to>7 min) revealed t hat receptor phosphorylation was not the rate limiting step for receptor de sensitization. Upon removal of dopamine, the receptor was rapidly dephospho rylated (t(1/2) similar to 10 min) and this was not blocked by agents (i.e. , concanavalin A or hypertonic sucrose) that inhibit D-1 receptor internali zation. Using specific inhibitors, the phosphatase involved in D-1 receptor dephosphorylation was shown not to correlate with the recently identified "G protein-coupled receptor phosphatase" (Proc Natl Acad Sci USA 92:8343-83 47, 1995). These results suggest that the phosphorylated D-1 receptor is pr ocessed through a novel recovery pathway and that internalization is not re quired for receptor dephosphorylation.