Sites in the third intracellular loop of the α(2A)-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism

To investigate the mechanisms of agonist-promoted desensitization of the α₂-adrenergic receptor (α₂AR), the human α(2A)AR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type α(2A)AR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by ~78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with ³²P(i), exposed to agonist, and α(2A)AR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the α(2A)AR in vivo. The β-adrenergic receptor kinase (βARK) is known to phosphorylate purified α(2A)AR in vitro. We found that heparin, a βARK inhibitor, ablated short term agonist-induced desensitization of α(2A)AR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of βARK, we constructed a mutated α(2A)AR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated α(2A)AR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in α(2A)AR desensitization. After 24 h of agonist exposure, an even more profound desensitization of α(2A)AR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular G(i), which was not dependent on receptor third loop phosphorylation sites.