Four consecutive serines in the third intracellular loop are the sites for β-adrenergic receptor kinase-mediated phosphorylation and desensitization of the α2A-adrenergic receptor

Abstract

During short term agonist exposure, the α2A-adrenergic receptor (α2AAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the β-adrenergic receptor kinase (βARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to βARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for βARK phosphorylation are not known. To delineate such a motif in the human α2AAR (α2C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 ± 7% and 57 ± 8% of the phosphorylation found for wild type α2C10. Additional substitution mutants (SSSA and SAAA) underwent 77 ± 1% and 27 ± 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type α2C10 by ∼25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonist-promoted desensitization. Thus, βARK-mediated phosphorylation of α2C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonist-promoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for βARK-mediated phosphorylation of α2C10, suggesting that these receptors may also be substrates for βARK.