CHARACTERIZATION OF THE PHOSPHORYLATION SITES INVOLVED IN G-PROTEIN-COUPLED RECEPTOR-KINASE-MEDIATED AND PROTEIN-KINASE-C-MEDIATED DESENSITIZATION OF THE ALPHA(1B)-ADRENERGIC RECEPTOR

Catecholamines as well as phorbol esters can induce the phosphorylatio n and desensitization of the alpha(1B)-adrenergic receptor (alpha(1B)A R). In this study, phosphoamino acid analysis of the phosphorylated al pha(1B)AR revealed that both epinephrine-and phorbol ester-induced pho sphorylation predominantly occurs at serine residues of the receptor, The findings obtained with receptor mutants in which portions of the C -tail were truncated or deleted indicated that a region of 21 amino ac ids (393-413) of the carboxyl terminus including seven serines contain s the main phosphorylation sites involved in agonist-as well as phorbo l ester-induced phosphorylation and desensitization of the alpha(1B)AR . To identify the serines invoved in agonist-versus phorbol ester-depe ndent regulation of the receptor, two different strategies were adopte d, the seven serines were either substituted with alanine or reintrodu ced into a mutant lacking all of them, Our findings indicate that Ser( 394) and Ser(400) were phosphorylated following phorbol ester-induced activation of protein kinase C, whereas Ser(404), Ser(408), and Ser(41 0) were phosphorylated upon stimulation of the alpha(1B)AR with epinep hrine, The observation that overexpression of G protein-coupled kinase 2 (GRK2) could increase agonist-induced phosphorylation of Ser(404), Ser(408), and Ser(410), strongly suggests that these serines are the p hosphorylation sites of the alpha(1B)AR for kinases of the GRK family, Phorbol ester-induced phosphorylation of the Ser(394) and Ser(400) as well as GRK2-mediated phosphorylation of the Ser(404), Ser(408), and Ser(410), resulted in the desensitization of cu,BAR-mediated inositol phosphate response, This study provides generalities about the biochem ical mechanisms underlying homologous and heterologous desensitization of G protein-coupled receptors linked to the activation of phospholip ase C.