Sites phosphorylated in human α_1B-adrenoceptors in response to noradrenaline and phorbol myristate acetate

Phosphorylation of the human α_1B-adrenergic receptor (fused with the green fluorescent protein) was studied employing the inducible Flp-ln HEK293 T-Rex system for expression. Serine/alanine substitutions were performed in five sites corresponding to those previously identified as phosphorylation targets in the hamster ortholog. Desensitization was decreased in these mutants but receptor phosphorylation was still clearly detected. The protein phosphorylation of the wild-type receptor (fused to the green fluorescent protein) was studied, using mass spectrometry, under baseline and stimulated conditions (noradrenaline or phorbol myristate acetate). Basal phosphorylation was detected at sites located at the intracellular loop 3 and carboxyl terminus, and the number of sites detected increased under agonist activation and stimulation of protein kinase C. The phosphorylation patterns differed under the distinct conditions. Three of the phosphorylation sites detected in this work corresponded to those observed in the hamster receptor. The phosphorylation sites detected included the following: a) at the intracellular loop 3: serines 246, 248, 257, 267, and 277; and threonines 252, 264, and 268, and b) at the carboxyl terminus: serines 396, 400, 402, 406, 423, 425, 427, 455, and 470, and threonines 387, 392, 420, and 475. Our data indicate that complex phosphorylation patterns exist and suggest the possibility that such differences could be relevant in receptor function and subcellular localization.