Agonist-induced phosphorylation of the angiotensin AT1a receptor is localized to a serine/threonine-rich region of its cytoplasmic tail

The agonist-induced phosphorylation sites of the rat AT(1a) angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin If-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta 342) but was abolished by truncation at Ser325 (Delta 325). Truncation at Ser335 (Delta 335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by similar to 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta 335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta 325 < Delta 335 < ST-AA < Delta 342 wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta 325 > Delta 335 > ST-AA = Delta 342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT(1a) receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.