Agonist-induced phosphorylation of the angiotensin AT(1a) receptor is localized to a serine/threonine-rich region of its cytoplasmic tail

The agonist-induced phosphorylation sites of the rat AT(1a) angiotensin rec eptor 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 Ser33 5 (Delta 335), or double-point mutations of Ser335 and Thr336 to alanine (S T-AA), reduced receptor phosphorylation by similar to 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 s egment are also phosphorylated. Agonist-induced phosphorylation of the ST-A A and Delta 335 receptors was partially inhibited by staurosporine, suggest ing that the single protein kinase C consensus site in the Ser326-Thr332 se gment (Ser331) is phosphorylated. The impairment of receptor phosphorylatio n was broadly correlated with the attenuation of agonist-induced internaliz ation rates (Delta 325 < Delta 335 < ST-AA < Delta 342 wild-type) and with the increasing rank order of magnitude of inositol phosphate production nor malized to an equal number of receptors (Delta 325 > Delta 335 > ST-AA = De lta 342 > wild-type). These results demonstrate that agonist-induced phosph orylation of the AT(1a) receptor is confined to an 11-amino-acid serine/thr eonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitizat ion.