Role of Cytoplasmic Tail of the Type 1A Angiotensin II Receptor in Agonist– and Phorbol Ester–Induced Desensitization

Abstract

Abstract —To investigate mechanisms underlying the agonist-induced desensitization of the type 1A angiotensin II receptor (AT 1A -R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild-type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5-trisphosphate (IP 3 ) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with G q protein with an efficiency similar to that of full-length receptors, whereas coupling of G q protein was abolished in the T310 truncated mutant devoid of the carboxyl-terminal 50 amino acids. Exposure of CHO/AT 1A -R cells expressing the wild-type AT 1A -R to angiotensin II resulted in rapid and dose-dependent homologous desensitization of receptor-mediated IP 3 formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein–coupled receptor kinase (GRK), induced desensitization of the AT 1A -R. The agonist-induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)-specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr-rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT 1A -R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12-myristate 13-acetate–induced desensitization by 30%. Moreover, we found an agonist-induced translocation of a heparin-sensitive kinase activity. The angiotensin II–stimulated heparin-sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT 1A -R cytoplasmic tail (N 295 to E 359 ), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin-sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT 1A -R cells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S 328 and S 347 in the cytoplasmic tail of AT 1A -R seem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin-sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist-induced homologous desensitization of the AT 1A -R.