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

From the Department of Biochemistry (H.T., D.F.G., Y.W., T.I.), Vanderbilt University School of Medicine, Nashville, Tenn, and the Department of Physiology and Biophysics (J.P.P.), University of Louisville (Ky).

Correspondence to Tadashi Inagami, PhD, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.

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 (IP₃) 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 IP₃ 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 (N²⁹⁵ to E³⁵⁹), 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 S³²⁸ and S³⁴⁷ 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.

Key Words: angiotensin II • desensitization • AT_1A receptor • phorbol ester

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