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The Role of a Conserved Region of the Second Intracellular Loop in AT₁ Angiotensin Receptor Activation and Signaling

Department of Physiology (Z.G., L.H.) and Laboratory of Cellular and Molecular Physiology (A.S.), Semmelweis University, Faculty of Medicine, H-1088 Budapest, Hungary; Endocrinology and Reproduction Research Branch (M.Z., K.J.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510; Division of Cardio-Renal Drug Products (G.J.), Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20857

Address all correspondence and requests for reprints to: László Hunyady, Department of Physiology, Semmelweis University, Faculty of Medicine, H-1444 Budapest, 8 P.O. Box 259, Hungary. E-mail: hunyady{at}puskin.sote.hu.

The pleiotropic actions of angiotensin II are mediated by the primarily G_q protein-coupled type 1 angiotensin (AT₁) receptor. In this study a mutational analysis of the function of the conserved DRYXXV/IXXPL domain in the second intracellular loop of the rat AT_1A receptor was performed in COS7 cells. Alanine substitution studies showed that single replacement of the highly conserved Asp¹²⁵ and Arg¹²⁶, but not Tyr¹²⁷, moderately impaired angiotensin II-induced inositol phosphate signaling. However, concomitant substitution of both Asp¹²⁵ and Arg¹²⁶ caused marked reduction of both inositol phosphate signaling and receptor internalization. Alanine scanning of the adjacent residues showed that substitution of Ile¹³⁰, His¹³², and Pro¹³³ reduced agonist-induced inositol phosphate signal generation, whereas mutations of Met¹³⁴ also impaired receptor internalization. Expression of the D125A mutant AT_1A receptor in COS7 cells endowed the receptor with moderate constitutive activity, as indicated by its enhanced basal Elk1 promoter activity and inositol phosphate response to partial agonists. Angiotensin II-induced stimulation of the Elk1 promoter showed parallel impairment with inositol phosphate signal generation in receptors containing mutations in this region of the AT_1A receptor. These data confirm that Ca²⁺ signal generation is required for the nuclear effects of angiotensin II-induced ERK activation. They are also consistent with the role of the conserved DRY sequence of the AT_1A receptor in receptor activation, and of Asp¹²⁵ in constraining the receptor in its inactive conformation. Furthermore, in the cytoplasmic helical extension of the third helix, an apolar surface that includes Ile¹³⁰ and Met¹³⁴ appears to have a direct role in G protein coupling.

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