The pleiotropic actions of angiotensin II are mediated by the primarily Gq protein-coupled type I 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 AT1A 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 AT1A 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 AT1A 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 AT1A 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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