G-protein binding domains of the angiotensin II AT_1A receptors mapped with synthetic peptides selected from the receptor sequence

The vascular angiotensin II type 1 receptor (AT_1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT_1AR using synthetic peptides selected from the receptor sequence, which interfere with AT_1AR-G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125-137), the N-terminal region of the third intracellular loop (217-227) and the juxtamembranous region of the C-terminal tail (304-316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217-227 and 304-316) are predicted to form amphiphilic α-helices. Only the peptide representing residues 217-227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT_1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT_1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.