Polar residues in the transmembrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by co-expression of two deficient mutants.

Type 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism. Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.