DIFFERENTIATION BETWEEN BINDING-SITES FOR ANGIOTENSIN-II AND NONPEPTIDE ANTAGONISTS ON THE ANGIOTENSIN-II TYPE-1 RECEPTORS

To characterize binding sites for nonpeptide angiotensin antagonists o n tile human angiotensin II receptor type 1 (AT(1) receptor) we have s ystematically exchanged segments of the human receptor with correspond ing segments from a homologous Xenopus laevis receptor, which does not bind the nonpeptide compounds. Substitution of transmembrane segment VII of the human AT(1) receptor dramatically reduced the binding affin ity of all of the 11 nonpeptide antagonists tested (55- to >2000-fold) with no effect on the binding of angiotensin. The affinity for the no npeptide compounds decreased additionally one order of magnitude when transmembrane segment VI and the connecting extracellular loop 3 from the Xenopus receptor were also introduced into the human AT(1) recepto r. Exchanges of smaller segments and single residues in transmembrane segments VI and VII and extracellular loop 3 revealed that the binding of nonpeptide antagonists was dependent on nonconserved residues loca ted deep within the transmembrane segments VI and VII, in particular A sn(295) in transmembrane segment VII. Surprisingly, all exchanges in t ransmembrane segment VII, including the Asn(295) to Ser substitution, had a more pronounced effect on the binding of the competitive antagon ists relative to the insurmountable antagonists. It is concluded that the binding mode for peptide and nonpeptide ligands an the ATI recepto r is rather different and that competitive and insurmountable antagoni sts presumably bind to overlapping but distinct sites located in trans membrane segments VI and VII.