MUTATIONAL ANALYSIS OF THE ANGIOTENSIN TYPE-2 RECEPTOR - CONTRIBUTIONOF CONSERVED AMINO-ACIDS IN THE REGION OF THE 6TH TRANSMEMBRANE DOMAIN

Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT(1)) and Type 2 (AT(2)) receptors. Th e subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT(1) receptor, ident ifying residues important for AngII binding. In constrast, relatively little is known of the binding mechanism of the AT(1) receptor. It has been hypothesized that residues that are conserved between the two su btypes that have been shown to be important in the AT(1) receptor may also contribute to AngII binding in the AT(1) receptor as well. To tes t this hypothesis, the role of two conserved residues in the sixth tra nsmembrane domain of the AT(1) receptor in ligand binding were investi gated: tryptophan 269 and aspartate 279. In contrast to the AT(1) rece ptor, mutation of Trp(259) in the AT(1) receptor to an alanine had no effect on AngII binding, while mutation of Asp(279) to alanine similar ly impaired AngII binding in both receptors, However, the more sterica lly conservative substitution of Asp(279) to asparagine in the AT(2) r eceptor showed near wild type affinity. Based on this finding, we muta ted Asp(263) in the AT(1) receptor to asparagine. Subsequent studies i ndicated that this more conservative mutation had no effect on AngII b inding to the AT(1) receptor. Collectively, these results demonstrate that although there may be commonalities in ligand binding between the AT(1) and AT(1) AngII receptors, there are also clear differences. (C ) 1998 Elsevier Science B.V.