Site-directed mutagenesis of the rat β₁-adrenoceptor. Involvement of Tyr^{356 (7.43)} in (+/-)cyanopindolol but not (+/-)[¹²⁵Iodo]cyanopindolol binding

To determine the role played by Tyr^{356 (7.43)} in the rat β₁-adrenoceptor in binding the antagonists (+/-)cyanopindolol (4-[3-(t-butylamino]-3-(2′-cyano-indoloxy)-2-propanolol) and its iodinated analogue (+/-)[¹²⁵Iodo]cyanopindolol (1-(t-butylamino]-3-(2′- cyano-3′-iodo-indoloxy)-2-propanolol), Tyr^{356 (7.43)} was mutated to either Phe or Ala and binding affinities determined for wild type and mutant rat β₁-adrenoceptors. Our results indicate that Tyr ^{356 (7.43)} is important for (+/-)cyanopindolol, but not (+/-)[ ¹²⁵Iodo]cyanopindolol, binding and that (+/-)cyanopindolol adopts a "reverse" binding orientation whereas (+/-)[¹²⁵Iodo] cyanopindolol cannot be accommodated in this binding mode. We define a "reverse" antagonist binding mode as one where the aryloxy moiety interacts with residues on transmembrane helices 1, 2, 3 and 7. The β₁-adrenoceptor site-directed mutagenesis results are the first to support a "reverse" antagonist binding orientation and the involvement of Tyr^{356 (7.43)} in this binding mode.