Role of Tyr^356(7.43) and Ser^190(4.57) in antagonist binding in the rat β₁-adrenergic receptor

Site-directed mutagenesis and photoaffinity labeling experiments suggest the existence of at least two distinct binding orientations for aryloxypropanolamine competitive antagonists in the β-adrenergic receptor (β-AR), one where the aryloxy moiety is located near transmembrane α-helix 7 (tm 7) and another where it is near tm 5. To explore a hydrophobic pocket involving tms 1, 2, 3, and 7 for potential aryloxy interaction sites, we selected Tyr^356(7.43) and Trp ^134(3.28) in the rat β₁-AR for site-directed mutagenesis studies. Ser^190(4.57) was also investigated, as the equivalent residues are known antagonist interaction sites in the muscarinic M₁ and the dopamine D₂ receptors. Binding affinities (pK_i) of a series of structurally diverse aryloxypropanolamine competitive antagonists were determined for wild type and Y356A, Y356F, W134A, and S190A mutant rat β₁-ARs stably expressed in Chinese hamster ovary cells. To visualize possible antagonist/receptor interactions, the compounds were docked into a three-dimensional model of the wild-type rat β₁-AR. The results indicate that Tyr^356(7.43) is an important aromatic interaction site for five of the eight competitive antagonists studied, whereas none of the compounds appeared to interact directly with Trp^134(3.28). Only two of the competitive antagonists interacted with Ser^190(4.57) on tm 4. Overall, the results extend our understanding of how β₁-AR competitive antagonists bind to the hydrophobic pocket involving tms 1, 2, 3, and 7; highlight the importance of Tyr^356(7.43) in this binding pocket; and demonstrate the involvement of tm 4 in competitive antagonist binding.