Agonist binding and activation of the rat β₁-adrenergic receptor: Role of Trp^{134 (3.28)}, Ser^{190 (4.57)} and Tyr^{356 (7.43)}

We investigated the role of Trp^{134 (3.28)}, Ser ^{190 (4.57)} and Tyr^{356 (7.43)} in agonist binding to, and activation of, the rat β₁-adrenergic receptor by comparing pK_is and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD₂ and pK_i (P > 0.4), suggesting that changes in pK_i were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr^{356 (7.43)}. For the other agonists, the change in pK_i values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat β₁-adrenergic receptor, Trp^{134 (3.28)} and Tyr^{356 (7.43)} form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp^{134 (3.28)} and Tyr^{356 (7.43)}, together with Trp^{353 (7.40)}, are able to interact via π-π interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser^{190 (4.57)} appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.