Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, beta(2)-adrenoceptor (beta(2)-AR) couples dually to G(s) and G(i) proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of beta(2)-AR causes a switch in receptor coupling from G(s) to G(i). More recent studies have demonstrated that phosphorylation of beta(2)-AR byGproteincoupled receptor kinases, particularly GRK2, markedly enhances the G(i) coupling. Wehave previously shown that although most beta(2)-AR agonists cause both G(s) and G(i) activation, (R,R')fenoterol preferentially activates beta(2)-AR-G(s) signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on beta(2)-AR essential for G(s)-biased signaling. Following stimulation with a beta(2)-AR-G(s)-biased agonist (R,R')-4'-aminofenoterol, the G(i) disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type beta(2)-AR. Interestingly, Y308F substitution on beta(2)-AR enabled (R,R')-4'-aminofenoterol to activate G(i) and to produce these responses in a pertussis toxin-sensitive manner without altering beta(2)-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of beta(2)-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of beta(2)-AR stabilize receptor conformations favoring the receptor-G(s) protein coupling and subsequently result in G(s)-biased agonism.