Tyrosine 308 is Necessary for Ligand-directed Gs-biased Signaling of {beta}2-Adrenoceptor [Signal Transduction]

May 15th, 2014 by Woo, A. Y.-H., Jozwiak, K., Toll, L., Tanga, M. J., Kozocas, J. A., Jimenez, L., Huang, Y., Song, Y., Plazinska, A., Pajak, K., Paul, R. K., Bernier, M., Wainer, I. W., Xiao, R.-P.

Interaction of a given G protein-coupled receptor (GPCR) to multiple different G proteins is a wide-spread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by GPCR kinases (GRKs) particularly GRK2 markedly enhances the Gi-coupling. We have previously shown that while most β2-AR agonists cause both Gs and Gi activation, (R,R′)-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here using docking simulation and site-directed mutagenesis, we defined Y308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R′)-4'-aminofenoterol, the Gi disruptor pertussis toxin (PTX) produced no effects on the receptor-mediated ERK phosphorylation in HEK-293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F-substitution on β2-AR enabled (R,R′)-4'-aminofenoterol to activate Gi and to produce these responses in a PTX-sensitive manner without altering β2-AR phosphorylation by PKA or GRKs. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Y308 residue of β2-AR stabilizes receptor conformations favoring the receptor-Gs protein coupling and subsequently results in Gs-biased agonism.