Abstract 3393: A Modified β1-adrenergic Receptor Demonstrates Bias Towards G Protein Receptor Kinase Phosphorylation
Beta 1-adrenergic receptor (β1AR) activation initiates both classical G protein-mediated signaling and β-arrestin dependent signaling. We previously described a β-arrestin dependent pathway in which phosphorylation of the β1AR by G protein-coupled receptor kinase (GRK) isoforms 5 and 6 (but not GRK 2) leads to biased non G protein signaling mediated by the transactivation of the epidermal growth factor receptor. To investigate the molecular mechanisms for β-arrestin mediated signaling by the β1AR, we engineered a β1AR mutant containing amino acid substitutions in the putative G protein-binding domain (β1ARY244A+Y366A). As measured by cAMP assays, β1ARY244A+Y366A has markedly diminished capacity to activate G protein signaling compared with the wild type receptor (β1ARWT), but maintains ability to recruit β-arrestin after catecholamine stimulation. Using CFP-tagged receptors and β-arrestin-YFP, we measured β-arrestin recruitment to β1ARWT and mutant β1ARY244A+Y366A by fluorescence resonance energy transfer (FRET). Non-ionic transfer of energy from CFP to YFP suggests close proximity or complex formation between receptor and β-arrestin and is measured as percentage FRET. In both overexpression and knock down experiments we show that while β1ARWT recruits β-arrestin primarily after phosphorylation by GRK 2, whereas the recruitment of β-arrestin to the mutant β1ARY244A+Y366A requires GRK 5 (figure⇓). Our findings suggest that altering critical amino acids in the β1AR can result in a receptor conformation that favors GRK 5 dependent phosphorylation which triggers unique signaling pathways in a G- protein independent β-arrestin mediated manner.