Agonist dose-dependent phosphorylation by protein kinase A and G protein-coupled receptor kinase regulates β₂ adrenoceptor coupling to G_i proteins in cardiomyocytes

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during β₂ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G_s and G_i proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that β₂AR coupling to G_i proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both β₂AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G_i inhibitor pertussis toxin (PTX). Further studies revealed that β ₂ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the β₂AR blocked receptor/G_i coupling. However, a sufficient β₂AR/G_i coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (≥100 nM). Disruption of the GRK phosphorylation sites on the β₂AR blocked receptor internalization and coupling to G_i proteins, probably by preventing the receptor's transportation to access G_i proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G_i-specific inhibitor, G_iCT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of β₂AR for agonist dose-dependent coupling to G_i proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.