Abstract
Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. beta(2)ARs, 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 the beta(2)AR coupling to G(i) proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the beta(2)AR-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 activated beta(2)ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the beta(2)AR blocked receptor/G(i) coupling. However, a sufficient beta(2)AR/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 (> or = 100 nm). Disruption of the GRK phosphorylation sites on the beta(2)AR 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(i)CT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the beta(2)AR for agonist dose-dependent coupling to G(i) proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.
Highlights
2ARs are prototypical G protein-coupled receptors (GPCRs) expressed in animal hearts, which mediate the increases in cardiac contraction upon agonist stimulation through the Gs-adenylyl cyclase-cAMPdependent protein kinase A (PKA) pathway [1]. 2ARs are known to uniquely couple to inhibitory Gi proteins, which inhibits adenylyl cyclases to reduce cardiac contraction and initiates anti-apoptotic and cell growth signaling [2, 3]
Assay with a biosensor for PKA activity and a physiological cardiac contraction rate assay, we show for the first time that 2AR/Gi coupling occurs only at a saturated concentration of isoproterenol (Iso) in cardiac cells
These data indicate that PKA phosphorylation is necessary but not sufficient for the activated 2AR coupling to Gi in cardiac myocytes
Summary
Gi inhibitor pertussis toxin (PTX) at 0.3 g/ml, 3 h before stimulation. GFP-GiCT virus was co-infected at a multiplicity of infection of 100 or, as indicated, along with the wild type or mutant 2AR viruses. Contraction rate assays were performed the day as described previously [16]. Receptor Phosphorylation—Neonatal cardiac myocytes were infected with FLAG-tagged wild type. Mutant 2ARs at a multiplicity of infection of 100. After 48 h of expression, cells were serum-starved for 2 h and stimulated with different doses of Iso for 5 min. For the time course experiments, cells were stimulated with 10 M Iso for different times as indicated. GRK-mediated phosphorylation and subsequent trafficking into lysis buffer
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