Abstract
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 Gs and Gi proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that β2AR coupling to Gi 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 Gi 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/Gi coupling. However, a sufficient β2AR/Gi 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 Gi proteins, probably by preventing the receptor's transportation to access Gi proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the Gi-specific inhibitor, GiCT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of β2AR for agonist dose-dependent coupling to Gi proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 32279-32287 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 284 |
| Issue number | 47 |
| DOIs | |
| State | Published - Nov 20 2009 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Cell Biology
- Molecular Biology
Cite this
Agonist dose-dependent phosphorylation by protein kinase A and G protein-coupled receptor kinase regulates β2 adrenoceptor coupling to Gi proteins in cardiomyocytes. / Liu, Ruijie; Ramani, Biswarathan; Soto, Dagoberto; De Arcangelis, Vania; Xiang, Yang Kevin.
In: Journal of Biological Chemistry, Vol. 284, No. 47, 20.11.2009, p. 32279-32287.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Agonist dose-dependent phosphorylation by protein kinase A and G protein-coupled receptor kinase regulates β2 adrenoceptor coupling to Gi proteins in cardiomyocytes
AU - Liu, Ruijie
AU - Ramani, Biswarathan
AU - Soto, Dagoberto
AU - De Arcangelis, Vania
AU - Xiang, Yang Kevin
PY - 2009/11/20
Y1 - 2009/11/20
N2 - 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 Gs and Gi proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that β2AR coupling to Gi 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 Gi 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/Gi coupling. However, a sufficient β2AR/Gi 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 Gi proteins, probably by preventing the receptor's transportation to access Gi proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the Gi-specific inhibitor, GiCT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of β2AR for agonist dose-dependent coupling to Gi proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.
AB - 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 Gs and Gi proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that β2AR coupling to Gi 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 Gi 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/Gi coupling. However, a sufficient β2AR/Gi 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 Gi proteins, probably by preventing the receptor's transportation to access Gi proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the Gi-specific inhibitor, GiCT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of β2AR for agonist dose-dependent coupling to Gi proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.
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U2 - 10.1074/jbc.M109.021428
DO - 10.1074/jbc.M109.021428
M3 - Article
VL - 284
SP - 32279
EP - 32287
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 47
ER -