Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes
Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes
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DOI：
10.1074/jbc.m109.021428
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发表时间：
2009-11-20
影响因子：
4.8
通讯作者：
Xiang, Yang
中科院分区：
文献类型：
--
作者：
Liu, Ruijie;Ramani, Biswarathan;Xiang, Yang
肾上腺素能受体（AR）在调节应激时心血管对儿茶酚胺的反应中起着关键作用。β（2）AR是在动物心脏中表达的原型G蛋白偶联受体（GPCR），显示与G（s）和G（i）蛋白的双重偶联，以控制腺苷酸环化酶-cAMP依赖性蛋白激酶A（PKA）途径来调节收缩反应。在这里，我们发现β 2 AR与G（i）蛋白的偶联是激动剂剂量依赖性的，并且仅在小鼠心肌细胞中高浓度时发生。通过荧光共振能量转移（FRET）成像测量的β（2）AR诱导的PKA活性和肌细胞收缩率的增加都显示出对G（i）抑制剂百日咳毒素（PTX）的敏感性。进一步的研究表明，激活的β 2 AR在广泛的激动剂浓度下经历PKA磷酸化。β 2 AR上PKA磷酸化位点的破坏阻断了受体/G（i）偶联。然而，充分的β 2AR/G（i）偶联也依赖于G蛋白偶联受体激酶（GRK）介导的受体磷酸化，这仅发生在高浓度激动剂（>= 100 nM）下。β 2 AR上GRK磷酸化位点的破坏阻断了受体的内化和与G（i）蛋白的偶联，可能是通过阻止受体转运至G（i）蛋白。此外，PKA和GRK位点突变的受体都没有表现出对G（i）特异性抑制剂G（i）CT的敏感性。总之，我们的研究揭示了PKA和GRK磷酸化β（2）AR的不同作用，激动剂剂量依赖性偶联到心肌细胞中的G（i）蛋白，这可能会保护细胞免受高浓度儿茶酚胺过度刺激。
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 2AR/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 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.