Murthy Karnam S, Mahavadi Sunila, Huang Jiean, Zhou Huiping, Sriwai Wimolpak
Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA.
Am J Physiol Cell Physiol. 2008 Feb;294(2):C477-87. doi: 10.1152/ajpcell.00229.2007. Epub 2007 Dec 12.
The smooth muscle of the gut expresses mainly G(s) protein-coupled vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptors (VPAC(2) receptors), which belong to the secretin family of G protein-coupled receptors. The extent to which PKA and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among the secretin family of receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC(2) receptors via the phosphorylation of GRK2 at Ser(685). VIP induced phosphorylation of GRK2 in a concentration-dependent fashion, and the phosphorylation was abolished by blockade of PKA with cell-permeable myristoylated protein kinase inhibitor (PKI) or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to G betagamma. VIP-induced phosphorylation of VPAC(2) receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC(2) receptor internalization (determined from residual (125)I-labeled VIP binding and receptor biotinylation after a 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC(2) receptor degradation (determined from residual (125)I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC(2) receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after a 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). These results demonstrate that in gastric smooth muscle VPAC(2) receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC(2) receptor phosphorylation, internalization, desensitization, and degradation.
肠道平滑肌主要表达G(s)蛋白偶联的血管活性肠肽（VIP）/垂体腺苷酸环化酶激活肽受体（VPAC(2)受体），这些受体属于G蛋白偶联受体的促胰液素家族。蛋白激酶A（PKA）和G蛋白偶联受体激酶（GRKs）参与同源脱敏的程度在促胰液素家族受体中差异很大。本研究确定了PKA通过在Ser(685)位点磷酸化GRK2在VPAC(2)受体同源脱敏中的新作用。VIP以浓度依赖的方式诱导GRK2磷酸化，用细胞可渗透的肉豆蔻酰化蛋白激酶抑制剂（PKI）阻断PKA或在表达PKA磷酸化位点缺陷的GRK2(S685A)的细胞中，这种磷酸化被消除。GRK2的磷酸化增加了其活性以及与Gβγ的结合。在表达激酶缺陷的GRK2(K220R)的肌肉细胞中，VIP诱导的VPAC(2)受体磷酸化被消除，在表达GRK2(S685A)的细胞中或用PKI处理后则减弱。VPAC(2)受体内化（通过在30分钟暴露于VIP后残留的(125)I标记的VIP结合和受体生物素化来确定）在表达GRK2(K220R)的细胞中被阻断，在表达GRK2(S685A)的细胞中或用PKI处理后则减弱。最后，VPAC(2)受体降解（通过在长时间暴露于VIP后残留的(125)I标记的VIP结合和受体表达来确定）和功能性VPAC(2)受体脱敏（通过在30分钟暴露于VIP后腺苷酸环化酶活性和cAMP形成的降低来确定）在表达GRK2(K220R)的细胞中被消除，在表达GRK2(S685A)的细胞中则减弱。这些结果表明，在胃平滑肌中，VPAC(2)受体磷酸化由GRK2介导。PKA对GRK2的磷酸化增强了GRK2的活性及其诱导VPAC(2)受体磷酸化、内化、脱敏和降解的能力。
