Submitted on June 1, 2007
Accepted on December 7, 2007
Phosphorylation of GRK2 by PKA Augments GRK2-Mediated Phosphorylation, Internalization, and Desensitization of VPAC2 Receptors in Smooth Muscle
Karnam S. Murthy1*, Sunila Mahavadi1, Jiean Huang1, Huiping Zhou1, and Wimolpak Sriwai1
1 Physiology, Virginia Commonwealth University, Richmond, Virginia, United States
* To whom correspondence should be addressed. E-mail: skarnam{at}vcu.edu.
Smooth muscle of the gut expresses mainly Gs-coupled VPAC2 receptors, which belong to secretin family of G protein-coupled receptors. The extent to which cAMP-dependent protein kinase (PKA) and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among secretin family receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC2 receptors via phosphorylation of GRK2 at Ser685. Vasoactive intestinal peptide (VIP) induced phosphorylation of GRK2 in a concentration-dependent fashion and the phosphorylation was abolished by the blockade of PKA with cell-permeable myristoylated PKI or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to Gbg. VIP-induced phosphorylation of VPAC2 receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC2 receptor internalization (determined from residual 125I-VIP binding, receptor biotinylation, and confocal microscopy after 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC2 receptor degradation (determined from residual 125I-VIP binding and receptor expression after prolonged exposure to VIP) and functional VPAC2 receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). The results demonstrate that in gastric smooth muscle VPAC2 receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA greatly enhances GRK2 activity and its ability to induce VPAC2 receptor phosphorylation, internalization, desensitization and degradation.
