title: Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle.
abstract: 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.
authors: Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA. skarnam@vcu.edu
authors: Karnam S
authors: KS
authors: Murthy
authors: Sunila
authors: S
authors: Mahavadi
authors: Jiean
authors: J
authors: Huang
authors: Huiping
authors: H
authors: Zhou
authors: Wimolpak
authors: W
authors: Sriwai
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D025461:Feedback, Physiological / Q000187:drug effects / Q000502:physiology; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011817:Rabbits; D011869:Radioligand Assay; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013270:Stomach / Q000166:cytology; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology
chemical_list: D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054769:G-Protein-Coupled Receptor Kinase 2