Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia Submitted 1 June 2007 ; accepted in final form 7 December 2007 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 Gβ . 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. homologous desensitization; vasoactive intestinal peptide; G protein-coupled receptor kinase; gastric muscle; G protein signaling; pituitary adenylate cyclase-activating peptide Address for reprint requests and other correspondence: K. S. Murthy, Medical College of Virginia Campus, Virginia Commonwealth Univ., PO Box 9