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This Article Full Text Full Text (PDF) All Versions of this Article: 294/2/C477    most recent 00229.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Murthy, K. S. Articles by Sriwai, W. PubMed PubMed Citation Articles by Murthy, K. S. Articles by Sriwai, W.

RECEPTORS AND SIGNAL TRANSDUCTION

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 (VPAC₂ 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 VPAC₂ receptors via the phosphorylation of GRK2 at Ser⁶⁸⁵. 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 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-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, VPAC₂ receptor degradation (determined from residual ¹²⁵I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC₂ 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 VPAC₂ receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC₂ 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