Cross-regulation of VPAC(2) receptor desensitization by M(3) receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2.
In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.
Huang
Jiean
J
Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Mahavadi
Sunila
S
Sriwai
Wimolpak
W
Grider
John R
JR
Murthy
Karnam S
KS
D000109:Acetylcholine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D003959:Diamines / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D049488:Phosphatidylethanolamine Binding Protein / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011817:Rabbits; D020239:Receptor Cross-Talk; D043585:Receptor, Muscarinic M2 / Q000037:antagonists & inhibitors; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000378:metabolism / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism*
D003959:Diamines; D007211:Indoles; D008301:Maleimides; D018727:Muscarinic Antagonists; D049488:Phosphatidylethanolamine Binding Protein; D047428:Protein Kinase Inhibitors; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D000262:Adenylyl Cyclases; C088060:bisindolylmaleimide; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate; C054938:methoctramine