Cross-regulation of VPAC₂ receptor desensitization by M₃ receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

Abstract

.-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 VPAC2 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 M3 receptors) inhibited VPAC2 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 Gq-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2. internalization; signaling; smooth muscle IN THE GASTROINTESTINAL TRACT, the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are colocalized in a subset of myenteric neurons that innervate the smooth muscle, and their release is functionally linked to smooth muscle relaxation (1, 13). The biological actions of VIP and PACAP are mediated by a family of G proteincoupled receptors, which are designated as VPAC 1 , VPAC 2 , and PAC 1 receptors (9, 26). These receptors belong to class II of the G protein-coupled receptors known as the secretin receptor family that include receptors for secretin, gastric inhibitory peptide, glucagon, glucagon-like peptide 1, calcitonin, parathyroid hormone, growth hormone-releasing factor, and corticotrophin-releasing hormone (9,23,24,26,27). PAC 1 receptors exhibit high affinity for PACAP and low affinity for VIP, whereas VPAC 1 and VPAC 2 receptors exhibit equal affinity for VIP and PACAP (9).Smooth muscle cells of the gut express VPAC 2 receptors, but not VPAC 1 and PAC 1 receptors (16,25). We have recently shown that VPAC 2 receptors are desensitized and internalized by a mec...

Gender differences in the regulation of MLC20 phosphorylation and smooth muscle contraction in rat stomach

“…This phosphorylation is initiated and regulated by activation of the Ca 2+ /calmodulin-dependent myosin light-chain kinase (MLCK), which transfers the phosphate group from adenosine triphosphate (ATP) to the Ser19 hydroxyl group of MLC 20 (16). This phosphorylation activates the actin-activated myosin ATPase and actin-myosin interaction, thereby initiating smooth muscle contraction (15,16).…”

Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/β-arrestin activation of ERK1/2 and Src kinase

“…We had previously shown that the initial transient contraction induced by ACh is mediated by G␣ qdependent activation of PLC-␤1 and terminated by RGS4 (9,11). In this study, we identified a consensus sequence ( 102 KSP-SKLSP 109 ) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a phosphorylation-deficient RGS4 mutant lacking Ser 103 and Ser 108 blocked the ability of AEA to inhibit of ACh-stimulated PI hydrolysis or enhance G␣ q :RGS4 association.…”

Raf Kinase Inhibitor Protein (RKIP) Dimer Formation Controls Its Target Switch from Raf1 to G Protein-coupled Receptor Kinase (GRK) 2

“…Furthermore, RKIP was suggested to be involved in GPCR signaling (5) and was eventually identified as a physiological and direct inhibitor of GRK2 (4), which is an important regulator of GPCR signaling. RKIP-mediated GRK2 inhibition is involved in cross-regulation of GPCRs (35), neuronal differentiation (36), and cardiomyocyte contractility (4). Taken together, RKIP controls a complex variety of signaling networks in mammalian cells.…”