The aim of the study was to examine the mechanisms by which ACh, acting via m2 receptors, regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. VIP induced VPAC(2) receptor phosphorylation and internalization in freshly dispersed smooth muscle cells. Co-stimulation with acetylcholine (ACh), in the presence of m3 receptor antagonist, 4-DAMP, augmented VPAC(2) receptor phosphorylation and internalization. The m2 receptor antagonist methoctramine or the c-Src inhibitor PP2 blocked the effect of ACh, suggesting that the augmentation was mediated by c-Src, derived from m2 receptor activation. ACh induced activation of c-Src and phosphorylation of GRK2 and the effects of ACh were blocked by methoctramine, PP2, or by uncoupling of m2 receptors from G(i3) with pertussis toxin. In conclusion, we identified a novel mechanism of cross-regulation of GRK2-mediated phosphorylation and internalization of G(s)-coupled VPAC(2) receptors by G(i)-coupled m2 receptors via tyrosine phosphorylation of GRK2 and stimulation of GRK2 activity.