Synaptic Scaffolding Molecule Binds to and Regulates Vasoactive Intestinal Polypeptide Type-1 Receptor in Epithelial Cells

Background & Aims: Vasoactive intestinal polypeptide (VIP) is a principal regulator of fluid and electrolyte secretion in the gastrointestinal system. The VIP type-1 receptor (VPAC₁), a class II G-protein-coupled receptor, contains a putative C-terminal PDZ-binding motif. A yeast 2-hybrid screen indicated that the C-terminus of VPAC₁ bound to the PDZ domain of synaptic scaffolding molecule (S-SCAM, also known as membrane-associated guanylate kinase inverted-2 [MAGI-2]). We analyzed the association between S-SCAM and VPAC₁. Methods: The biochemical properties and physiologic significance of the interaction between VPAC₁ and S-SCAM were examined in heterologous expression systems, T84 colonic epithelial cells, and human pancreas and colon tissues using an integrated molecular and physiologic approach. Results: The physical interaction between VPAC₁ and S-SCAM was confirmed by immunoprecipitation in HEK 293 mammalian cells and human pancreatic and colonic tissues. Immunocytochemical analysis indicated that S-SCAM recruited VPAC₁ to the junctional area near the apical end of the lateral membrane in T84 cells. Several lines of evidence revealed that S-SCAM inhibits VPAC₁ activation. Overexpression of S-SCAM inhibited VPAC₁-mediated cAMP production and agonist-induced VPAC₁ internalization in HEK 293 and HeLa cells. In addition, S-SCAM decreased the VPAC₁-mediated current through the cystic fibrosis transmembrane conductance regulator in Xenopus oocytes, especially at low concentrations of VIP. Importantly, loss of S-SCAM increased VIP-induced short-circuit currents in T84 monolayers, which endogenously express VPAC₁ and S-SCAM. Conclusions: S-SCAM/MAGI-2 interacts with and regulates VPAC₁ intracellular localization in epithelial cells and inhibits VPAC₁ agonist-induced activation and internalization.