Synaptic scaffolding molecule binds to and regulates vasoactive intestinal polypeptide type-1 receptor in epithelial cells.
Authors
HEON YUNG GEE ; YOUNG WOONG KIM ; MIN JAE JO ; WAN NAMKUNG ; JOO YOUNG KIM ; HYUN WOO PARK ; KYUNG SIK KIM ; HOGUEN KIM ; AKEMICHI BABA ; JINHEE YANG ; EUNJOON KIM ; KYUNG HWAN KIM ; MIN GOO LEE
Analysis of Variance ; Carrier Proteins ; Cell Communication/genetics ; Cell Communication/physiology ; Cells, Cultured ; Epithelial Cells/metabolism* ; Epithelial Cells/physiology ; Gene Expression Regulation ; Humans ; Immunoblotting ; Immunohistochemistry ; Probability ; Protein Binding/genetics ; Protein Binding/physiology* ; Receptors, Vasoactive Intestinal Polypeptide, Type I/genetics ; Receptors, Vasoactive Intestinal Polypeptide, Type I/metabolism* ; Signal Transduction/genetics ; Two-Hybrid System Techniques
Abstract
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.