Synaptic scaffolding molecule binds to and regulates vasoactive intestinal polypeptide type-1 receptor in epithelial cells.
OBJECTIVE
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.
Gee
Heon Yung
HY
Department of Pharmacology and Brain Korea 21 Project for Medical Science, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea.
Kim
Young Woong
YW
Jo
Min Jae
MJ
Namkung
Wan
W
Kim
Joo Young
JY
Park
Hyun Woo
HW
Kim
Kyung Sik
KS
Kim
Hoguen
H
Baba
Akemichi
A
Yang
Jinhee
J
Kim
Eunjoon
E
Kim
Kyung Hwan
KH
Lee
Min Goo
MG
D048868:Adaptor Proteins, Signal Transducing; D000704:Analysis of Variance; D002352:Carrier Proteins; D002450:Cell Communication / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation; D051528:Guanylate Kinases; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D011336:Probability; D011485:Protein Binding / Q000235:genetics / Q000502:physiology*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; C495506:VIPR1 protein, human; D051528:Guanylate Kinases; C485777:MAGI2 protein, human