Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

Abstract

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01203-13.

ACKNOWLEDGMENTS

We thank Bob White and Theo Kantidakis for help setting up the PLA, Frances Wason, Ewan Ramsay, Theo Laftsoglou, Luke Chamberlain, Lindsay Carpp, and Fiona Brandie for technical assistance and providing plasmids, and Chris MacDonald for critical reading of the manuscript.

This work was supported by a studentship from the BBRSC to D.K. and a project grant from Diabetes UK (11/0004289) to G.W.G. and N.J.B. N.J.B. is a Prize Fellow of the Lister Institute of Preventive Medicine.