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J. Biol. Chem., Vol. 280, Issue 3, 1944-1952, January 21, 2005

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A Direct Interaction between Cdc42 and Vesicle-associated Membrane Protein 2 Regulates SNARE-dependent Insulin Exocytosis^*

Angela K. Nevins and Debbie C. Thurmond

From the Department of Biochemistry and Molecular Biology and the Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.

Received for publication, August 18, 2004 , and in revised form, October 18, 2004.

^* This work was supported by a predoctoral fellowship from National Institutes of Health Diabetes Training Grant T32 DK064466-01 (to A. K. N.) and by American Diabetes Association Career Development Award 1-03-CD-10 (to D. C. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 317-274-1551; Fax: 317-274-4686; E-mail: dthurmon{at}iupui.edu.

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