A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis.
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-GTPgammaS 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.
Nevins
Angela K
AK
Department of Biochemistry and Molecular Biology and the Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Thurmond
Debbie C
DC
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005089:Exocytosis / Q000502:physiology*; D005947:Glucose / Q000008:administration & dosage; D007328:Insulin / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins / Q000502:physiology*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D007328:Insulin; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D020764:cdc42 GTP-Binding Protein; D005947:Glucose