A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis

Angela K. Nevins, Debbie C. Thurmond

Research output: Contribution to journalArticle

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Abstract

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-GTPγS 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.

Original languageEnglish
Pages (from-to)1944-1952
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number3
DOIs
StatePublished - Jan 21 2005

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Vesicle-Associated Membrane Protein 2
SNARE Proteins
Exocytosis
Insulin
Syntaxin 1
Glucose
Cell membranes
Cell Membrane
R-SNARE Proteins
rho GTP-Binding Proteins
GTP Phosphohydrolases
Insulin-Secreting Cells
Fractionation
Guanosine Triphosphate
Actins
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis. / Nevins, Angela K.; Thurmond, Debbie C.

In: Journal of Biological Chemistry, Vol. 280, No. 3, 21.01.2005, p. 1944-1952.

Research output: Contribution to journalArticle

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