Doc2β is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis

Ban Ke, Eunjin Oh, Debbie C. Thurmond

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2β as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2β was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2β-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2β in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2β. The second C2 domain (C2B) of Doc2β and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2β complexes by addition of the Doc2β binding domain of Munc18c (residues 173-255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2β enhanced glucose-stimulated insulin secretion by ∼40%, whereas siRNA-mediated depletion of Doc2β attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2β at the plasma membrane to facilitate exocytosis.

Original languageEnglish
Pages (from-to)21786-21797
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number30
DOIs
StatePublished - Jul 27 2007

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Qa-SNARE Proteins
Exocytosis
Munc18 Proteins
Insulin
SNARE Proteins
Cell membranes
Islets of Langerhans
Adipocytes
Vesicle-Associated Membrane Protein 2
Cell Membrane
Association reactions
Glucose
Immunoprecipitation
Small Interfering RNA
Muscle
Assays
Carrier Proteins
Skeletal Muscle
Switches

ASJC Scopus subject areas

  • Biochemistry

Cite this

Doc2β is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis. / Ke, Ban; Oh, Eunjin; Thurmond, Debbie C.

In: Journal of Biological Chemistry, Vol. 282, No. 30, 27.07.2007, p. 21786-21797.

Research output: Contribution to journalArticle

Ke, Ban ; Oh, Eunjin ; Thurmond, Debbie C. / Doc2β is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 30. pp. 21786-21797.
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abstract = "The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2β as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2β was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2β-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2β in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2β. The second C2 domain (C2B) of Doc2β and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2β complexes by addition of the Doc2β binding domain of Munc18c (residues 173-255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2β enhanced glucose-stimulated insulin secretion by ∼40{\%}, whereas siRNA-mediated depletion of Doc2β attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2β at the plasma membrane to facilitate exocytosis.",
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