Doc2beta is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis

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 Doc2beta as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2beta was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2beta-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2beta in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2beta. The second C2 domain (C2B) of Doc2beta and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2beta complexes by addition of the Doc2beta binding domain of Munc18c (residues 173-255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2beta enhanced glucose-stimulated insulin secretion by approximately 40%, whereas siRNA-mediated depletion of Doc2beta 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 Doc2beta at the plasma membrane to facilitate exocytosis.