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- Title
SNARE-Dependent Exocytosis Is Facilitated by Association of Munc18c with Doc2β.
- Authors
Ice, Ban; Thurmond, Debbie C.
- Abstract
Our laboratory has recently shown that stimulus-induced tyrosine-phosphorylation of Munc18c specifically at the plasma membrane induces dissociation of the Mune18c-Syntaxin 4 complex to facilitate increases in both insulin granule exocytosis from pancreatic beta cells as well as GLUT4 vesicle translocation in skeletal muscle and adipocytes. Since Munc18c is a soluble protein it was expected that dissociation from Syntaxin 4 would result in loss of Mnnc18c from the plasma membrane compartment, however this was not the case. In this study we have determined that Munc18c released from Syntaxin 4 can bind to a newly identified Munc18c binding protein Doc2β, in a 'switch mechanism'. Doc2β belongs to double C2 domain protein family and has previously been characterized as a neuronal protein which interacts with Munc13 and Munc18a (nSec 1). Immunoblot analyses showed that Doc2β is expressed in islet beta cells as well as in 3T3L1 adipocytes, and is principally localized to the plasma membrane in each cell type. Specifically in the plasma membrane fraction, Doc2β was found to associate with Munc18c. GST-interaction assays and immunoprecipitation analyses further revealed that the Munc18eDoc21β complex associated in a manner mutually exclusive of Syntaxin 4, and in vitro binding assays showed the Munc18c-Doc2β interaction to be direct. Deletion analyses of each protein revealed that this direct interaction was mediated by residues 173-255 of Munc18c, and the second C2 domain of Doc2β. Expression of the 173-255 residue region of Munc18c resulted in inhibition of glucose-stimulated insulin secretion from MIN6 beta cells, indicating a functional importance for the Munc18c-Doc2β interaction. Consistent with the notion that this complex plays a positive role in vesicle exocytosis, increased expression of Doc2β enhanced glucose-stimulated insulin secretion by ∼40%, while siRNA-mediated depletion of Dec2β attenuated insulin release. Moreover, increased expression of Doc2β coordinately increased glucose-induced SNARE complex formation by 30%, and could also restore secretion to cells with inhibited secretion. Taken together, these data support a model wherein Munc18c switches from association with Syntaxin 4 upon stimulation to association with Doc2L3 at the plasma membrane to facilitate exocytosis.
- Subjects
EXOCYTOSIS; MEMBRANE proteins; CARRIER proteins; PANCREATIC beta cells; FAT cells
- Publication
Diabetes, 2007, Vol 56, pA4
- ISSN
0012-1797
- Publication type
Article