Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells involves soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for first- and second-phase GSIS respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate target (t-)SNAREs, Syntaxin 1A and Syntaxin 4 respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via its distinct C2A and C2B domains respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Towards this, macromolecular complexes composed of Munc18c, Doc2b and Munc18-1 were detected in β-cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST–Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly.
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December 2014
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Research Article|
November 14 2014
Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet β-cells
Latha Ramalingam;
Latha Ramalingam
*Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
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Jingping Lu;
Jingping Lu
1
†Department of Pediatrics, Herman B Wells Center, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
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Andy Hudmon;
Andy Hudmon
*Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
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Debbie C. Thurmond
Debbie C. Thurmond
2
*Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
†Department of Pediatrics, Herman B Wells Center, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
‡Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
2To whom correspondence should be addressed (email dthurmon@iu.edu).
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Publisher: Portland Press Ltd
Received:
July 03 2014
Revision Received:
August 27 2014
Accepted:
September 05 2014
Accepted Manuscript online:
September 05 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 464 (2): 251–258.
Article history
Received:
July 03 2014
Revision Received:
August 27 2014
Accepted:
September 05 2014
Accepted Manuscript online:
September 05 2014
Citation
Latha Ramalingam, Jingping Lu, Andy Hudmon, Debbie C. Thurmond; Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet β-cells. Biochem J 1 December 2014; 464 (2): 251–258. doi: https://doi.org/10.1042/BJ20140845
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