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.
- insulin exocytosis
- islet β-cell
- soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein
Abbreviations: GSIS, glucose-stimulated insulin secretion; HRP, horseradish peroxidase; IIDP, Integrated Islet Distribution Program; Ni-NTA, Ni2+-nitrilotriacetate; NP40, Nonidet P40; PM, plasma membrane; SNAP, synaptosome-associated protein; SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor; T2D, Type 2 diabetes; t-SNARE, target SNARE; VAMP, vesicle-associated membrane protein; V-SNARE, vesicle/granule SNARE
- © The Authors Journal compilation © 2014 Biochemical Society