title: Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet β-cells.
abstract: 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.
authors.affiliation: *Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
authors.forename: Latha
authors.initials: L
authors.lastname: Ramalingam
authors.affiliation: †Department of Pediatrics, Herman B Wells Center, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
authors.forename: Jingping
authors.initials: J
authors.lastname: Lu
authors.affiliation: *Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
authors.forename: Andy
authors.initials: A
authors.lastname: Hudmon
authors.forename: Debbie C
authors.initials: DC
authors.lastname: Thurmond
mesh_terms: D000818:Animals; D061265:Biphasic Insulins / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005089:Exocytosis; D005947:Glucose / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes; D051938:Munc18 Proteins / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism
chemical_list: D061265:Biphasic Insulins; D002135:Calcium-Binding Proteins; C490421:Doc2b protein, mouse; D046912:Multiprotein Complexes; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D005947:Glucose