VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion

EMBO J. 1995 Jun 15;14(12):2723-30.

Abstract

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Botulinum Toxins / pharmacology
  • Brain Chemistry
  • Calcium / pharmacology*
  • Cell Fractionation
  • Cell Line
  • Cloning, Molecular
  • Cytoplasmic Granules / metabolism
  • Exocytosis / drug effects
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
  • Insulin / analysis
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / chemistry
  • Islets of Langerhans / metabolism*
  • Membrane Proteins / analysis
  • Membrane Proteins / biosynthesis*
  • Membrane Proteins / genetics
  • Nerve Tissue Proteins / analysis
  • Nerve Tissue Proteins / biosynthesis*
  • Nerve Tissue Proteins / genetics
  • R-SNARE Proteins
  • Rats
  • Sequence Analysis, DNA
  • Tetanus Toxin / pharmacology
  • Vesicle-Associated Membrane Protein 3

Substances

  • Insulin
  • Membrane Proteins
  • Nerve Tissue Proteins
  • R-SNARE Proteins
  • Tetanus Toxin
  • Vesicle-Associated Membrane Protein 3
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Botulinum Toxins
  • Calcium

Grant support