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.
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.
Regazzi
R
R
Department of Medicine, University of Geneva, Switzerland.
Wollheim
C B
CB
Lang
J
J
Theler
J M
JM
Rossetto
O
O
Montecucco
C
C
Sadoul
K
K
Weller
U
U
Palmer
M
M
Thorens
B
B
D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology*; D002458:Cell Fractionation; D002460:Cell Line; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D050683:R-SNARE Proteins; D051381:Rats; D017422:Sequence Analysis, DNA; D013744:Tetanus Toxin / Q000494:pharmacology; D051040:Vesicle-Associated Membrane Protein 3
D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; D051040:Vesicle-Associated Membrane Protein 3; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001905:Botulinum Toxins; D002118:Calcium