The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium.
The Ca2+- and phospholipid-binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+-affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 microM cytosolic free Ca2+. The well-characterized synaptotagmin isoforms I and II are present in pancreatic beta-cell lines RINm5F, INS-1 and HIT-T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin-containing secretory granules whereas only minor amounts were found on synaptic vesicle-like microvesicles. Antibodies or Fab-fragments directed against the Ca2+-dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not GTPgammaS-induced exocytosis from streptolysin-O-permeabilized INS-1 and HIT-T15 cells. Transient expression of wild-type synaptotagmin II did not alter exocytosis in HIT-T15 cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C2 domain (Delta180-183, D231S) again inhibited only Ca2+-, but not GTPgammaS-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C2 domain (Delta325-341; K327,328, 332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Delta180-183/K327,328,332Q) induced greater inhibition than mutant Delta180-183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.
Lang
J
J
Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland. Jochen.Lang@medecine.hcuge.ch
Fukuda
M
M
Zhang
H
H
Mikoshiba
K
K
Wollheim
C B
CB
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D007328:Insulin; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D050863:Synaptotagmin I; D050857:Synaptotagmins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium