Expression and localisation of synaptotagmin isoforms in endocrine beta-cells: their function in insulin exocytosis.
Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic beta-cells and derived cell lines is mainly controlled by Ca(2+). Several lines of evidence have demonstrated a role of the Ca(2+)- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca(2+) determined by their two C(2) domains (C(2)A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca(2+) sensors in LDCV exocytosis, syt III was not expressed in primary beta-cells, whereas syt IV was only found adjacent to the TGN. However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary beta-cells the introduction of recombinant peptides (100 nM) corresponding to the C(2) domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca(2+)-evoked insulin exocytosis by 30% without altering GTP gamma S-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary beta-cells whereas V, VII and VIII may mediate Ca(2+)-regulation of exocytosis.
Gut
A
A
Division de Biochimie Clinique, Département de Médecine, Université de Genève, CH-1211 Genève 4, Switzerland.
Kiraly
C E
CE
Fukuda
M
M
Mikoshiba
K
K
Wollheim
C B
CB
Lang
J
J
D000818:Animals; D002135:Calcium-Binding Proteins; D002460:Cell Line; D005089:Exocytosis; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D007328:Insulin; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D050857:Synaptotagmins