Mol Pharmacol

Mol Pharmacol. Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of NBD-556 Syt isoforms, suggests that the secretory machinery contains a vast DLEU2 repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly. INTRODUCTION Synaptotagmins (Syts) are a large family of vesicle proteins implicated in neurotransmitter release from neural and neuroendocrine tissues. All Syt isoforms are characterized by an amino-terminal intravesicular domain, a single transmembrane domain, and a large cytoplasmic region containing two homologous repeats termed C2 domains (C2A and C2B). A role for NBD-556 the C2 domains of Syt I in modulating neurotransmitter release is well established (OConnor DH5 or JM109, all fusion proteins were purified from bacterial lysates by glutathioneCagarose chromatography (Guan and Dixon, 1991 ). Oligomerization of Syt I and IV Syt I (amino acids 95C421) and Syt IV (amino acids 54C425) were in vitro translated using the TnT-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturers instructions. Five microliters of in vitroCtranslated Syt I or Syt IV were incubated with 10 g of GST-Syt IV C2A, GST-Syt IV C2B, GST-Syt I C2B, or GST alone in binding buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1% nonfat dry-milk, and 0.05% NBD-556 Tween 20) supplemented with 1 mM CaCl2 or 2 mM EGTA for 1 h at 4C. The samples were washed three times in binding buffer, solubilized in 1% SDS, passed over a Spin-30 column (310 laser scanning microscope (Wayne State University, School of Medicine). RESULTS The Specificity of SytCStx Interactions Differs for Syt I and Syt IV Syt I has been postulated to function in exocytosis through a mechanism involving interactions with the plasma membrane protein Stx 1a through the C2A domain (Sdhof and Rizo, 1996 ). To determine whether Syt IV is also capable of interacting with native Stx 1a, recombinant Syt IV C2A was immobilized on glutathioneCagarose and incubated with detergent-solubilized rat brain synaptosomes in the absence and presence of calcium. Recombinant proteinCsynaptosome complexes were detected by Western blot analysis using the anti-Stx 1a antibody HPC-1. As shown in Figure ?Figure1A,1A, the Syt IV C2A domain binds Stx 1a in the absence and presence of calcium. In contrast, the Syt I C2A domain bound Stx 1a in a calcium-dependent manner, consistent with previous studies (Chapman Syt I C2B domain abolish the formation of Syt I dimers, thereby decreasing the calcium responsiveness of the secretory machinery (Littleton synaptotagmin mutants. Proc Natl Acad Sci USA. 1994;91:10888C10892. [PMC free article] [PubMed] [Google Scholar]Mikoshiba K, Fukuda M, Moreira JE, Lewis FMT, Sugimora M, Niinobe M, Llins R. Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal. Proc Natl Acad Sci USA. 1995;92:10703C10707. [PMC free article] [PubMed] [Google Scholar]Niinobe M, Yamaguchi Y, Fukuda M, Mikoshiba K. Synaptotagmin is an inositol polyphosphate binding protein: isolation and characterization as an Ins 1,3,4,5-P4 binding protein. Biochem Biophys Res Commun. 1994;205:1036C1042. [PubMed] [Google Scholar]OConnor V, Augustine GJ, Betz H. Synaptic vesicle exocytosis: molecules and models. Cell. 1994;76:785C787. [PubMed] [Google Scholar]Ohara-Imaizumi M, Fukuda M, Niinobe M, Misonou H, Ikeda K, Murakami T, Kawasaki M, Mikoshiba K, Kumakura K. Distinct roles of C2A and C2B domains of synaptotagmin in the regulation of exocytosis in adrenal chromaffin cells. Proc Natl Acad Sci USA. 1997;94:287C291. [PMC free article] [PubMed] [Google Scholar]Perin MS, Fried VA, Mignery GA, Jahn R, Sdhof TC. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 1990;345:260C263. [PubMed] [Google Scholar]Plant PJ, Yeger H, Staub O, Howard P, Rotin D. The C2 domain of the ubiquitin protein ligase Nedd4 mediates Ca2+-dependent plasma membrane localization. J Biol Chem. 1997;272:32329C32336. [PubMed] [Google Scholar]Rosenthal L, Zacchetti D, Madeddu L, Meldolesi J. Mode of action of -latrotoxin: role.