? a-Latrotoxin is a presynaptic neurotoxin that stimulates massive synaptic vesicle exocytosis. a-latrotoxin acts by binding to high-affinity neuronal cell-surface receptors that are thought to represent specific markers of presynaptic neurotransmitter release sites. Our previous studies identified three a- latrotoxin receptors that belong to different signaling pathways: The CIRLs are G protein-coupled receptors, neurexins are cell adhesion-like proteins with a single transmembrane domain and short cytoplasmic tail, and PTP6 is a receptor-like protein tyrosine phosphatase. To understand how a-latrotoxin receptors couple synaptic adhesion to neuronal exocytosis, we propose to identify receptor-interacting proteins and to study the mechanisms of their interactions. Our experimental approach is based on the use of a unique tool, a-latrotoxin, that binds the receptors with high affinity, thus allowing the one-stage isolation of the receptors together with associated proteins. We propose two sets of experiments. The first one is to identify less abundant protein components present in the preparations of affinity-purified a-latrotoxin receptors, and to analyze the specificity of their interaction with the receptors. This will be achieved by modern proteomics techniques complemented by immunoprecipitation with receptor-specific and associated protein-specific antibodies. As a key negative control, receptor preparations obtained from brains of knockout mice lacking a-latrotoxin receptors will be used. The second set of experiments is to study the mechanism of the interaction of individual a-latrotoxin receptors with synaptotagmin and syntaxin, proteins, involved in exocytosis that bind to a-latrotoxin receptors. In particular, we will identify to which receptors these proteins bind, and test if this interaction is direct. We will also determine the interacting domains of the receptors and their associated proteins. Obtaining a better biochemical definition of the a-latrotoxin receptor complexes is important because it promises to provide insight into the signaling mechanisms involved in their function, and may provide insight into presynaptic cell adhesion. This research will be performed primarily in Russia at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in collaboration with Alexander Petrenko as an extension of NIH grant R37 MH5280. ? ?

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW007210-03
Application #
7282030
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Program Officer
Michels, Kathleen M
Project Start
2005-09-15
Project End
2008-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
3
Fiscal Year
2007
Total Cost
$38,231
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Genetics
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Popova, Nadezhda V; Deyev, Igor E; Petrenko, Alexander G (2011) Association of adaptor protein TRIP8b with clathrin. J Neurochem 118:988-98
Deyev, Igor E; Sohet, Fabien; Vassilenko, Konstantin P et al. (2011) Insulin receptor-related receptor as an extracellular alkali sensor. Cell Metab 13:679-89
Serova, Oxana V; Popova, Nadezhda V; Petrenko, Alexander G et al. (2010) Association of the subunits of the calcium-independent receptor of ?-latrotoxin. Biochem Biophys Res Commun 402:658-62
Deyev, Igor E; Petrenko, Alexander G (2010) Regulation of CIRL-1 proteolysis and trafficking. Biochimie 92:418-22
Krasnoperov, Valery; Deyev, Igor E; Serova, Oxana V et al. (2009) Dissociation of the subunits of the calcium-independent receptor of alpha-latrotoxin as a result of two-step proteolysis. Biochemistry 48:3230-8
Serova, O V; Deyev, I E; Petrenko, A G (2009) Novel GPS-containing G protein-coupled receptor from Monosiga brevicollis. Dokl Biochem Biophys 427:191-4
Popova, N V; Plotnikov, A N; Ziganshin, R Kh et al. (2008) Analysis of proteins interacting with TRIP8b adapter. Biochemistry (Mosc) 73:644-51
Popova, N V; Plotnikov, A; Deev, I E et al. (2007) Interaction of calcium-independent latrotoxin receptor with intracellular adapter protein TRIP8b. Dokl Biochem Biophys 414:149-51