Isolation and biochemical characterization of a Ca2+-independent alpha-latrotoxin-binding protein.
alpha-Latrotoxin, a black widow spider neurotoxin, can bind to high affinity receptors on the presynaptic plasma membrane and stimulate massive neurotransmitter release in the absence of Ca2+. Neurexins, previously isolated as alpha-latrotoxin receptors, require Ca2+ for their interaction with the toxin and, thus, may not participate in the Ca2+-independent alpha-latrotoxin activity. We now report the isolation of a novel protein that binds alpha-latrotoxin with high affinity in the presence of various divalent cations (Ca2+, Mg2+, Ba2+, and Sr2+) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized alpha-latrotoxin and concentrated on a wheat germ agglutinin affinity column. The single polypeptide chain of latrophilin is N-glycosylated and has an apparent molecular weight of 120,000. Sucrose gradient centrifugations demonstrated that latrophilin and alpha-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-alpha-latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 nM. This presumably membrane-bound protein is localized to and differentially distributed among neuronal tissues, with about four times more latrophilin expressed in the cerebral cortex than in the cerebellum; subcellular fractionation showed that the protein is highly enriched in synaptosomal plasma membranes. Our data suggest that latrophilin may represent the Ca2+-independent receptor and/or molecular target for alpha-latrotoxin.
Davletov
B A
BA
Department of Biochemistry, Imperial College, London, SW7 2AY, United Kingdom.
Shamotienko
O G
OG
Lelianova
V G
VG
Grishin
E V
EV
Ushkaryov
Y A
YA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D018000:Receptors, Peptide / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017421:Sequence Analysis; D013045:Species Specificity; D013111:Spider Venoms / Q000378:metabolism*; D013574:Synaptosomes / Q000737:chemistry; D014018:Tissue Distribution
D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; C020763:alpha-latrotoxin; D002118:Calcium