ISOLATION AND BIOCHEMICAL-CHARACTERIZATION OF A CA2-INDEPENDENT ALPHA-LATROTOXIN-BINDING PROTEIN()

alpha-Latrotoxin, a black widow spider neurotoxin, can bind to high af finity receptors on the presynaptic plasma membrane and stimulate mass ive neurotransmitter release in the absence of Ca2+. Neurexins, previo usly isolated as Lu-latrotoxin receptors, require Ca2+ for their inter action with the toxin and, thus, may not participate in the Ca2+-indep endent cu-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 i n EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatograph y on immobilized alpha-latrotoxin and concentrated on a wheat germ agg lutinin affinity column. The single polypeptide chain of latrophilin i s N-glycosylated and has an apparent molecular weight of 120,000. Sucr ose gradient centrifugations demonstrated that latrophilin and alpha-l atrotoxin form a stable equimolar complex, In the presence of the toxi n, anti-alpha-latrotoxin antibodies precipitated iodinated latrophilin , whose binding to immobilized toxin was characterized by a dissociati on constant of 0.5-0.7 nM. This presumably membrane-bound protein is l ocalized to and differentially distributed among neuronal tissues, wit h about four times more latrophilin expressed in the cerebral cortex t han in the cerebellum; subcellular fractionation showed that the prote in is highly enriched in synaptosomal plasma membranes, Our data sugge st that latrophilin may represent the Ca2+-independent receptor and/or molecular target for alpha-latrotoxin.