Isolation and Biochemical Characterization of a Ca2+-independent α-Latrotoxin-binding Protein

Abstract

␣-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 Ca 2؉. Neurexins, previously isolated as ␣-latrotoxin receptors, require Ca 2؉ for their interaction with the toxin and, thus, may not participate in the Ca 2؉ -independent ␣-latrotoxin activity. We now report the isolation of a novel protein that binds ␣-latrotoxin with high affinity in the presence of various divalent cations (Ca 2؉ , Mg 2؉ , Ba 2؉ , and Sr 2؉ ) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized ␣-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 ␣-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-␣-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 Ca 2؉ -independent receptor and/or molecular target for ␣-latrotoxin.␣-Latrotoxin (LTX), 1 a neurotoxin from the black widow spider venom, potently stimulates neurotransmitter release from all vertebrate synapses tested (1). The toxin causes a massive discharge of synaptic vesicles (2) by acting upon nerve terminals hypothetically in two stages. Initially, it binds to a high affinity presynaptic receptor and then forms nonselective cation-permeable channels in the plasma membrane (reviewed in Ref.3). The subsequent entry of Ca 2ϩ through these channels triggers fast neurotransmitter release (4). However, accumulating evidence suggests that the mode of LTX action is more complex.First, studies in neuronal and PC12 cells demonstrate that the toxin-receptor interaction does not require Ca 2ϩ , although the removal of Ca 2ϩ appreciably decreases the binding (5, 6). This result is best explained by the existence of two classes of LTX receptors, Ca 2ϩ -dependent and independent, both presumably active when Ca 2ϩ is present. These receptor types possess similar high affinities to LTX (7) but display different toxin binding properties (e.g. the Ca 2ϩ -independent binding is more sensitive to high salt) (8). Furthermore, in at least one PC12 cell line only the Ca 2ϩ -independent binding was detectable (7), indicating that the heterogeneous LTX receptors are probably differentially regulated and, thus, may also be structurally different. Ca 2ϩ is also not essential during the second p...

α-Latrotoxin Receptor, Latrophilin, Is a Novel Member of the Secretin Family of G Protein-coupled Receptors

“…In good agreement with our previous results on the tissue distribution of latrophilin (12), LPH1 mRNA has been found in the brain but not in non-neuronal tissues (Fig. 3A).…”

“…Moreover, LTX potently causes dopamine release in a PC12 cell line that completely lacks Ca 2ϩ -dependent receptors (6), thus ruling out the importance of neurexins in the toxin action. Recently, we purified latrophilin, a synaptic protein that binds LTX independently of Ca 2ϩ (12). The isolation of the same protein was later also described by others (13).…”

“…Latrophilin from rat brain was isolated by LTX affinity chromatography (12) and further purified by SDS-PAGE. The protein was digested in gel pieces with endoproteinases Lys-C or trypsin (Wako).…”

“…Latrophilin preparations enriched in heterotrimeric G proteins were made as published (12), except that 20 M Mg 2ϩ and 2 mM GDP were included during purification. Antibodies against G protein ␣-subunits and synaptotagmin were from Santa Cruz and Affiniti.…”

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells

“…For example, gliomedin is a type II transmembrane protein containing the olfactomedin domain in the carboxy-terminal extracellular region [10]. Latrophilins are calcium-independent, seven-transmembrane receptors for latrotoxin (CIRL1-CIRL3) with a large N-terminal extracellular part containing the olfactomedin domain [11][12][13][14][15].…”