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

doi:10.1074/jbc.271.38.23239

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

From the Department of Biochemistry, Imperial College, London, SW7 2AY, United Kingdom and the
^‡Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117871, Russia

^§ To whom correspondence and reprint requests should be addressed. Tel.: 44-171-594-5237; Fax: 44-171-225-0960.

Abstract

α-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 α-latrotoxin receptors, require Ca²⁺ for their interaction with the toxin and, thus, may not participate in the Ca²⁺-independent α-latrotoxin activity. We now report the isolation of a novel protein that binds α-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 α-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 α-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-α-latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 n. 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 α-latrotoxin.

Footnotes

↵* This work was supported by the Wellcome Senior European Research Fellowship (to Y. A. U.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

LTX

α-latrotoxin

BSA

bovine serum albumin

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

ECL

enhanced chemiluminescence

PAGE

polyacrylamide gel electrophoresis

WGA

wheat germ agglutinin.
↵²Yu. Ushkaryov, unpublished observation (based on peptide sequencing and partial cloning).
↵³B. A. Davletov, O. G. Shamotienko, and Yu. A. Ushkaryov, manuscript in preparation.
- Received April 19, 1996.
- Revision received July 8, 1996.