©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
High Affinity Binding of -Latrotoxin to Recombinant Neurexin I (*)

(Received for publication, July 6, 1995; and in revised form, August 21, 1995)

Bazbek A. Davletov (1)(§) Valery Krasnoperov (2) Yutaka Hata (1)(¶) Alexander G. Petrenko (2) Thomas C. Südhof (1)(**)

From the  (1)Department of Molecular Genetics and Howard Hughes Medical Institute, The University of Texas Southwestern Medical School, Dallas, Texas 75235 and the (2)Department of Environmental Medicine, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity alpha-latrotoxin binding proteins were isolated and demonstrated to contain neurexin Ialpha as a major component. Neurexin Ialpha is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin Ialpha binds alpha-latrotoxin directly with high affinity (K approx 4 nM). Binding of alpha-latrotoxin to recombinant neurexin Ialpha is dependent on Ca (EC approx 30 µM). Our data suggest that neurexin Ialpha is a Ca-dependent high affinity receptor for alpha-latrotoxin.


INTRODUCTION

alpha-Latrotoxin, a component of black widow spider venom, is one of the most potent excitatory neurotoxins known. alpha-Latrotoxin stimulates neurotransmitter release from vertebrate nerve terminals by triggering massive exocytosis of small synaptic vesicles(1, 2) . alpha-Latrotoxin-stimulated neurotransmitter release is accompanied by presynaptic membrane depolarization and the influx of Ca through ion channels induced by the toxin(3, 4) . Purified alpha-latrotoxin forms cation channels in black lipid membranes, leading to the hypothesis that the toxin may act as an ionophore although the channel characteristics differ from those observed in intoxicated PC12 cells(5, 6, 7) . alpha-Latrotoxin binds to specific membrane receptors that are found only in the nervous system(8, 9) . Immunofluorescence localization of bound alpha-latrotoxin at the neuromuscular junction suggested that the binding sites are localized to the presynaptic plasma membrane(10) . Together, these studies suggest that alpha-latrotoxin acts by binding to presynaptic receptors, which it either activates directly or which serves to target its insertion into the presynaptic plasma membrane.

The binding sites for alpha-latrotoxin in brain membranes are of low abundance (approx0.3 nmol/g of protein) and of high affinity (approx10M). Affinity purification of alpha-latrotoxin-binding proteins from brain resulted in the isolation of a protein fraction that bound alpha-latrotoxin with high affinity (11) and contained two classes of proteins (12, 13, 14) : (^1)a family of high molecular mass proteins (180-220 kDa) that were shown by molecular cloning to be composed of variants of neurexin Ialpha, and a distinct low molecular mass protein (29 kDa) named neurexophilin. The purification of these proteins suggested that they represent components of the alpha-latrotoxin receptor. However, it was impossible to define the exact binding partner because direct binding of recombinant proteins to alpha-latrotoxin was not achieved.

Neurexin Ialpha and its isoforms, IIalpha and IIIalpha, structurally resemble cell surface proteins(13, 15) . The neurexins are highly polymorphic due to extensive alternative splicing(16) . Each neurexin gene not only generates alpha-neurexins but also beta-neurexins that have a distinct N terminus but share the C-terminal sequences with alpha-neurexin(13, 17) . The discovery of neurexin Ialpha as a component of the protein complex that binds alpha-latrotoxin with high affinity raised the question of whether neurexin Ialpha represents an alpha-latrotoxin receptor or is only purified indirectly. We have now studied the interaction of alpha-latrotoxin with recombinant neurexins and determined the requirements for high affinity binding. Our data demonstrate that neurexin Ialpha represents a high affinity, Ca-dependent cell surface binding molecule for alpha-latrotoxin.


EXPERIMENTAL PROCEDURES

Construction and Transfection of Expression Vectors

Vectors directing expression of extracellular domains of neurexins fused to the Fc domain of human IgG were obtained by an adaptation of the method of Aruffo et al.(18) utilizing pCD5-IgG(1) as the starting vector as described (17, 19) and the rat and bovine neurexin cDNAs(13, 15, 16) . The vectors used in the current study encode the following residues and splice variants of neurexins (all numbers correspond to the numbering of the rat proteins in (13) and (15) ; splice variants are given in parentheses for splice sites 1 to 4 in the terminology of (16) ): pCMVIGbNIalpha-1 (B/C/A/B), -2 (B/B/A/B), -3 (E/B/A/B), -4 (G/C/A/B), and -7 (G/A/A/B) encode residues 1 to 1339 or bovine neurexin Ialpha and pCMVIGNIalpha-1 (G/C/A/A) of rat neurexin Ialpha; pCMVIGbNIalpha-15 and -17 encode residues 1-588 and 1-655 (splice variants for splice sites 1 and 2 for both: B/B); pCMVIGNIbeta-1 and -3 encode residues 1 to 300 from rat neurexin Ibeta without or with an insert in splice site 4, respectively; and pCMVIGNIIIalpha-2 encodes residues 1-1499 of rat neurexin IIIalpha with no insert in splice sites 1 and 5 and full inserts in 3 and 4. The proteins encoded by the vectors are depicted schematically in Fig. 1. Plasmid DNA was transfected into COS cells using DEAE-dextran(20) , and expressed proteins were purified from the medium as described(17, 19) . As controls, media from COS cells transfected with salmon sperm DNA or with control IgG vector were used.


Figure 1: Structures of the neurexin-IgG fusion proteins used in the current study. Four types of recombinant proteins containing C-terminally fused human IgG were produced for the current study: fusions of the extracellular domains of neurexins I and III containing the complete extracellular sequences up to the O-linked sugar domain with different splice site inserts at the numbered sites of alternative splicing; fusions of C-terminally truncated forms of neurexin Ialpha; fusions of neurexin Ialpha containing or lacking an insert in splice site 4; and a fusion protein containing only the signal sequence and 18 amino acids of neurexin Ialpha (control IgG).



alpha-Latrotoxin Binding to Recombinant Neurexins

alpha-Latrotoxin was purified from the glands of Latrodectus mactans as described(12) . Activity was assayed using release of labeled noradrenaline from synaptosomes. For some experiments, alpha-latrotoxin was iodinated using chloramine T(12) . alpha-Latrotoxin was incubated in buffer A (50 mM Tris-HCl, pH 7.7, 2 g/liter bovine serum albumin, and 150 mM NaCl) containing the indicated concentrations of EGTA, Ca, and Mg with protein A-Sepharose beads to which the recombinant proteins had been attached. Incubations were for 15 min under vigorous shaking at room temperature. Beads were then washed with buffer A containing the additions described in the figure legends, and bound proteins were analyzed by SDS-PAGE (^2)followed by Coomassie Blue staining or immunoblotting and/or determination of radioactivity. For determinations of binding affinities, aliquots of the COS cell medium were spotted onto nitrocellulose, and binding of I-labeled alpha-latrotoxin to the immobilized neurexins was analyzed as described(12) .

Miscellaneous Procedures

SDS-PAGE, Coomassie staining, and immunoblotting were performed as described(14) . Immunoreactive bands were detected by enhanced chemiluminescence (Amersham). The antibody against alpha-latrotoxin (X751) was raised against purified protein in rabbits.


RESULTS AND DISCUSSION

We constructed a series of fusion proteins of bovine neurexin Ialpha with IgG in order to take advantage of the recent cloning of a large number of independent neurexin Ialpha cDNAs(16) . The neurexin Ialpha IgG fusion proteins are depicted schematically in Fig. 1together with the other IgG fusion proteins used for the current study. Incubation of neurexin Ialpha-IgG fusion protein immobilized on protein A-Sepharose with purified alpha-latrotoxin demonstrated stoichiometric and specific binding of alpha-latrotoxin only in the presence of Ca (Fig. 2, lanes 1-4). Binding was reversible since alpha-latrotoxin that was bound to neurexin Ialpha-IgG in the presence of Ca could be readily dissociated by EGTA (Fig. 2, lane 6). Thus, alpha-latrotoxin binds to the extracellular domains of recombinant neurexin Ialpha in a Ca-dependent manner.


Figure 2: Ca-dependent binding of alpha-latrotoxin to neurexin Ialpha-IgG fusion protein. Protein A-Sepharose preincubated with medium from COS cells transfected with control DNA (lanes 1, 3, and 5) or with the bovine neurexin Ialpha-IgG expression vector pCMVIGbN1alpha-1 (lanes 2, 4, and 6) was incubated with 5 µg of purified alpha-latrotoxin in the presence of 10 mM EGTA (lanes 1 and 2) or 1 mM Ca (lanes 3-6). Beads were washed with 50 mM Tris, pH 7.7, 1 M NaCl in the presence of either 1 mM Ca (lanes 1-4) or 10 mM EGTA (lanes 5 and 6). Bound proteins were analyzed by SDS-PAGE and Coomassie Blue staining. Bovine serum albumin (BSA) was present in all buffers to block nonspecific binding. Immunoglobulin G (IgG) was bound to the protein A from the serum used for cell culture.



Previous studies using recombinant rat neurexin Ialpha were unsuccessful in detecting binding. Therefore, we studied the potential dependence of binding on splice variants by analyzing a series of independent cDNAs. Four different neurexin Ialpha-IgG fusion proteins containing a variety of inserts in the first three splice sites of alpha-neurexins bound alpha-latrotoxin, whereas the recombinant proteins corresponding to neurexin IIIalpha and the previously studied rat neurexin Ialpha did not (lanes 1-8 versus 13-16, Fig. 3). Furthermore, C-terminal truncations of cDNAs that bound alpha-latrotoxin as full-length protein abolished binding (lanes 9-12), and the two splice variants of neurexin Ibeta were also unable to bind (lanes 17-20, Fig. 3; see Fig. 1for an overview of the structures of the neurexin-IgG fusion proteins). Thus, several recombinant neurexin Ialpha proteins with different splice site variants bind alpha-latrotoxin. Both N-terminal alpha-specific sequences of neurexin Ialpha and its C-terminal half are required for binding.


Figure 3: Binding of alpha-latrotoxin to different alpha- and beta-neurexin IgG fusion proteins. Eight alpha-neurexin IgG (lanes 1-16) and two beta-neurexin IgG fusion proteins (lanes 17-20) immobilized on protein A-Sepharose were incubated with purified alpha-latrotoxin in the presence of 10 mM EGTA or 1 mM Ca. Bound proteins were analyzed by SDS-PAGE and Coomassie Blue staining (top panel) or immunoblotting for alpha-latrotoxin (bottom panel). The different constructs used for production of IgG fusion proteins were: lanes 1 and 2, pCMVIGbNIalpha-2; lanes 3 and 4, pCMVIGbNIalpha-3; lanes 5 and 6, pCMVIGbNIalpha-4; lanes 7 and 8, pCMVIGbNIalpha-7; lanes 9 and 10, pCMVIGbNIalpha-15; lanes 11 and 12, pCMVIGbNIalpha-17; lanes 13 and 14, pCMVIGbNIIIalpha-2; lanes 15 and 16, pCMVIGNIalpha-1; lanes 17 and 18, pCMVIGbNIbeta-1; lanes 19 and 20, pCMVIGbNIbeta-3. Different neurexin IgG fusion constructs vary widely in expression levels, and experiments were normalized for the concentrations of the neurexin IgG fusion proteins. Since neurexin Ibeta constructs express much better than the alpha constructs, no immunoglobulin heavy chain (IgG-HC) is detected in the lanes with neurexin Ibeta-IgG constructs because fewer protein A-beads were used.



The nearly stoichiometric binding of alpha-latrotoxin to neurexin Ialpha suggests a stable interaction of high affinity. To test this, the binding of radiolabeled alpha-latrotoxin to recombinant neurexin Ialpha was measured (Fig. 4). A binding affinity of approximately 4 nM was determined, suggesting that neurexin Ialpha is indeed a high affinity alpha-latrotoxin-binding protein. The affinity of recombinant neurexin Ialpha was compared with that of the high affinity binding proteins that were purified by affinity chromatography on immobilized alpha-latrotoxin(11, 12, 14) . Recombinant neurexin Ialpha had an almost identical affinity as the purified protein, confirming that the alpha-latrotoxin binding observed in the purified receptor corresponds to neurexin Ialpha (Fig. 4).


Figure 4: Affinity of alpha-latrotoxin for neurexin Ialpha-IgG. Immobilized neurexin Ialpha-IgG (squares, IG-Nx) or alpha-latrotoxin binding proteins purified by affinity chromatography on alpha-latrotoxin (diamonds, LTR) (11, 12, 14) were incubated with radiolabeled alpha-latrotoxin at the indicated concentrations. The amount of bound and free alpha-latrotoxin was determined and analyzed in a Scatchard plot as shown. Note that the affinity of recombinant neurexin is virtually identical with that of the purified protein complex.



The experiment in Fig. 2suggested that alpha-latrotoxin binding to neurexin Ialpha may be Ca-dependent. To investigate this further, we studied the effect of different Ca concentrations on binding in the presence of a saturating concentration of Mg (Fig. 5). Mg alone was unable to trigger binding. Ca acted in a concentration-dependent manner with an EC of approx35 µM and with a single apparent binding site. This result suggests that alpha-latrotoxin and/or neurexins contain a structural Ca binding site which has to be occupied in order for the two proteins to interact. Since previous studies demonstrated that neuroligin 1, the ligand for beta-neurexins, also requires Ca for binding(19) , it is tempting to speculate that the extracellular domains of neurexins contain structural Ca binding sites that are required to keep the molecule in an active conformation.


Figure 5: Ca dependence of alpha-latrotoxin binding to neurexin Ialpha. Protein A-beads coated with neurexin Ialpha-IgG (closed circles) or with neurexin Ibeta-IgG (open circles) were incubated with radiolabeled alpha-latrotoxin in the presence of 2 mM Mg and the indicated Ca concentrations. Binding of alpha-latrotoxin was analyzed by SDS-PAGE and Coomassie staining (shown in the top panel for neurexin Ialpha-IgG) and quantified by determining the radioactivity of the bound alpha-latrotoxin (bottom panel). The curve drawn in the bottom panel was generated with the GraphInPlot program and corresponds to a single binding site with an EC of 35 µM and a Hill coefficient of 0.9.



The goal of the current study was to investigate the candidacy of neurexin Ialpha as the alpha-latrotoxin receptor. This receptor is interesting because binding to it may mediate the ability of alpha-latrotoxin to trigger massive neurotransmitter release. Synaptotagmin, a nerve terminal Ca sensor(21) , co-purifies with this receptor on an alpha-latrotoxin column, suggesting a possible role of the alpha-latrotoxin receptor in regulating synaptic vesicle fusion with the plasma membrane(22) . The current study demonstrates that the extracellular domains of neurexin Ialpha bind alpha-latrotoxin with high affinity in a Ca-dependent manner. This binding is specific since it was observed with only a subset of neurexin Ialpha-IgG fusion proteins and not with control proteins or other IgG fusion proteins.

The affinity of the interaction between neurexin Ialpha and alpha-latrotoxin agrees well with the alpha-latrotoxin concentrations required for toxic actions(1, 2, 3, 4) . However, the Ca dependence of the interaction is puzzling, even though the Ca concentration required for binding is low. Although the alpha-latrotoxin receptor purified by affinity chromatography also requires Ca for binding, alpha-latrotoxin binding to brain membranes is decreased but not abolished in the absence of Ca(23) . Furthermore, alpha-latrotoxin is capable of triggering neurotransmitter release in the absence of extracellular Ca if Mg is present. Thus, it is possible that a second high affinity binding protein for alpha-latrotoxin exists that is distinct from neurexin Ialpha and binds the toxin in the absence of Ca. Since Scatchard plots of alpha-latrotoxin binding demonstrated only a single class of binding sites, any putative receptor would have to bind alpha-latrotoxin with the same affinity as neurexin Ialpha. Alternatively, a neurexin isoform may exist that does not require Ca for alpha-latrotoxin binding. Future experiments will have to address these possibilities.


FOOTNOTES

*
This study was supported by a fellowship from the Human Frontiers Science Program (to Y. H.) and by a grant from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, Imperial College of Science, Technology and Medicine, London SW72AY, United Kingdom.

Present address: Research Development Corp., Kobe 651-22, Japan.

**
To whom correspondence should be addressed.

(^1)
A. G. Petrenko and T. C. Südhof, unpublished observation.

(^2)
The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We wish to thank I. Leznicki, E. Borowicz, H. Tripoli, and A. Roth for excellent technical support and Drs. D. W. Russell, B. Seed, J. Dixon, and K. Zinn for supplying us with reagents.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.