(Received for publication, July 6, 1995; and in revised form, August 21, 1995)
From the
-Latrotoxin is a potent neurotoxin from black widow spider
venom that stimulates neurotransmitter release.
-Latrotoxin is
thought to act by binding to a high affinity receptor on presynaptic
nerve terminals. In previous studies, high affinity
-latrotoxin
binding proteins were isolated and demonstrated to contain neurexin
I
as a major component. Neurexin I
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 I
binds
-latrotoxin directly with high affinity (K
4 nM). Binding of
-latrotoxin to recombinant neurexin I
is dependent on
Ca
(EC
30 µM). Our
data suggest that neurexin I
is a Ca
-dependent
high affinity receptor for
-latrotoxin.
-Latrotoxin, a component of black widow spider venom, is
one of the most potent excitatory neurotoxins known.
-Latrotoxin
stimulates neurotransmitter release from vertebrate nerve terminals by
triggering massive exocytosis of small synaptic
vesicles(1, 2) .
-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
-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) .
-Latrotoxin binds to specific membrane receptors that are found
only in the nervous system(8, 9) . Immunofluorescence
localization of bound
-latrotoxin at the neuromuscular junction
suggested that the binding sites are localized to the presynaptic
plasma membrane(10) . Together, these studies suggest that
-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
-latrotoxin in brain membranes are of low abundance (
0.3
nmol/g of protein) and of high affinity (
10
M). Affinity purification of
-latrotoxin-binding
proteins from brain resulted in the isolation of a protein fraction
that bound
-latrotoxin with high affinity (11) and
contained two classes of proteins (12, 13, 14) : (
)a family of high
molecular mass proteins (180-220 kDa) that were shown by
molecular cloning to be composed of variants of neurexin I
, and a
distinct low molecular mass protein (29 kDa) named neurexophilin. The
purification of these proteins suggested that they represent components
of the
-latrotoxin receptor. However, it was impossible to define
the exact binding partner because direct binding of recombinant
proteins to
-latrotoxin was not achieved.
Neurexin I and
its isoforms, II
and III
, structurally resemble cell surface
proteins(13, 15) . The neurexins are highly
polymorphic due to extensive alternative splicing(16) . Each
neurexin gene not only generates
-neurexins but also
-neurexins that have a distinct N terminus but share the
C-terminal sequences with
-neurexin(13, 17) . The
discovery of neurexin I
as a component of the protein complex that
binds
-latrotoxin with high affinity raised the question of
whether neurexin I
represents an
-latrotoxin receptor or is
only purified indirectly. We have now studied the interaction of
-latrotoxin with recombinant neurexins and determined the
requirements for high affinity binding. Our data demonstrate that
neurexin I
represents a high affinity,
Ca
-dependent cell surface binding molecule for
-latrotoxin.
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 I; fusions of neurexin
I
containing or lacking an insert in splice site 4; and a fusion
protein containing only the signal sequence and 18 amino acids of
neurexin I
(control IgG).
We constructed a series of fusion proteins of bovine neurexin
I with IgG in order to take advantage of the recent cloning of a
large number of independent neurexin I
cDNAs(16) . The
neurexin I
IgG fusion proteins are depicted schematically in Fig. 1together with the other IgG fusion proteins used for the
current study. Incubation of neurexin I
-IgG fusion protein
immobilized on protein A-Sepharose with purified
-latrotoxin
demonstrated stoichiometric and specific binding of
-latrotoxin
only in the presence of Ca
(Fig. 2, lanes
1-4). Binding was reversible since
-latrotoxin that was
bound to neurexin I
-IgG in the presence of Ca
could be readily dissociated by EGTA (Fig. 2, lane
6). Thus,
-latrotoxin binds to the extracellular domains of
recombinant neurexin I
in a Ca
-dependent manner.
Figure 2:
Ca-dependent binding of
-latrotoxin to neurexin I
-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 I
-IgG expression vector pCMVIGbN1
-1 (lanes 2, 4, and 6) was incubated with 5
µg of purified
-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 I 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 I
-IgG fusion proteins
containing a variety of inserts in the first three splice sites of
-neurexins bound
-latrotoxin, whereas the recombinant
proteins corresponding to neurexin III
and the previously studied
rat neurexin I
did not (lanes 1-8 versus
13-16, Fig. 3). Furthermore, C-terminal truncations
of cDNAs that bound
-latrotoxin as full-length protein abolished
binding (lanes 9-12), and the two splice variants of
neurexin I
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
I
proteins with different splice site variants bind
-latrotoxin. Both N-terminal
-specific sequences of neurexin
I
and its C-terminal half are required for binding.
Figure 3:
Binding of -latrotoxin to different
- and
-neurexin IgG fusion proteins. Eight
-neurexin IgG (lanes 1-16) and two
-neurexin IgG fusion proteins (lanes 17-20) immobilized on protein A-Sepharose were
incubated with purified
-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
-latrotoxin (bottom panel). The
different constructs used for production of IgG fusion proteins were: lanes 1 and 2, pCMVIGbNI
-2; lanes 3 and 4, pCMVIGbNI
-3; lanes 5 and 6,
pCMVIGbNI
-4; lanes 7 and 8, pCMVIGbNI
-7; lanes 9 and 10, pCMVIGbNI
-15; lanes 11 and 12, pCMVIGbNI
-17; lanes 13 and 14, pCMVIGbNIII
-2; lanes 15 and 16,
pCMVIGNI
-1; lanes 17 and 18, pCMVIGbNI
-1; lanes 19 and 20, pCMVIGbNI
-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 I
constructs express much better than the
constructs, no immunoglobulin heavy chain (IgG-HC) is
detected in the lanes with neurexin I
-IgG constructs because fewer
protein A-beads were used.
The nearly
stoichiometric binding of -latrotoxin to neurexin I
suggests
a stable interaction of high affinity. To test this, the binding of
radiolabeled
-latrotoxin to recombinant neurexin I
was
measured (Fig. 4). A binding affinity of approximately 4 nM was determined, suggesting that neurexin I
is indeed a high
affinity
-latrotoxin-binding protein. The affinity of recombinant
neurexin I
was compared with that of the high affinity binding
proteins that were purified by affinity chromatography on immobilized
-latrotoxin(11, 12, 14) . Recombinant
neurexin I
had an almost identical affinity as the purified
protein, confirming that the
-latrotoxin binding observed in the
purified receptor corresponds to neurexin I
(Fig. 4).
Figure 4:
Affinity of -latrotoxin for neurexin
I
-IgG. Immobilized neurexin I
-IgG (squares, IG-Nx) or
-latrotoxin binding proteins purified by
affinity chromatography on
-latrotoxin (diamonds, LTR) (11, 12, 14) were incubated
with radiolabeled
-latrotoxin at the indicated concentrations. The
amount of bound and free
-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 -latrotoxin
binding to neurexin I
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
35 µM and with a single apparent
binding site. This result suggests that
-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
-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
-latrotoxin binding to neurexin I
. Protein A-beads coated
with neurexin I
-IgG (closed circles) or with neurexin
I
-IgG (open circles) were incubated with radiolabeled
-latrotoxin in the presence of 2 mM Mg
and the indicated Ca
concentrations. Binding of
-latrotoxin was analyzed by SDS-PAGE and Coomassie staining (shown
in the top panel for neurexin I
-IgG) and quantified by
determining the radioactivity of the bound
-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 I as the
-latrotoxin receptor. This
receptor is interesting because binding to it may mediate the ability
of
-latrotoxin to trigger massive neurotransmitter release.
Synaptotagmin, a nerve terminal Ca
sensor(21) , co-purifies with this receptor on an
-latrotoxin column, suggesting a possible role of the
-latrotoxin receptor in regulating synaptic vesicle fusion with
the plasma membrane(22) . The current study demonstrates that
the extracellular domains of neurexin I
bind
-latrotoxin with
high affinity in a Ca
-dependent manner. This binding
is specific since it was observed with only a subset of neurexin
I
-IgG fusion proteins and not with control proteins or other IgG
fusion proteins.
The affinity of the interaction between neurexin
I and
-latrotoxin agrees well with the
-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
-latrotoxin receptor purified by affinity
chromatography also requires Ca
for binding,
-latrotoxin binding to brain membranes is decreased but not
abolished in the absence of Ca
(23) .
Furthermore,
-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
-latrotoxin exists that is
distinct from neurexin I
and binds the toxin in the absence of
Ca
. Since Scatchard plots of
-latrotoxin binding
demonstrated only a single class of binding sites, any putative
receptor would have to bind
-latrotoxin with the same affinity as
neurexin I
. Alternatively, a neurexin isoform may exist that does
not require Ca
for
-latrotoxin binding. Future
experiments will have to address these possibilities.