Activation of alpha-latrotoxin receptors in neuromuscular synapses leads to a prolonged splash acetylcholine release.

The mechanisms of acetylcholine release in presynaptic terminals of motoneurons induced by mutant alpha-latrotoxin (LT(N4C)) were analyzed. In contrast to wild-type alpha-latrotoxin that causes both continuous and splash secretion of acetylcholine and necessarity block neuromuscular transmission, LT(N4C) causes only splash release lasting over many hours. Thus, activation of alpha-latrotoxin receptors controls long-lasting enhanced secretion of acetylcholine.

Insecticidal toxins from black widow spider venom.

The biological effects of Latrodectus spider venom are similar in animals from different phyla, but these symptoms are caused by distinct phylum-specific neurotoxins (collectively called latrotoxins) with molecular masses ranging from 110 to 140 kDa. To date, the venom has been found to contain five insecticidal toxins, termed alpha, beta, gamma, delta and epsilon-latroinsectotoxins (LITs). There is also a vertebrate-specific neurotoxin, alpha-latrotoxin (alpha-LTX), and one toxin affecting crustaceans, alpha-latrocrustatoxin (alpha-LCT). These toxins stimulate massive release of neurotransmitters from nerve terminals and act (1) by binding to specific receptors, some of which mediate an exocytotic signal, and (2) by inserting themselves into the membrane and forming ion-permeable pores. Specific receptors for LITs have yet to be identified, but all three classes of vertebrate receptors known to bind alpha-LTX are also present in insects. All LTXs whose structures have been elucidated (alpha-LIT, delta-LIT, alpha-LTX and alpha-LCT) are highly homologous and have a similar domain architecture, which consists of a unique N-terminal sequence and a large domain composed of 13-22 ankyrin repeats. Three-dimensional (3D) structure analysis, so far done for alpha-LTX only, has revealed its dimeric nature and an ability to form symmetrical tetramers, a feature probably common to all LTXs. Only tetramers have been observed to insert into membranes and form pores. A preliminary 3D reconstruction of a delta-LIT monomer demonstrates the spatial similarity of this toxin to the monomer of alpha-LTX.

The multiple actions of black widow spider toxins and their selective use in neurosecretion studies.

The black widow spider venom contains several large protein toxins--latrotoxins--that are selectively targeted against different classes of animals: vertebrates, insects, and crustaceans. These toxins are synthesised as large precursors that undergo proteolytic processing and activation in the lumen of the venom gland. The mature latrotoxins demonstrate strong functional structure conservation and contain multiple ankyrin repeats, which mediate toxin oligomerisation. The three-dimensional structure has been determined for alpha-latrotoxin (alphaLTX), a representative venom component toxic to vertebrates. This reconstruction explains the mechanism of alphaLTX pore formation by showing that it forms tetrameric complexes, harbouring a central channel, and that it is able to insert into lipid membranes. All latrotoxins cause massive release of neurotransmitters from nerve terminals of respective animals after binding to specific neuronal receptors. A G protein-coupled receptor latrophilin and a single-transmembrane receptor neurexin have been identified as major high-affinity receptors for alphaLTX. Latrotoxins act by several Ca(2+)-dependent and -independent mechanisms based on pore formation and activation of receptors. Mutant recombinant alphaLTX that does not form pores has been used to dissect the multiple actions of this toxin. As a result, important insights have been gained into the receptor signalling and the role of intracellular Ca(2+) stores in the effect of alphaLTX.

alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles.

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.

alpha-latrotoxin forms calcium-permeable membrane pores via interactions with latrophilin or neurexin.

In order to explore the mechanisms by which alpha-latrotoxin activates neurotransmitter release, we have characterized its effects by patch-clamp methods on cells heterologously expressing its receptors, latrophilin-1 or neurexin-Ialpha. Application of alpha-latrotoxin (1 nM) to cells expressing rat latrophilin or neurexin, but not mock-transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, alpha-latrotoxin did not elicit currents in the absence of Ca2+. The toxin-induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single-channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of alpha-latrotoxin binding to latrophilin and neurexin, the pores induced by alpha-latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, alpha-latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.

Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

Tetramerisation of alpha-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca(2+)-dependent vesicular exocytosis from synaptosomes.

A novel procedure of alpha-latrotoxin (alpha LTX) purification has been developed. Pure alpha LTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca(2+), Mg(2+) or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified alpha LTX loses the ability to tetramerise spontaneously; the addition of Mg(2+) or Ca(2+) restores this ability. This suggests that alphaLTX has some intrinsically bound divalent cation(s) that normally support its tetramerisation. Single-particle cryoelectron microscopy and statistical image analysis have shown that: 1) the toxin has a non-compact, branching structure; 2) the alpha LTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 A-diameter channel in the centre. Both alpha LTX oligomers bind to the same receptors in synaptosomes and rat brain sections. To study the effects of the dimers and tetramers on norepinephrine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a preparation correlates with alpha LTX pore formation, suggesting that the tetramers are the pore-forming species of alpha LTX. The toxin actions mediated by the pore include: 1) Ca(2+) entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca(2+). The Ca(2+)-dependent alpha LTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca(2+) stores and functional phospholipase C (PLC). The Ca(2+)-dependent effect of the toxin is stronger when dimeric alpha LTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca(2+)-independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations.

Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores.

We report here the first three-dimensional structure of alpha-latrotoxin, a black widow spider neurotoxin, which forms membrane pores and stimulates secretion in the presence of divalent cations. We discovered that alpha-latrotoxin exists in two oligomeric forms: it is dimeric in EDTA but forms tetramers in the presence of Ca2+ or Mg2+. The dimer and tetramer structures were determined independently at 18 A and 14 A resolution, respectively, using cryo-electron microscopy and angular reconstitution. The alpha-latrotoxin monomer consists of three domains. The N- and C-terminal domains have been identified using antibodies and atomic fitting. The C4-symmetric tetramers represent the active form of alpha-latrotoxin; they have an axial channel and can insert into lipid bilayers with their hydrophobic base, providing the first model of alpha-latrotoxin pore formation.

Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C.

alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G-protein-coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha z, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca(2+)-dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2(+)-independent component of LTX-stimulated release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non-selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca2+ provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion.

The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution.

Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. We now report the finding of two novel latrophilin homologues. All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha-latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain-specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.

Functional expression of alpha-latrotoxin in baculovirus system.

To facilitate the study of the mechanism of alpha-latrotoxin action, it is necessary to create a biologically active recombinant toxin. Mature alpha-latrotoxin is naturally produced by post-translational cleavage, probably at two furin sites located at the N- and C-termini of the precursor. A recombinant baculovirus has now been constructed, which encodes the melittin signal peptide fused to the 130-kDa mature toxin between the furin sites. Insect cells, infected with this baculovirus, secreted recombinant alpha-latrotoxin. This was partially purified and proved indistinguishable from the natural toxin with respect to its molecular mass, immunostaining, toxicity to mice, binding to alpha-latrotoxin receptors (latrophilin or neurexin Ialpha) and electrophysiological recording in the mouse diaphragm. The successful expression of recombinant alpha-latrotoxin permits mutational analysis of the toxin.

Enhancement of spontaneous transmitter release at neonatal mouse neuromuscular junctions by the glial cell line-derived neurotrophic factor (GDNF).

1. The acute effects of neurotrophic factors on the frequency of spontaneous transmitter release (miniature endplate potentials (MEPPs)) from motor nerve terminals has been examined in skeletal muscles of neonatal mice aged between 9 and 20 days. The following factors were tested at a concentration of 50 ng ml-1: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neuronotrophic factor (CNTF), leukaemia inhibitory factor (LIF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), and glial cell line-derived neurotrophic factor (GDNF). In some experiments, the responses to 2 microM LaCl3 and 10 mM K+, or to 2-5 nM purified alpha-latrotoxin (alpha-LTX) were also measured. 2. Neither BDNF, NT-3, NT-4, LIF, IGF-1 or IGF-2 - singly or in combination - caused any significant change in MEPP frequency. GDNF, however, produced a highly significant, 2-fold increase in neurotransmitter release that was reproduced in fourteen muscles. 3. Potentiation of MEPP frequency in GDNF was of the same order as that induced by tetanic stimulation or substitution of the bathing medium with hypertonic saline; but substantially less than that induced either by lanthanum ions or alpha-latrotoxin. 4. The data suggest that concentrations of GDNF that produce maximal enhancement of motoneurone survival in vitro and in vivo also produce acute, non-saturating enhancement in transmitter release at immature mammalian neuromuscular synapses. Taken together with other reports, these findings suggest that GDNF may mediate both functional and structural plasticity of neonatal neuromuscular junctions.

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.

alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

Alpha-latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors.

alpha-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca2+-independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a Galphao subunit of heterotrimeric G proteins, because the two proteins co-purify in a two-step affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

Modification of rat brain Kv1.4 channel gating by association with accessory Kvbeta1.1 and beta2.1 subunits.

We have examined the effects of co-expression of Kvbeta1.1 and Kvbeta2.1 subunits on the gating of rat brain Kv1.4 channels, expressed in Xenopus oocytes. Expression of Kv1.4 subunits alone produced a rapidly inactivating "A" type current, which activated at potentials beyond -60 mV in a solution containing high levels of rubidium. Current activation curves obtained from tail current measurements were fitted with a Boltzmann function, with V1/2 = -47 mV and k = 10 mV. Neither the Kvbeta1.1 nor Kvbeta2.1 subunits altered the voltage dependence of activation. Both subunits accelerated the activation time constant of Kv1.4, without affecting its voltage dependence. Surprisingly, the Kvbeta2.1 subunit, which lacks an N-terminal inactivation domain, was almost as effective as the Kvbeta1.1 subunit in speeding up Kv1.4. Steady-state inactivation of Kv1.4 was unchanged upon co-expression with either Kvbeta1.1 or Kvbeta2.1 subunits. Kv1.4 recovered from inactivation with two time constants; apart from an approximately 50% lengthening of the slow time constant with a high Kvbeta2.1 injection ratio, neither time constant was altered by either the Kvbeta1.1 or Kvbeta2.1 subunits, suggesting little interaction with recovery from C-type inactivation. Clearly, beta subunits have the potential to modify the gating of Kv1.4 channels in the brain more subtly than has been suggested previously.

Isolation and biochemical characterization of a Ca2+-independent alpha-latrotoxin-binding protein.

alpha-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 alpha-latrotoxin receptors, require Ca2+ for their interaction with the toxin and, thus, may not participate in the Ca2+-independent alpha-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 in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized alpha-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 alpha-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-alpha-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 Ca2+-independent receptor and/or molecular target for alpha-latrotoxin.

Cartography of neurexins: more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons.

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. In situ hybridization revealed differential but overlapping distributions of neurexin isoforms in different classes of neurons. PCRs demonstrated that alpha-neurexins are alternatively spliced at five canonical positions, and beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells.

Conserved domain structure of beta-neurexins. Unusual cleaved signal sequences in receptor-like neuronal cell-surface proteins.

Neurexins, a family of neuronal cell-surface proteins, consist of the longer alpha-neurexins (I alpha, II alpha, and III alpha) and the shorter beta-neurexins (I beta and II beta) with identical C termini but distinct N termini. alpha-Neurexins have the structure of cell surface receptors, but the membrane topology and conservation of beta-neurexins is unknown. We have now characterized cDNA clones encoding bovine neurexins I beta and III beta, thereby demonstrating the presence of a beta-form for neurexin III and the evolutionary conservation of beta-neurexins in mammals. Similar to alpha-neurexins, beta-neurexins were found to be highly O-glycosylated after expression by transfection in COS cells, suggesting that alpha- and beta-neurexins utilize the same O-glycosylation cassette and have similar transmembrane orientations. To determine if beta-neurexins contain a cleaved or uncleaved signal sequence for membrane translocation, beta-neurexin-IgG fusion proteins were expressed in COS cells, and their N termini were directly sequenced. This revealed that the N terminus of all three beta-neurexins contains an unusual cleaved signal sequence. Together our data show that all known neurexin genes generate alpha and beta forms with similar transmembrane organizations and receptor-like structures. Due to the presence of a long atypical cleaved signal peptide, beta-neurexins contain only a short unique sequence before splicing into the alpha-neurexin sequence. Thus, beta-neurexins are essentially N terminally truncated alpha-neurexins.