alpha-Latrotoxin is a potent neurotoxin from black widow spider venom, stimulating neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. High affinity alpha-latrotoxin binding proteins contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms, belonging to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, it is shown that recombinant neurexin I alpha binds alpha-Latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). These data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin (Davletov, 1995).

Alpha-latrotoxin is a potent neurotoxin that triggers synaptic exocytosis. Two distinct neuronal receptors for alpha-latrotoxin have been described: CIRL/latrophilin 1 (CL1) and neurexin-1alpha. Alpha-latrotoxin is thought to trigger exocytosis by binding to CL1, while the role of neurexin 1alpha is uncertain. Using PC12 cells, neurexins are demonstrated to indeed function as alpha-latrotoxin receptors that are at least as potent as CL1. Both alpha- and beta-neurexins represent autonomous alpha-latrotoxin receptors that are regulated by alternative splicing. Similar to CL1, truncated neurexins without intracellular sequences are fully active; therefore, neurexins and CL1 recruit alpha-latrotoxin but are not themselves involved in exocytosis. Thus, alpha-latrotoxin is unique among neurotoxins, because it utilizes two unrelated receptors, probably to amplify recruitment of alpha-latrotoxin to active sites (Sugita, 1999).

The potential role of contactin and contactin-associated protein (Caspr) has been examined in the axonal-glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homolog of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy has demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia (Einheber, 1997).

alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. Recombinant alpha-latrotoxin (LtxWT) has been produced that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. Three alpha-latrotoxin mutants were generated with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants are unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibits receptor-binding affinities identical to wild-type LtxWT; binds to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulates synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocks release triggered by LtxWT, whereas Cd2+ enhances it. Both cations, however, have no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. These data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+ (Ichtchenko, 1998).

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description of two distinct cell surface proteins that bind alpha-latrotoxin with nanomolar affinities: Neurexin I alpha binds alpha-latrotoxin in a Ca(2+)-dependent manner, and CIRL/latrophilin binds in a Ca(2+)-independent manner. Mice that lack neurexin I alpha were generated and analyzed to test the importance of neurexin I alpha in alpha-latrotoxin action. alpha-Latrotoxin binding to brain membranes from mutant mice is decreased by almost 50% compared with wild type membranes; the decrease is almost entirely due to a loss of Ca(2+)-dependent alpha-latrotoxin binding sites. In cultured hippocampal neurons, alpha-latrotoxin is still capable of activating neurotransmission in the absence of neurexin I alpha. Direct measurements of [3H]glutamate release from synaptosomes, however, shows a major decrease in the amount of release triggered by alpha-latrotoxin in the presence of Ca2+. Thus neurexin I alpha is not essential for alpha-latrotoxin action but contributes to alpha-latrotoxin action when Ca2+ is present. Viewed as a whole, these results show that mice contain two distinct types of alpha-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of alpha-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin I alpha pathway contributes to the Ca(2+)-dependent action of alpha-latrotoxin (Geppert, 1998).

Neuroligin1 is a neuronal cell surface protein. It is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds only to beta-neurexins that lack an insert in the alternatively spliced sequence of the G domain; put another way, if the beta-neurexins have an insert, Neuroligin will not bind. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. Alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. A model proposed for this regulation suggests that the alternative splicing of neurexins creates a family of cell surface receptors that confer interactive specificity onto adjacent resident neurons (Ichtchenko, 1995).

Neuroligin 1 is a neuronal cell surface protein that binds to a subset of neurexins, polymorphic cell surface proteins that are also localized on neurons. Two novel neuroligins, neuroligins 2 and 3, are similar in structure and sequence to neuroligin 1. All neuroligins contain an N-terminal hydrophobic sequence with several characteristics in common: a cleaved signal peptide followed by a large esterase homology domain, a highly conserved single transmembrane region, and a short cytoplasmic domain. The three neuroligins are alternatively spliced at the same position and expressed at high levels only in the brain. Binding studies demonstrate that all three neuroligins bind to beta-neurexins both as native brain proteins and as recombinant proteins. Tight binding of the three neuroligins to beta-neurexins is observed only for beta-neurexins lacking an insert in splice site 4. Thus, neuroligins constitute a multigene family of brain-specific proteins with distinct isoforms that may have overlapping functions in mediating recognition processes between neurons (Ichtchenko, 1996).

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 binds to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins (Irie, 1997).

beta-Neurexins and neuroligins are plasma membrane proteins that are displayed on the neuronal cell surface. The interaction of neurexin 1beta with neuroligin 1 was examined to evaluate their potential to function as heterophilic cell adhesion molecules. Using detergent-solubilized neuroligins and secreted neurexin 1beta-IgG fusion protein, the binding of these proteins to each other was observed only in the presence of Ca2+ and in no other divalent cation tested. Only neurexin 1beta, lacking an insert in splice site 4, binds neuroligins, whereas neurexin 1beta containing an insert is inactive. Half-maximal binding requires 1-3 microM free Ca2+, which probably acts by binding to neuroligin 1 but not to neurexin 1beta. To determine if neurexin 1beta and neuroligin 1 can also interact with each other when present in a native membrane environment on the cell surface, transfected cell lines expressing neuroligin 1 and neurexin 1beta were generated. Upon mixing different cell populations, cells are found to aggregate only if cells expressing neurexin 1beta are mixed with cells expressing neuroligin 1. Aggregation is dependent on Ca2+ and is inhibited by the addition of soluble neurexin 1beta lacking an insert in splice site 4 but not by the addition of neurexin 1beta containing an insert in splice site 4. It is concluded that neurexin 1beta and neuroligin 1 (and, by extension, other beta-neurexins and neuroligins) function as heterophilic cell adhesion molecules in a Ca2+-dependent reaction that is regulated by alternative splicing of beta-neurexins (Nguyen, 1997).

At the synapse, presynaptic membranes specialized for vesicular traffic are linked to postsynaptic membranes specialized for signal transduction. The mechanisms that connect pre- and postsynaptic membranes into synaptic junctions are unknown. Neuroligins and beta-neurexins are neuronal cell-surface proteins that bind to each other and form asymmetric intercellular junctions. To test whether the neuroligin/beta-neurexin junction is related to synapses, monoclonal antibodies to neuroligin 1 were generated and characterized. With these antibodies, neuroligin 1 has been shown to be synaptic. The neuronal localization, subcellular distribution, and developmental expression of neuroligin 1 are all similar to those of the postsynaptic marker proteins PSD-95 and NMDA-R1 receptor. Quantitative immunogold electron microscopy demonstrates that neuroligin 1 is clustered in synaptic clefts and postsynaptic densities. Double immunofluorescence labeling reveals that neuroligin 1 colocalizes with glutamatergic but not gamma-aminobutyric acid (GABA)ergic synapses. Thus neuroligin 1 is a synaptic cell-adhesion molecule that is enriched in postsynaptic densities where it may recruit receptors, channels, and signal-transduction molecules to synaptic sites of cell adhesion. In addition, the neuroligin/beta-neurexin junction may be involved in the specification of excitatory synapses (Song, 1999).

Accumulated evidence attributes one or more noncatalytic morphogenic activities to acetylcholinesterase (AChE). Despite sequence homologies, functional overlaps between AChE and catalytically inactive AChE-like cell surface adhesion proteins have been demonstrated only for the Drosophila protein Neurotactin. Furthermore, no mechanism had been proposed to enable signal transduction by AChE, an extracellular enzyme. Impaired neurite outgrowth and loss of neurexin Ialpha mRNA has been demonstrated under antisense suppression of AChE in PC12 cells (AS-ACHE cells). Neurite growth is partially rescued by the addition of recombinant AChE to the solid substrate or by transfection with various catalytically active and inactive AChE variants. Moreover, overexpression of the homologous neurexin I ligand, neuroligin-1, restores both neurite extension and expression of neurexin Ialpha. Differential PCR display reveals expression of a novel gene, nitzin, in AS-ACHE cells. Nitzin displays 42% homology to the band 4.1 protein superfamily capable of linking integral membrane proteins to the cytoskeleton. Nitzin mRNA is high throughout the developing nervous system; is partially colocalized with AChE, and increases in rescued AS-ACHE cells. These findings demonstrate redundant neurite growth-promoting activities for AChE and neuroligin and implicate interactions of AChE-like proteins and neurexins as potential mediators of cytoarchitectural changes supporting neuritogenesis (Grifman, 1998).

Most neurons form synapses exclusively with other neurons, but little is known about the molecular mechanisms mediating synaptogenesis in the central nervous system. Using an in vitro system, it has been demonstrated that neuroligin-1 and -2, both of which are postsynaptically localized proteins, can trigger the de novo formation of presynaptic structure. Nonneuronal cells engineered to express neuroligins induce morphological and functional presynaptic differentiation in contacting axons. This activity can be inhibited by addition of a soluble version of beta-neurexin, a receptor for neuroligin. Furthermore, addition of soluble beta-neurexin to a coculture of defined pre- and post-synaptic CNS neurons inhibits synaptic vesicle clustering in axons contacting target neurons. These results suggest that neuroligins are part of the machinery employed during the formation and remodeling of CNS synapses (Scheiffele, 2000).

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic beta-neurexins, but not with presynaptic alpha-neurexins. Unexpectedly, neuroligins were also found to bind alpha-neurexins and that alpha- and beta-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines alpha-neurexin binding but leaves beta-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only beta-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both alpha- and beta-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to alpha- and beta-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code (Boucard, 2005).

Formation of synapses requires specific cellular interactions that organize pre- and postsynaptic compartments. The neuroligin-neurexin complex mediates heterophilic adhesion and can trigger assembly of glutamatergic and GABAergic synapses in cultured hippocampal neurons. Both neuroligins and neurexins are encoded by multiple genes. Alternative splicing generates large numbers of isoforms, which may engage in selective axo-dendritic interactions. This study explored whether alternative splicing of the postsynaptic neuroligins modifies their activity toward glutamatergic and GABAergic axons. It was found that small extracellular splice insertions restrict the function of neuroligin-1 and -2 to glutamatergic and GABAergic contacts and alter interaction with presynaptic neurexins. The neuroligin isoforms associated with GABAergic contacts bind to neurexin-1alpha and a subset of neurexin-1betas. In turn, these neurexin isoforms induce GABAergic but not glutamatergic postsynaptic differentiation. These findings suggest that alternative splicing plays a central role in regulating selective extracellular interactions through the neuroligin-neurexin complex at glutamatergic and GABAergic synapses (Chih, 2006).

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. In cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits (Chubykin, 2007).

The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a β-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an α/β-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism (Fabrichny, 2007).

Neurexins and neuroligins provide trans-synaptic connectivity by the Ca²⁺-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. This study presents the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1β. Neuroligin-1 forms a constitutive dimer, and two neurexin-1β monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1β complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca²⁺. Alternatively spliced sites in neurexin-1β and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1β, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1β complex. These results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function (AraÁ, 2007).