Interactive Fly, Drosophila

Carbohydrates are ligands for Neurexins

Neurexins are expressed in hundreds of isoforms on the neuronal cell surface, where they may function as cell recognition molecules. Neurexins contain LNS domains, folding units found in many proteins like the G domain of laminin A, agrin, and slit. LNS domains are found in the G domain of Laminin A, Neurexins, and serum proteins, such as Sex hormone binding globulins. alpha-neurexins contain three EGF-like repeats and six LNS domains in their extracellular region, while beta-neurexins contain only a single LNS domain that is identical with the sixth LNS domain of alpha-neurexins. The crystal structure of neurexin Ibeta, a single LNS domain, reveals two seven-stranded beta sheets forming a jelly roll fold with unexpected structural similarity to lectins. The LNS domains of neurexin and agrin undergo alternative splicing that modulates their affinity for protein ligands in a neuron-specific manner. These splice sites are localized within loops at one edge of the jelly roll, suggesting a distinct protein interaction surface in LNS domains that is regulated by alternative splicing (Rudenko, 1999).

A large number of ligands for the LNS domains in the G domain of alpha-laminins has been reported, including heparin, sulfatides, integrins, dystroglycan, nidogen, and fibulin, but the specificity of most of these interactions is unknown. In neurexins, the functions of two LNS domains have been defined via their ligands neurexophilins, alpha-latrotoxin, and neuroligins, and binding is tightly regulated by alternative splicing. The structure of the LNS domain is very similar to lectin(-like) proteins, in particular, serum amyloid protein, S-lectin, and glucanase, all shown to bind sugars. In agrin and alpha-laminins, LNS domains bind to heparin and other glycosaminoglycan components of the extracellular matrix. The LNS domains in neurexins, however, are not known to bind carbohydrates. The structural homology between neurexin Ibeta and lectins raises the possibility that LNS domains may have a general function as carbohydrate-binding modules, and that protein:carbohydrate interactions might contribute to neurexin cell-adhesive properties at neuronal junctions. While it remains to be investigated whether LNS domains in neurexins do indeed bind sugars, the interactions of these domains with protein ligands are well characterized. There is no question that at least some of these domains are involved in high-affinity protein:protein interactions. For example, neurexin Ibeta binds alpha-latrotoxin with picomolar affinity. This interaction is not mediated by sugars because alpha-latrotoxin is not glycosylated (Rudenko, 1999).

Neurexophilins are secreted ligands for neurexins

A novel 29 kDa protein, neurexophilin, has been purified in a complex with neurexin l alpha. Cloning reveals that rat and bovine neurexophilins are composed of N-terminal signal peptides, nonconserved N-terminal domains (20% identity over 80 residues), and highly homologous C-terminal sequences (85% identity over 169 residues). There are two distinct neurexophilin genes in mice, one of which is more homologous to rat neurexophilin and the other to bovine neurexophilin. The first neurexophilin gene is expressed abundantly in adult rat and mouse brain, whereas no mRNA corresponding to the second gene is detected in rodents, despite its abundant expression in bovine brain, suggesting that rodents and cattle primarily express distinct neurexophilin genes. Neurexophilin is expressed in adult rat brain at high levels only in a scattered subpopulation of neurons that probably represent inhibitory interneurons; in contrast, neurexins are expressed in all neurons. Neurexophilin contains a signal sequence and is N-glycosylated at multiple sites, suggesting that it is secreted and binds to the extracellular domain of neurexin l alpha. This hypothesis has been confirmed by binding recombinant neurexophilin to the extracellular domains of neurexin l alpha. Together these data suggest that neurexophilin constitutes a secreted glycoprotein that is synthesized in a subclass of neurons and may be a ligand for neurexins (Petrenko, 1996).

Neurexophilin was discovered as a neuronal glycoprotein that is copurified with neurexin Ialpha during affinity chromatography on immobilized alpha-latrotoxin. An investigation has been carried out into how neurexophilin interacts with neurexins: whether neurexophilin is post-translationally processed by site-specific cleavage similar to neuropeptides, and whether related neuropeptide-like proteins are expressed in brain. Mammalian brains contain four genes for neurexophilins, whose products share a common structure composed of five domains: an N-terminal signal peptide, a variable N-terminal domain, a highly conserved central domain that is N-glycosylated, a short linker region, and a conserved C-terminal domain that is cysteine-rich. When expressed in pheochromocytoma (PC12) cells with a replication-deficient adenovirus, neurexophilin 1 is rapidly N-glycosylated and then slowly processed to a smaller mature form, probably by endoproteolytic cleavage. Similar expression experiments in other neuron-like cells and in fibroblastic cells revealed that N-glycosylation of neurexophilin 1 occurs in all cell types tested, whereas proteolytic processing is observed only in neuron-like cells. Finally, only recombinant neurexin Ialpha and IIIalpha but not neurexin Ibeta interact with neurexophilin 1 and are preferentially bound to the processed mature form of neurexophilin. Together these data demonstrate that neurexophilins form a family of related glycoproteins that are proteolytically processed after synthesis and bind to alpha-neurexins. The structure and characteristics of neurexophilins indicate that they function as neuropeptides that may signal via alpha-neurexins (Missler, 1998a).

alpha-Neurexins (Ialpha, IIalpha, and IIIalpha) are receptor-like proteins expressed in hundreds of isoforms on the neuronal cell surface. The extracellular domains of alpha-neurexins are composed of six LNS repeats, named after homologous sequences in the Laminin A G domain, Neurexins, and Sex hormone-binding globulin, with three interspersed epidermal growth factor-like domains. Purification of neurexin Ialpha reveals that it is tightly complexed to a secreted glycoprotein called neurexophilin 1. Neurexophilin 1 is a member of a family of at least four genes and resembles a neuropeptide, suggesting a function as an endogenous ligand for alpha-neurexins. Recombinant proteins and knockout mice have been used to investigate which isoforms and domains of different neurexins and neurexophilins interact with each other. Neurexophilins 1 and 3 but not 4 (neurexophilin 2 is not expressed in rodents) bind to a single individual LNS domain, the second overall LNS domain in all three alpha-neurexins. Although this domain is alternatively spliced, all splice variants bind, suggesting that alternative splicing does not regulate binding. Using homologous recombination to disrupt the neurexophilin 1 gene, mutant mice were generated that do not express detectable neurexophilin 1 mRNA. Mice lacking neurexophilin 1 are viable with no obvious morbidity or mortality. However, homozygous mutant mice exhibit male sterility, probably because homologous recombination resulted in the co-insertion into the neurexophilin gene of herpes simplex virus thymidine kinase, which is known to cause male sterility. In the neurexophilin 1 knockout mice, neurexin Ialpha is complexed with neurexophilin 3 but not neurexophilin 4, suggesting that neurexophilin 1 is redundant with neurexophilin 3 and that neurexophilins 1 and 3 but not 4 bind to neurexins. This hypothesis was confirmed using expression experiments. These data reveal that the six LNS and three epidermal growth factor domains of neurexins are independently folding ligand-binding domains that may interact with distinct targets. The results support the notion that neurexophilins represent a family of extracellular signaling molecules that interact with multiple receptors, including all three alpha-neurexins (Missler, 1998b).

Protein 4.1, the vertebrate homolog of Drosophila Coracle, interacts with Neurexin

Protein 4.1 (P4.1) is a multifunctional protein with heterogeneity in molecular weight, intracellular localization, tissue- and development-specific expression patterns. Mouse protein 4.1 gene, over 90 kilobases long, comprises at least 23 exons (13 constitutive exons, 10 alternative exons) interrupted by 22 introns. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. No significant sequence difference has been observed between splice junctions of alternative and constitutive exons. Apparently, most alternative exon-encoded peptides are located within particular functional domains of the P4.1 protein: two peptides encoded by alternative exons 4 and 5 are located near or within the glycophorin/calmodulin binding domain, whereas three other alternative exon-encoded peptides (19-amino acid encoded by exon 14, 14-amino acid encoded by exon 15, and 21-amino acid encoded by exon 16) are located near or within the spectrin-actin binding domain. Selective use of exon 2', which carries an upstream translation initiation codon (AUG), may produce an elongated P4.1 isoform (135 kDa) that is predominantly expressed in nonerythroid tissues. Combinatorial splicing of these exons may generate different isoforms that will exhibit complicated tissue-specific expression patterns (Huang, 1993).

Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the spectrin-actin binding domain of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette do not function in these assays. The 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 suggest different, yet to be defined functions (Discher, 1993).

Mints are adaptors that directly bind to neurexins and recruit of munc18

Mint1 (X11/human Lin-10) and Mint2 are neuronal adaptor proteins that bind to Munc18-1 (n/rb-sec1), a protein essential for synaptic vesicle exocytosis. Mint1 has previously been characterized in a complex with CASK, another adaptor protein which in turn interacts with neurexins. Neurexins are neuron-specific cell surface proteins that act as receptors for the excitatory neurotoxin alpha-latrotoxin. Hence, one possible function for Mint1 is to mediate the recruitment of Munc18 to neurexins. In agreement with this hypothesis, it has been shown that the cytoplasmic tail of neurexins captures Munc18 via a multiprotein complex that involves Mint1. Furthermore, both Mint1 and Mint2 can directly bind to neurexins in a PDZ domain-mediated interaction. Various Mint and/or CASK-containing complexes can be assembled on neurexins, and Mint1 can bind to Munc18 and CASK simultaneously. These data support a model whereby one of the functions of Mints is to localize the vesicle fusion protein Munc18 to those sites at the plasma membrane that are defined by neurexins, presumably in the vicinity of points of exocytosis (Biederer, 2000).

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. Alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, have now been identified as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. These data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells (Sudhof, 2001).

Protein 4.1 interaction with Glycophorin, a protein that shares a domain with Neurexin

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. Synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila Discs-large tumor suppressor protein (Marfatia, 1995)

The major attachment site for protein 4.1 on the human erythrocyte is glycophorin (GP) C/D. Purified protein 4.1 can bind to two distinct sites on glycophorin C/D. One of these interactions is direct, involving residues 82-98 on glycophorin C (61-77 on glycophorin D), while the other interaction is mediated by p55. The binding site for p55 on glycophorin C is localized to residues 112-128 (glycophorin D91-107). Band 3 is an additional and minor binding site for Protein 4-1. The binding sites for band 3, glycophorin C/D, and p55 are all located within the 30-kDa domain of protein 4.1. The relative utilization of the three sites in normal membranes comprises 40% to p55, 40% to GPC/D, and 20% to band 3. The same region of protein 4.1 binds GPC/D and band 3, while the p55 binding site is distinct. The interactions involving protein 4.1 with p55 and p55 with GPC/D are of high affinity (nM), while those involving GPC/D and band 3 are 100-fold lower (microM). These results suggest that the most significant interactions between protein 4.1 and the membrane are those involving p55 (Hemming, 1995).

Contactin, a protein that interacts with Neurexins

Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. Contactin belongs to the Ig superfamily and is expressed on the cell surface of neurons. A novel contactin-associated transmembrane receptor (p190/Caspr) has been cloned. Caspr contains a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, as well as a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs and unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains in signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development. The sequence and domain structure of Caspr show a close similarity to Drosophila Neurexin IV; it is suggested that these two proteins could recruit PDZ or SH3 domains containing signaling molecules to specific regions of cell-cell contacts thereby regulating intracellular events in the nervous system and in other tissues. Indeed, the intracellular domain of Drosophila NrxIV has a binding site for PDZ domain-containing proteins and is required for the localization of Coracle protein to pleated septate junctions (Peles, 1997a and b).

In myelinated fibers of the vertebrate nervous system, glial-ensheathing cells interact with axons at specialized adhesive junctions, the paranodal septate-like junctions. The axonal proteins paranodin/Caspr and contactin (see Drosophila Contactin) form a cis complex in the axolemma at the axoglial adhesion zone, and both are required to stabilize the junction. There has been intense speculation that an oligodendroglial isoform of the cell adhesion molecule neurofascin, NF155, expressed at the paranodal loop might be the glial receptor for the paranodin/Caspr-contactin complex, particularly since paranodin/Caspr and NF155 colocalize to ectopic sites in the CNS of the dysmyelinated mouse Shiverer mutant. The extracellular domain of NF155 binds specifically to transfected cells expressing the paranodin/Caspr-contactin complex at the cell surface. This region of NF155 also binds the paranodin/Caspr-contactin complex from brain lysates in vitro. In support of the functional significance of this interaction, NF155 antibodies and the extracellular domain of NF155 inhibit myelination in myelinating cocultures, presumably by blocking the adhesive relationship between the axon and glial cell. These results demonstrate that the paranodin/Caspr-contactin complex interacts biochemically with NF155 and that this interaction is likely to be biologically relevant at the axoglial junction (Charles, 2002).

Carbohydrate binding function of Discoidin, homologous to the extracellular domain of Drosophila Neurexin

One of the common characteristics observed in different families of sugar-binding proteins is the presence of aromatic residues in proximity to the functional sugar-binding site. This general property has made these proteins a very appropriate subject for studies using intrinsic fluorescence assays. The galactose binding of the lectin discoidin I has been estimated, with an affinity constant of 1.8.10(-7) M-1 in the absence of calcium. In the presence of 1 mM Ca2+, the Kd of galactose binding is lowered to 2.7.10(-8) M-1. Calcium binding, by itself, seems to occur as two components with Kd values of 10(-7) and 10(-6) M-1. From these data, and sequence comparison of discoidin I with other lectins, a general model for ligand binding has been proposed in which a sequence from position 176 to 188, together with another region close to an apolar tryptophan residue, most probably Trp-50, would participate in the calcium- and sugar-binding site(s) of this protein (Valencia, 1989).

Disruption of axo-glial junctions causes cytoskeletal disorganization and degeneration of Purkinje neuron axons

Axo-glial junctions (AGJs) play a critical role in the organization and maintenance of molecular domains in myelinated axons. Neurexin IV/Caspr1/paranodin (NCP1) is an important player in the formation of AGJs because it recruits a paranodal complex implicated in the tethering of glial proteins to the axonal membrane and cytoskeleton. Mice deficient in either the axonal protein NCP1 or the glial ceramide galactosyltransferase (CGT) display disruptions in AGJs and severe ataxia. In this article, these two phenotypes were correlated and it was shown that both NCP1 and CGT mutants develop large swellings accompanied by cytoskeletal disorganization and degeneration in the axons of cerebellar Purkinje neurons. alphaII spectrin was found to be part of the paranodal complex and that, although not properly targeted, this complex is still formed in CGT mutants. Together, these findings establish a physiologically relevant link between AGJs and axonal cytoskeleton and raise the possibility that some neurodegenerative disorders arise from disruption of the AGJs (Garcia-Fresco, 2006; full text of article).

back to Neurexin Evolutionary homologs part 1/3 | part 2/3

The Interactive Fly resides on the
Society for Developmental Biology's Web server.