Neurexophilin binding to alpha-neurexins. A single LNS domain functions as an independently folding ligand-binding unit.
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 revealed 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. We have now used recombinant proteins and knockout mice to investigate which isoforms and domains of different neurexins and neurexophilins interact with each other. We show that 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, we generated mutant mice 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. Our 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
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M
Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas, Texas 75235, USA.
Hammer
R E
RE
Südhof
T C
TC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D007248:Infertility, Male; D008024:Ligands; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism
D006023:Glycoproteins; D008024:Ligands; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D011993:Recombinant Fusion Proteins; C093175:neurexin II; C082354:neurexin IIIalpha; C112162:neurexophilin; D012697:Serine Endopeptidases; C026515:type I signal peptidase