Neurexophilin binding to α-neurexins -: A single LNS domain functions as an independently folding ligand-binding unit

alpha-Neurexins (I alpha, II alpha, and III alpha) 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 I alpha 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 I alpha 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.