Neurexophilins form a conserved family of neuropeptide-like glycoproteins.

Neurexophilin was discovered as a neuronal glycoprotein that is copurified with neurexin Ialpha during affinity chromatography on immobilized alpha-latrotoxin (Petrenko et al., 1996). We have now investigated how neurexophilin interacts with neurexins, whether it is post-translationally processed by site-specific cleavage similar to neuropeptides, and whether related neuropeptide-like proteins are expressed in brain. Our data show that mammalian brains contain four genes for neurexophilins the products of which 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 was 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 occurred in all cell types tested, whereas proteolytic processing was observed only in neuron-like cells. Finally, only recombinant neurexin Ialpha and IIIalpha but not neurexin Ibeta interacted with neurexophilin 1 and were preferentially bound to the processed mature form of neurexophilin. Together our 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.