Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.

Synaptotagmins (Syts) are brain-specific Ca2+/phospholipid-binding proteins. In hippocampal synapses, Syt I is essential for fast Ca(2+)-dependent synaptic vesicle exocytosis but not for Ca(2+)-independent exocytosis. In vertebrates and invertebrates, Syt may therefore participate in Ca(2+)-dependent synaptic membrane fusion, either by serving as the Ca2+ sensor in the last step of fast Ca(2+)-triggered neurotransmitter release, or by collaborating with an additional Ca2+ sensor. While Syt I binds Ca2+ (refs 10, 11), its phospholipid binding is triggered at lower calcium concentrations (EC50 = 3-6 microM) than those required for exocytosis. Furthermore, Syts bind clathrin-AP2 with high affinity, indicating that they may play a general role in endocytosis rather than being confined to a specialized function in regulated exocytosis. Here we resolve this apparent contradiction by describing four Syts, three of which (Syt VI, VII and VIII) are widely expressed in non-neural tissues. All Syts tested share a common domain structure, with a cytoplasmic region composed of two C2 domains that interacts with clathrin-AP2 (Kd = 0.1-1.0 nM) and with neural and non-neural syntaxins. The first C2 domains of Syt I, II, III, V and VII, but not of IV, VI or VIII, bind phospholipids with a similar Ca(2+)-concentration dependence (EC50 = 3-6 microM). The same C2 domains also bind syntaxin as a function of Ca2+ but the Ca(2+)-concentration dependence of Syt I, II and V (> 200 microM) differs from that of Syt III and VII (< 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. In situ hybridization revealed differential but overlapping distributions of neurexin isoforms in different classes of neurons. PCRs demonstrated that alpha-neurexins are alternatively spliced at five canonical positions, and beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells.

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Using affinity chromatography on immobilized alpha-latrotoxin, we have purified a novel 29 kDa protein, neurexophilin, in a complex with neurexin l alpha. Cloning revealed 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). Analysis of genomic clones from mice identified two distinct neurexophilin genes, 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 was detected in rodents despite its abundant expression in bovine brain, suggesting that rodents and cattle primarily express distinct neurexophilin genes. RNA blots and in situ hybridizations revealed that neurexophilin is expressed in adult rat brain at high levels only in a scattered subpopulation of neurons that probably represent inhibitory interneurons; by 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 was confirmed by binding recombinant neurexophilin to the extracellular domains of neurexin l alpha. Together our data suggest that neurexophilin constitutes a secreted glycoprotein that is synthesized in a subclass of neurons and may be a ligand for neurexins.

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