We have recently isolated a novel cytomatrix at the active zone (CAZ)–associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of ∼120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules and comprise a family of four members. At the mossy fiber terminals of hippocampus, nectin-1 and nectin-3 localize at the presynaptic and postsynaptic sides of synaptic junctions, respectively, and their trans-interactions play a role in formation of synapses in cooperation with N-cadherin. Nectins are associated with the actin cytoskeleton through afadin, a nectin- and actin-filament-binding protein. Five nectin-like molecules (Necls) which have domain structures similar to those of nectins have been identified and here we characterize Necl-1/TSLL1/SynCAM3, from now on referred to as Necl-1. Tissue distribution analysis showed that Necl-1 was specifically expressed in the neural tissue. Immunofluorescence and immunoelectron microscopy revealed that Necl-1 localized at the contact sites among axons, their terminals, and glia cell processes that cooperatively formed synapses, axon bundles and myelinated axons. Necl-1 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 from now on referred to as Necl-2, nectin-1 and nectin-3, but not with Necl-5 or nectin-2. The C-terminal cytoplasmic region of Necl-1 did not bind afadin but bound membrane-associated guanylate kinase subfamily members that contain the L27 domain, including Dlg3, Pals2 and CASK. These results indicate that Necl-1 is a neural-tissue-specific Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which potentially has membrane-associated guanylate kinase subfamily member-binding activity and localizes at the non-junctional cell-cell contact sites.

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

The cytomatrix at the active zone (CAZ) is thought to define the site of Ca 2+ -dependent exocytosis of neurotransmitters. We have recently identified a novel CAZ protein from rat brain which we have named CAST (CAZ-associated structural protein). CAST forms a large molecular complex with other CAZ proteins such as Bassoon, RIM1 and Munc13-1, at least through direct binding to RIM1. Here, we have identified a rat protein that is structurally related to CAST and named it CAST2. Subcellular fractionation analysis of rat brain shows that CAST2 is also tightly associated with the postsynaptic density fraction. Like CAST, CAST2 directly binds RIM1 and forms a heterooligomer with CAST. In primary cultured rat hippocampal neurones, CAST2 co-localizes with Bassoon at synapses. Furthermore, immunoelectron microscopy reveals that CAST2 localizes to the vicinity of the presynaptic membrane of synapses in mouse brain. Sequence analysis reveals that CAST2 is a rat orthologue of the human protein ELKS. ELKS has also recently been identified as Rab6IP2 and ERC1. Accordingly, the original CAST is tentatively re-named CAST1. These results indicate that CAST2 is a new component of the CAZ and, together with CAST1, may be involved in the formation of the CAZ structure.