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¹ KAN Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8815, Japan
² Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan

	Abstract

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The cytomatrix at the active zone (CAZ) is thought to define the site of Ca²⁺-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 hetero-oligomer 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.

	Introduction

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Neurotransmitter release is precisely regulated at a specific site beneath the presynaptic membrane, the so-called active zone (Landis et al. 1988; Burns & Augustine 1995). The cytomatrix associated with the active zone (CAZ) may be involved in determining the site of synaptic vesicle fusion (Gotow et al. 1991; Südhof 1995; Dresbach et al. 2001). So far, several CAZ proteins have been isolated and characterized. Bassoon is a 420 kDa protein containing two N-terminal zinc fingers and three coiled-coil domains (tom Dieck et al. 1998). Piccolo/aczonin is structurally related to Bassoon, but it has additional PDZ and C2 domains (Wang et al. 1999; Fenster et al. 2000). Munc13-1 is a mammalian orthologue of C. elegans UNC-13 (Brose et al. 1995) and Munc13-1 contains one C1 and three C2 domains. Recent studies have revealed that Munc13-1 plays a crucial role in synaptic vesicle cycling (Betz et al. 1998; Augustin et al. 1999). RIM1, a small G protein Rab3A effector, is a 180 kDa protein containing two zinc fingers, one PDZ, and two C2 domains, and regulates Ca²⁺-dependent exocytosis of neurotransmitters in a Rab3A-dependent manner (Wang et al. 1997; Castillo et al. 2002; Schoch et al. 2002). These relatively large proteins with multiple domains appear to function as scaffold molecules at the CAZ, but the mechanistic functions for CAZ assembly are largely unknown. Recently, we have isolated and characterized another CAZ protein and named it CAST (Ohtsuka et al. 2002). CAST is a 120 kDa protein containing four coiled-coil domains and a C-terminal consensus motif for binding to PDZ domains. CAST directly binds RIM1 and indirectly binds Munc13-1 to form a ternary complex. Moreover, Bassoon is also associated with this ternary complex in vivo, providing the first evidence that CAZ proteins form a network of protein–protein interactions at the CAZ (Ohtsuka et al. 2002). In addition, CAST is, at least in part, associated with the same vesicles as those transporting Bassoon during synapse formation (Ohtsuka et al. 2002). Because Bassoon and Piccolo are almost always found at nascent synapses during synapse formation (Zhai et al. 2001), CAST might also be involved in the formation of the CAZ.

During the course of the study, we have identified a molecule closely related to CAST in the GENBANK database. Here we have cloned its putative full-length cDNA from a rat brain cDNA library and named it CAST2. Accordingly, the original CAST is tentatively re-named here CAST1. Sequence analysis has revealed that CAST2 is a rat orthologue of the human protein ELKS (Nakata et al. 1999). Recently, ELKS has also been identified as Rab6-interacting protein 2 (Rab6IP2) (Monier et al. 2002) and ERC (Wang et al. 2002). ELKS has been identified as a gene whose 5'-terminus is fused to the RET oncogene in papillary thyroid carcinomas, but its function is unknown (Nakata et al. 1999); Rab6IP2 has been identified as a Rab6 small G protein-interacting protein and is implicated in the transport of endosomes to the Golgi complex (Monier et al. 2002); and ERCs have been isolated as RIM1-binding proteins with an unknown function (Wang et al. 2002). In this paper, we describe the biochemical and biological properties of CAST2 as a new component of the CAZ.

	Results

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Molecular cloning of the CAST2 cDNA

A homology search for CAST1 allowed us to find a closely related sequence to CAST1 in the GENBANK database from a partial amino acids (aa) sequence for a human cDNA (KIAA1081). With a probe designed from the cDNA, we cloned a full-length cDNA from a rat brain cDNA library. The encoded protein consisted of 948 amino acids (Fig. 1A) and was named CAST2. CAST2 showed 70% amino acid identity to CAST1. In human sequences, CAST2 showed the highest homology to KIAA1081 (data not shown), whereas CAST1 showed the highest homology to KIAA0378 (Ohtsuka et al. 2002). KIAA0378 and KIAA1081 are similar but different proteins and locate in distinct chromosomes (data not shown). Like CAST1, CAST2 had no transmembrane segment but had four coiled-coil domains. The last three aa (IWA) were conserved between CAST1 and CAST2. This C-terminal motif is required for binding to the PDZ domain of RIM1 (Ohtsuka et al. 2002). These sequence data also revealed that CAST2 was a rat orthologue of the human protein ELKS (Nakata et al. 1999) (data not shown), also recently identified as Rab6IP2 (Monier et al. 2002) and ERC1 (Wang et al. 2002) (see Discussion). In C. elegans, a single CAST protein has been found (Ohtsuka et al. 2002) and shows a similar domain structure to mammalian CASTs (Fig. 1B), suggesting that CAST is an evolutionally conserved protein. Accordingly, C. elegans CAST bound UNC10, a homologue of RIM, in vitro (data not shown). Thus, it is likely that the CAST-RIM system is conserved at least in C. elegans.

View larger version (86K): [in this window] [in a new window]   Figure 1  Deduced aa sequence of CAST2. (A) Alignment of CAST1 and CAST2 sequences. Identical residues are highlighted. These sequence data from CAST2 are available from GENBANK/EMBL/DDBJ under accession no. AY174115. (B) Comparison of the structures between , , and C. elegans CAST (CeCAST). CC, coiled-coil domain. The last three aa (IWA) are conserved among these three proteins.

To examine biochemical properties of CAST2, we first produced a polyclonal antibody (Ab) against CAST2. The anti-CAST2 Ab specifically detected exogenously expressed Myc-tagged CAST2, whereas the anti-Myc Ab detected both CAST1 and CAST2 (Fig. 2A). Western blot analysis showed that the anti-CAST2 Ab recognized a protein band of 120 kDa in rat brain and multiple protein bands of approximately 130 kDa in other rat tissues including the spleen, lung, liver, muscle, kidney, and testis (Fig. 2B). The protein bands in tissues other than the brain appeared to be multiple splice variants of CAST2 (see Discussion). Subcellular distribution analysis of the rat brain showed that, like CAST1 (Ohtsuka et al. 2002), CAST2 was tightly associated with the postsynaptic density fraction (Fig. 2C). Taken together, these results indicate that CAST2 is also a synaptic protein tightly associated with the synaptic junction.

View larger version (42K): [in this window] [in a new window]   Figure 2  Tissue and subcellular distribution of CAST2. (A) CAST2-specific Ab. HEK293 cells were transfected with the plasmid of Myc-CAST1 or Myc-CAST2. Each protein was extracted and the extract (10 µL each) was subjected to SDS-PAGE, followed by Western blotting using the anti-Myc or anti-CAST2 Ab. The anti-Myc Ab recognized both the proteins, whereas the anti-CAST2 Ab specifically recognized CAST2 but not CAST1. (B) Tissue distribution. The homogenates of various rat tissues (20 µg of protein each) were analysed by Western blotting using the anti-CAST2 Ab. (C) Subcellular distribution. The homogenate of rat brain was subjected to subcellular fractionation. An aliquot of each fraction (5 µg of protein each) was analysed by Western blotting using the anti-CAST2 Ab. Hom, homogenate; CSV, the crude synaptic vesicle fraction; CSM, the crude synaptic membrane fraction; and PSD, the postsynaptic density fraction. These results are representative of three independent experiments.

We next examined whether, like CAST1 (Ohtsuka et al. 2002), CAST2 binds RIM1. HEK293 cells were transfected with Myc-CAST2 and/or HA-RIM1. With immunoprecipitation by the anti-Myc Ab, HA-RIM1 was co-immunoprecipitated with Myc-CAST2 (Fig. 3A). Moreover, Myc-CAST2 specifically bound GST fusion protein containing the PDZ domain of RIM1, but did not bind GST alone (Fig. 3B). Because the last three aa (IWA), which are essential for binding to the PDZ domain of RIM1 (Ohtsuka et al. 2002), are conserved in CAST2 (Fig. 1A), this result indicates that both CAST1 and CAST2 directly bind RIM1 through this C-terminal motif.

View larger version (45K): [in this window] [in a new window]   Figure 3  Direct binding of CAST2 and RIM1. (A) Co-immunoprecipitation assay. Each expression plasmid of Myc-CAST2 or HA-RIM1 was transfected into HEK293 cells. Each protein was extracted and then mixed in the indicated combinations, followed by immunoprecipitation using the anti-Myc Ab. The immunoprecipitates were then analysed by Western blotting using the indicated Abs. IP, immunoprecipitation. (B) Pull-down assay. The GST fusion protein containing RIM1 PDZ domain as well as GST alone was bound to glutathione-Sepharose beads. The extract of HEK293 cells expressing Myc-CAST2 was then incubated with the beads. Proteins bound to the beads were analysed by Western blotting using the anti-Myc Ab. The GST fusion proteins were loaded equally, and assessed by protein staining with Coomassie Brilliant Blue (data not shown). Input contains 10% of the extract used for the assay. These results are representative of three independent experiments.

ELKS was first identified as a protein encoded by a gene fused to RET protein kinase in thyroid carcinomas by chromosomal translocation (Nakata et al. 1999). ELKS is thought to form a homo-oligomer, which activates RET protein kinase (Nakata et al. 1999; Yokota et al. 2000). Because CAST2 corresponds to ELKS, and CAST1 and CAST2/ELKS are highly homologous to each other, we speculated that CAST1 and CAST2 form a hetero-oligomer. To address this issue, we first performed an immunoprecipitation assay using HEK293 cells. HEK293 cells were transfected with EGFP-CAST1 and/or Myc-CAST2. With immunoprecipitation by the anti-Myc Ab, EGFP-CAST1 was co-immunoprecipitated with Myc-CAST2 (Fig. 4A). Next, to determine the domain responsible for oligomerization, we performed a pull-down assay using GST fusion proteins containing various regions of CAST1 (Fig. 4B). The extract of HEK293 cells expressing EGFP-CAST2 was incubated with the beads containing the GST fusion proteins. EGFP-CAST2 bound GST-CAST1-1 and GST-CAST1-4, but did not bind other GST fusion proteins (Fig. 4Ca). In addition, EGFP-CAST1 bound GST-CAST1-1 and GST-CAST1-4, but did not bind other GST fusion proteins (Fig. 4Cb). These results suggest that CAST2 forms a hetero-oligomer with CAST1. Moreover, CAST1 has the potency to form a homo-oligomer. Although its precise mode of oligomerization is unclear, at least the N- and C-terminal regions of CASTs may be responsible for their oligomerization.

View larger version (35K): [in this window] [in a new window]   Figure 4  Hetero-oligomerization of CAST1 and CAST2. (A) Co-immunoprecipitation assay. Each expression plasmid of EGFP-CAST1 or Myc-CAST2 was transfected into HEK293 cells in the indicated combinations. Proteins were extracted and subjected to immunoprecipitation using the anti-Myc Ab. The immunoprecipitates were then analysed by Western blotting using the indicated Abs. IP, immunoprecipitation. (B) GST constructs of CAST1. CC, coiled-coil domain. (C) Pull-down assay. The GST fusion proteins containing various regions of CAST1, as well as GST alone, were immobilized to glutathione-Sepharose beads. The extract of HEK293 cells expressing EGFP-CAST1 or EGFP-CAST2 was then incubated with the beads. Protein bound to the beads was analysed by Western blotting using the anti-GFP Ab. The GST fusion proteins were loaded equally, which was assessed by protein staining with Coomassie Brilliant Blue (data not shown). Input contains 15% of the extract used for the assay. These results are representative of three independent experiments.

Finally, we examined the localization of CAST2 at synapses. We first examined the localization of CAST2 in primary cultured rat hippocampal neurones. Under the same conditions, CAST1 co-localized with Bassoon (data not shown) and CAST2 co-localized with Bassoon and the synaptic vesicle protein, synaptophysin (Fig. 5). It may also be noted that all the signals of Bassoon or synaptophysin do not co-localize with those of CAST2. Probably, CAST1 may localize with such CAST2-negative signals of Bassoon or synaptophysin. Using immunohistochemistry, we next determined the localization of CAST2 in the mouse cerebellum. CAST1 was expressed in both the granular and molecular layers whereas CAST2 was expressed predominantly in the molecular layer (Fig. 6A). The signal of CAST2 was strongly co-localized with Bassoon. To further determine the subsynaptic localization of CAST2, we performed immunoelectron microscopic analysis using the anti-CAST2 Ab. At the ultrastructural level, CAST2 localized close to the presynaptic membrane (Fig. 6B). Taken all together, these results indicate that CAST2 is a component of the CAZ.

View larger version (57K): [in this window] [in a new window]   Figure 5  Localization of CAST2 in cultured neurones. Primary cultured rat hippocampal neurones after 21 days of culture were fixed and double stained using the anti-CAST2 and anti-Bassoon Abs or anti-synaptophysin Ab. Bars = 10 µm. These results are representative of three independent experiments.

View larger version (98K): [in this window] [in a new window]   Figure 6  Localization of CAST2 in the mouse cerebellum. (A) Immunohistochemistry. Gr, granular layer; Pj, Purkinje cell layer; Mo, molecular layer. Bars, 30 µm (B) Immunoelectron microscopy. The sections were reacted with the anti-CAST2 or anti-Bassoon Ab, incubated with immnogold particles (1.4 nm) conjugated with goat IgG against rabbit and mouse IgGs, and silver enhanced, for analysis by electron microscopy. Bars = 100 nm. The results are representative of three independent experiments.

	Discussion

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CAST2 has been separately described as human ELKS, mouse Rab6IP2, and rat ERC1. Together with our present results, we could tentatively categorize the CAST family into at least three members as follows: (1) CAST1, originally identified as a novel CAZ protein (Ohtsuka et al. 2002), which corresponds to human KIAA0378 (Kikuno et al. 2002) and rat ERC2 (Wang et al. 2002); (2) CAST2: described in this paper as CAST2 and corresponds to human KIAA1081 (Kikuno et al. 2002), human ELKS (Nakata et al. 2002), mouse Rab6IP2A, and rat ERC1b (Wang et al. 2002); and (3) CAST2ß, an alternative splicing isofom of CAST2, corresponds to the other human ELKS isoforms (Nakata et al. 2002), mouse Rab6IP2B (Monier et al. 2002), and rat ERC1a (Wang et al. 2002), which lacks the last three aa (IWA). Indeed, we have cloned one of the isoforms from a mouse heart cDNA library as CAST2ß (GENBANK/EMBL/DDBJ accession no. AY316692) (data not shown). In summary, and are mainly expressed in the brain whereas ß, the splicing isoform of , which lacks the last three aa (IWA), is ubiquitously expressed outside of the brain.

Among these three proteins, ELKS, Rab6IP, and ERC, ELKS was first identified (Nakata et al. 1999). ELKS consists of at least five isoforms and its genomic organization has most extensively been studied (Nakata et al. 2002). Of these isoforms, ELKS is abundantly expressed in the brain, while most other isoforms are ubiquitously expressed (Nakata et al. 2002). Rab6IP2 was originally isolated and characterized as a small G protein Rab6-interacting protein (Monier et al. 2002). Rab6IP2 consists of at least two isoforms, Rab6IP2A and Rab6IP2B (Monier et al. 2002). CAST2/ELKS corresponds to Rab6IP2A, which contains the last three aa (IWA), whereas Rab6IP2B has a different C-terminal sequence from Rab6IP2A. ERCs have more recently been discovered (Wang et al. 2002). ERC consists of ERC1 and ERC2 and ERC1 further consists of ERC1a and ERC1b, which are splicing isoforms to each other. The name of ERC is a conjugation of three proteins (ELKS, Rab6IP2 and CAST) and it has been described as if they are the same protein derived from the same gene (Wang et al. 2002). However, the data from ours and other groups, it is clear that CAST1 and ELKS/Rab6IP2 are similar but distinctly different proteins (Nakata et al. 1999; Monier et al. 2002; Ohtsuka et al. 2002).

In a previous paper, we have shown that CAST1 plays a role in the localization of RIM1 (Ohtsuka et al. 2002). Consistent with this idea, the subcellular localization and protein expression of CAST1/ERC2 and CAST2/ERC1b were intact in RIM1-deficient mice (Wang et al. 2002). Because CAST2 also binds RIM1 (Fig. 3), not only CAST1 but also CAST2 plays a role in the localization of RIM1 in neurones. At present we cannot rule out the possibility that other proteins in addition to CASTs bind the PDZ domain of RIM1 at the CAZ. It would be of interest to find proteins which, in addition to CASTs, bind the PDZ domain of RIM1 and determine their relationship with CASTs at the CAZ.

We have, moreover, shown here that CAST2 forms a hetero-oligomer with CAST1. The physiological significance of the oligomerization is currently unknown but we speculate that CASTs form a nucleation site for the assembly of presynaptic proteins by their oligomerization, which could eventually capture more proteins such as RIM1 and Munc13-1 at the presynaptic active zone. The other CAZ proteins Bassoon and Piccolo have also coiled-coiled domains (tom Dieck et al. 1998; Fenster et al. 2000). Thus, these coiled-coil domains may be involved not only in the oligomerization of Bassoon and/or Piccolo themselves but also in the oligomerization with CASTs. Revealing the molecular interactions among the CAZ proteins step by step would definitely provide more insights into the molecular mechanisms underlying the assembly, maintenance, and function of the CAZ.

	Experimental procedures

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Molecular cloning and plasmid construction of CAST2

A search of the GENBANK database with the CAST1 sequence revealed that CAST1 showed a relatively high homology to a protein sequence deduced from a human cDNA for KIAA1081 (Kikuno et al. 2002) (data not shown). The cDNA for KIAA1081 was obtained from Kazusa DNA Research Institute (Chiba, Japan). With a probe designed from the cDNA, a rat brain cDNA library was screened and the full-length cDNA (CAST2) was obtained. The full-length CAST2 cDNA was subsequently subcloned into a pCIneo-Myc vector (Ohtsuka et al. 2002) and pEGFP-C1 (Clonetech) at SalI and NotI sites. pEGFP-CAST1 and pCMV-HA-RIM1 were obtained as previously described (Ohtsuka et al. 2002).

Antibodies

Rabbit antiserum against CAST2 was raised against a glutathione-S transferase-fusion protein containing 117–142 amino acids residues of CAST2. The antiserum was affinity purified as previously described (Ohtsuka et al. 2002). The monoclonal anti-Myc, anti-HA, and anti-GFP Abs were purchased from Roche, the monoclonal anti-synaptophysin Ab from Chemicon, and monoclonal anti-Bassoon Ab from StressGen Biotechnologies.

Immunoprecipitation

HEK293 cells were transfected with pCIneo-Myc-CAST2 or pCMV-HA-RIM1 by lipofectAMINE 2000 reagent (Invitrogen). 48 h after transfection, the cells were collected and immunoprecipitated using the anti-Myc Ab as described (Ohtsuka et al. 2002) and then analysed by Western blotting. For analysis of the hetero-oligomerization of CAST1 and CAST2, HEK293 cells were transfected with pEGFP-CAST1 or pCIneo-Myc-CAST2, and then analysed as described above.

Pull-down assay

HEK293 cells expressing EGFP-CAST1 or EGFP-CAST2 were lysed in 1 mL of a lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1%[wt/vol] Triton X-100, and 10 µg/mL leupeptin) at 4 °C for 30 min. The sample was centrifuged at 15 000 r.p.m. at 4 °C for 30 min to collect the supernatant. 500 µL of each extract was then incubated with 20 µL of glutathione-Sepharose beads containing the indicated GST fusion proteins (1 µg of protein each) at 4 °C for 1 h. After the beads were extensively washed with the lysis buffer, the bound proteins were eluted by boiling the beads in an SDS sample buffer (60 mM Tris-Cl, pH 6.7; 3% SDS; 2% (v/v) 2-mercaptoethanol; 5% glycerol) for 5 min. The samples were then analysed by Western blotting using the anti-GFP Ab.

Neurone culture, immunohistochemistry and immunoelectron microscopy

Primary culture of rat hippocampal neurones was done as previously described (Ohtsuka et al. 2002). For immunocytochemistry of the cultured neurones, coverslips were removed from culture wells and fixed in a 2% paraformaldehyde, 4% sucrose PBS solution (pH 7.4) for 20 min at room temperature. In the following experiments, all procedures were carried out at room temperature. After washing five times with PBS for 5 min each, the cells were permeabilized with 0.25% Triton X-100 in PBS for 15 min. Nonspecific binding was blocked with 25% Block Ace (Dainippon Pharmaceutical, Osaka, Japan) for 2 h. The cells were then incubated with the primary Abs diluted in 10% Block Ace and 0.25% Triton X-100 in PBS for 2 h. After washing five times with PBS for 5 min each, the cells were further incubated with secondary Abs, and rinsed again with PBS. The coverslips were mounted on slides using ProLong Antifade Kit (Molecular Probes, OR, USA). Fluorescence images were acquired with a confocal laser microscopy using a 63x or 100x oil immersion objective lens (LSM510, Carl Zeiss Microimaging Inc., Germany).

Immunohistochemistry of the mouse cerebellum was done as previously described (Ohtsuka et al. 2002). The sections were fixed with ethanol and acetone, incubated with the primary Abs and washed, and then incubated with rhodamine-conjugated donkey anti-rabbit IgG or FITC-conjugated donkey anti-mouse IgG. The samples were imaged by confocal microscopy using a 100x oil immersion objective lens as described above. Immunoelectron microscopy of the cerebellum was performed essentially as previously described (Kinoshita et al. 1998). Adult mice were perfused and fixed with 2% and 4% paraformaldehyde for CAST2 and Bassoon, respectively. After processing, ultrathin sections were examined by electron microscopy (H-7500, Hitachi, Tokyo, Japan).

	Acknowledgements

 
We thank Dr S. Seino (Chiba University, Japan) for providing us with pCMV-Tag3-human RIM1 expression vector to construct pCMV-HA-RIM1.

	Footnotes

 
Communicated by: Shoichiro Tsukita

^* Correspondence: Email: t-ohtsuka{at}kan.gr.jp

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Received: 8 October 2003
Accepted: 28 October 2003

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