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Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

	Abstract

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Rim1 and Rim2 were originally described as specific Rab3A-effector proteins involved in the regulation of secretory vesicle exocytosis. The putative Rab3A-binding domain (RBD) of Rim consists of two -helical regions (named RBD1 and RBD2) separated by two zinc finger motifs. Although alternative splicing in the RBD1 of Rim is known to produce long and short forms of RBD (named Rim1 and Rim156-105, and Rim2(+40A) and Rim2, respectively), with the long form of Rim1 and short form of Rim2 being dominant in mouse brain, the physiological significance of the alternative splicing in RBD1 has never been elucidated. In the present study I discovered that alternative splicing in Rim RBD1 alters Rab3A binding affinity to Rims, and found that insertion of 40 amino acids into the RBD1 of Rim2 (i.e. Rim2(+40A)) dramatically reduced its Rab3A binding activity (more than a 50-fold decrease in affinity). Similarly, Rim156-105 exhibited higher affinity binding to Rab3A than the long form of Rim1. Expression of the short forms of the Rim RBD in PC12 cells co-localized well with endogenous Rab3A, whereas expression of the long forms of the Rim RBD in PC12 cells resulted in cytoplasimc and nuclear localization. Moreover, I found that Caenorhabditis elegans Rim/UNC-10 (ce-Rim) and Drosophila Rim (dm-Rim) do not interact with ce-Rab3 and dm-Rab3, respectively, indicating that the Rab3-effector function of Rim has not been retained during evolution. Based on these findings, I propose that the Rab3A-effector function of Rim during secretory vesicle exocytosis is limited to the short form of the mammalian Rim RBD alone.

	Introduction

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Rim was originally identified as a protein that contains a putative Rab3A-effector domain at the N terminus (Wang et al. 1997), the same as rabphilin (Shirataki et al. 1993), and it has been shown to be involved in the regulation of secretory vesicle exocytosis and synaptic plasticity (Koushika et al. 2001; Castillo et al. 2002; Schoch et al. 2002). Two isoforms of Rim (named Rim1 and Rim2, designated simply Rim1 and Rim2, respectively, below) have been reported in mammals (Ozaki et al. 2000; Wang et al. 2000), and they share the same domain structures: a putative Rab3A-binding domain at the N terminus, a PDZ domain in the middle region, and two C2 Ca²⁺-binding motifs at the C terminus. Rim is a scaffold protein that binds a variety of molecules in vitro, including Munc13-1 (Betz et al. 2001; Wang et al. 2001), synaptotagmin I (Coppola et al. 2001; Schoch et al. 2002), SNAP-25 (Coppola et al. 2001), N-type Ca²⁺-channels (Coppola et al. 2001), Rim-BPs (Rim-binding proteins) (Wang et al. 2000; Hibino et al. 2002), cAMP-GEFII/Epac2 (Ozaki et al. 2000; Fujimoto et al. 2002), CAST/ERC2 (Ohtsuka et al. 2002; Wang et al. 2002), liprins (Wang et al. 2000), and 14-3-3 (Sun et al. 2003), and several of the interactions have been shown to be essential for the control of secretory vesicle exocytosis. As an example, the interaction between Rim1 and Munc13-1 is required for the secretory vesicle priming step (Betz et al. 2001).

Although Rim was first described as a putative Rab3A effector protein, the physiological role of the Rim·Rab3A interaction in secretory vesicle exocytosis remains unclear and is a matter of controversy. Iezzi et al. (2000) showed that expression of the N-terminal Rab-binding domain (RBD) of Rim1 in pancreatic ß-cell lines promotes human growth hormone secretion, and that this effect is reversed by coexpression with a dominant active form of Rab3A(Q81L). By contrast, Sun et al. (2001) argued that the Rab3A-binding and secretion-enhancing domains in Rim1 in chromaffin cells are separate. It is interesting that the expressed RBD of Rim1 was mainly localized in the cytosol and did not co-localize well with Rab3A (or granule markers) in either cell type (Iezzi et al. 2000; Sun et al. 2001), despite the fact that the Rim1 RBD interacts with Rab3A in vitro (Wang et al. 1997, 2000, 2001; Iezzi et al. 2000; Ozaki et al. 2000; Sun et al. 2001; Fukuda 2003a). The reason for the inability of Rim1 RBD to recruit to secretory granules in living cells through interaction with Rab3A, however, has never been elucidated.

In the present study, I investigated the functional relationship between alternative splicing in the RBD1 of Rims and Rab3A-binding activity and found that an amino acid insertion in the RBD1 of Rims dramatically reduced their Rab3A binding affinity both in vitro and in intact cells. I also found that invertebrate (Caenorhabditis elegans and Drosophila) Rims do not interact with invertebrate Rab3, whereas mouse Rim2 RBD interacted with both invertebrate and vertebrate Rab3. These findings indicate that Rim is not a Rab3 effector across phylogeny and that the Rab3 effector function of Rim is regulated by alternative splicing in the RBD1 of Rims.

	Results

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The RBD1 of Rim2 is essential, but not sufficient for high affinity Rab3A binding

Although the N-terminal RBD of rabphilin, Noc2, and Rim was originally described as a specific Rab3-effector domain (Shirataki et al. 1993; Wang et al. 1997; Ozaki et al. 2000; Haynes et al. 2001), the results of recent in vitro binding experiments on Rab1-40 and functional studies in PC12 cells have clearly indicated that the RBD of rabphilin and Noc2 also function as a Rab27-effector domain that controls regulated exocytosis (Fukuda 2003a; Cheviet et al. 2004; Fukuda et al. 2004). In addition, the three subdomains of the RBD (i.e. RBD1, zinc finger motifs, and RBD2) of Noc2 (or rabphilin) have been shown to make different contributions to the recognition of Rab3A and Rab27A (Fukuda et al. 2004): the Noc2 RBD1 alone is necessary and sufficient for Rab27A recognition, whereas all three subdomains are required for Rab3A recognition. A systematic deletion analysis was then performed to identify the mechanism of recognition of Rab3A by the Rim2 RBD (Fig. 1A). Similar to the RBD1 of Noc2, the Rim2 RBD1 interacted with Rab3, but much more weakly than the whole RBD (compare lanes 1 and 2 in the middle panel of Fig. 1B) (Wang et al. 2001). The RBD1 of Rim2 should be the central Rab3A binding domain, because deletion of the RBD1 (i.e. RBD1) or point mutations in the RBD1 (i.e, E36A/R37S) completely abrogated Rab3A binding activity (lane 3 in the middle panel of Fig. 1B and lane 2 in the middle panel of Fig. 1C). Interestingly, deletion of the RBD2 (i.e. RBD2) also abrogated the Rab3A-binding activity despite the presence of the RBD1 (lane 4 in the middle panel of Fig. 1B), although the RBD2 alone did not interact with Rab3A (data not shown). These results indicate that both the RBD1 and RBD2 of Rim2 are necessary for high-affinity Rab3A recognition and suggested that the zinc finger motifs may have an inhibitory role in regard to binding to Rab3A.

View larger version (32K): [in this window] [in a new window]   Figure 1  (A) Schematic representation of deletion mutants of the RBD of Rim2. The Rab3A binding activity of each mutant (–, +, or +++) is indicated after its name. The RBD of Rim2 consists of two -helical regions (RBD1(1) and RBD2(2)) (black box and shaded box, respectively) that are separated by two zinc finger motifs (indicated by ‘Cs’) (Ostermeier & Brunger 1999). The conserved SGAWF(Y/F) motif in the RBD2 is essential for Rab3A binding by rabphilin (Ostermeier & Brunger 1999). The amino acid positions are indicated on both sides. (B) Rab3A binding activity of deletion mutants of Rim2 (GST-RBD1, RBD1, and RBD2). Note that GST-RBD1, but not the RBD1 or RBD2, have weakly bound Rab3A (lane 2, middle panel), suggesting that the RBD1 of Rim2 is a major Rab3A-binding site and that the RBD2 is required in addition for high-affinity Rab3A binding, the same as the RBD of rabphilin (Fukuda et al. 2004). (C) Glu-36 and Arg-37 in the RBD1 of Rim2 are critical residues for Rab3A binding. Mutation of Glu-36 to Asn and of Arg-37 to Ser in the RBD1 completely impaired the Rab3A binding activity of Rim2 (lane 2, middle panel). pEF-T7-Rim2-RBD mutants (1 µg of plasmids) and pEF-FLAG-Rab3A (0.5 µg of plasmids) were co-transfected into COS-7 cells, and associations between the T7-Rim2-RBD mutant and FLAG-Rab3A were evaluated by co-immunoprecipitation assay as previously described (Fukuda et al. 1999, 2004). Co-immunoprecipitated FLAG-Rab3A and the immunoprecipitated (IP) T7-Rim2-RBD mutants were visualized with HRP-conjugated anti-FLAG tag antibody (1 : 10 000 dilution) (Blot: anti-FLAG, IP: anti-T7; middle panels) and HRP-conjugated anti-T7 tag antibody (1 : 10 000 dilution) (Blot: anti-T7, IP anti-T7; bottom panels), respectively. Input means 1/80 volume of the reaction mixture used for immunoprecipitation (top panels). The positions of the molecular mass markers (x 10–3) are shown on the left.

In a previous study I discovered that an alternative splicing site in the RBD1 of Rim1 that resulted in the production of three forms of the mouse Rim1 RBD (Fig. 2A) (Wang et al. 2000; Fukuda 2003a; Johnson et al. 2003; Wang & Südhof 2003), and in the present study I identified a similar alternative splicing event in the RBD1 of Rim2 (40-amino acid-insertion) and a novel 4-amino acid-insertion (i.e. EDKV) in the second zinc finger motif of Rim2 (Fig. 2A,B). The same EDKV sequence was also found in the corresponding position of all three forms of Rim1 (Wang et al. 2000; Fukuda 2003a). Because of the presence of two independent alternative splicing sites, four forms of Rim2 were produced (named Rim2, Rim2(+4A), Rim2(+40A), and Rim2(+44A)) (Fig. 2A), making it of interest to investigate whether these amino acid insertions in the Rim2 RBD affect Rab3A binding activity. To do so, a T7-tagged Rim-RBD and FLAG-tagged Rab3A coexpression assay in COS-7 cells was performed as previously described (Fukuda et al. 1999; Fukuda 2003a). Since the expression level of FLAG-Rab3A in COS-7 cells was extremely high and recombinant FLAG-Rab3A protein in the total cell lysates was often visible even with Coomassie Brilliant Blue staining on SDS-polyacrylamide gel (data not shown), a much smaller amount of plasmids was used for co-transfection assay (0.5 µg of pEF-FLAG-Rab3A and 1 µg of pEF-T7-Rim2-RBD) than under normal conditions (2 µg each of pEF-T7 and pEF-FLAG plasmids) to highlight the difference in Rab3A binding capacity between the Rim2-RBDs. As shown in Fig. 3A, the interaction between the short forms of Rim1 and Rim2 (Rim1-RBD56-105, Rim2-RBD, and Rim2-RBD(+4A)) and Rab3A was easily detected (lanes 3-5 in the middle panel), but hardly any the interaction of the long forms of Rim1 or Rim2 (Rim1-RBD, Rim1-RBD83-105, Rim2-RBD(+40A), and Rim2-RBD(+44A)) with Rab3A was detected under the reduced Rab3A conditions (insets in the middle panel; prolonged X-ray film exposure). Similar results were obtained when the full-length Rim2(+4A) and Rim2(+44A) were used for the co-transfection assay (Fig. 3F). Competition experiments between the long form of Rim (Rim1-RBD or Rim2-RBD(+40A)) and the short form of Rim2 clearly indicated that the short form of Rim has much higher affinity for Rab3A than the long form of Rim (open and closed arrowheads in the middle panels of Fig. 3B,C) (Fukuda et al. 2004). I then investigated the relative affinity of the Rim2-RBDs for Rab3A (Fig. 3D,E). The Rim2-RBD exhibited highest affinity for Rab3A (open circles in Fig. 3E), and insertion of the 4-amino acids into the second zinc finger motif (closed circles) caused an approximately 2-fold decrease in affinity compared to the calculated EC₅₀ values (0.057/0.025, Rim2(+4A)/Rim2). The B_max value for the Rim2-RBD(+4A)·Rab3A interaction was also lower (approximately 75% of that for the Rim2-RBD·Rab3A interactions (Fig. 3E). Although the binding of the Rim2-RBD(+40A) and (+44A) to Rab3A was not saturated under my experimental conditions, insertion of the 40-amino acids into the RBD1 (open and closed squares) was estimated to reduce affinity by at least more than 50-fold in compared to the Rim2-RBD.

View larger version (28K): [in this window] [in a new window]   Figure 2  Novel alternative splicing isoforms of mouse Rim2. (A) Schematic representation of alternative splicing isoforms of mouse Rim1 and Rim2. The RBD of Rim1 and Rim2 consists of two -helical regions (RBD1(1) and RBD2(2)) (black boxes and shaded boxes, respectively) that are separated by two zinc finger motifs (indicated by ‘Cs’) (Ostermeier & Brunger 1999). The conserved SGAWF(Y/F) motif in the RBD2 is essential for Rab3A binding by rabphilin (Ostermeier & Brunger 1999). Amino acid insertions (or deletions) by alternative splicing were found in the C terminus of the RBD1 and the second zinc finger motifs (indicted by cross-hatched and hatched boxes, respectively). At least three forms of the Rim1-RBD and four forms of the Rim2-RBD can be produced by alternative splicing, but the longest form of Rim1 (i.e. Rim1) and the shortest form of Rim2 (i.e. Rim2) were predominant in mouse brain (Fukuda 2003a). The amino acid positions are indicated on both sides. (B) Reverse transcriptase-PCR analysis of the Rab-binding domain of Rim2 in mouse brain, kidney, and testis. Sequence analysis indicated that the shortest form of Rim2 is dominant in the mouse brain (Fukuda 2003a), whereas similar amounts of the long (Rim2(+44A) > Rim2(+40A)) and short forms of Rim2 (Rim2(+4A) > Rim2) are expressed in the mouse testis. The size of the molecular weight markers (100-bp marker) is shown at the left. The gels shown here are representative of two independent experiments.

View larger version (43K): [in this window] [in a new window]   Figure 3  Distinct Rab3A binding affinity of alternative splicing isoforms of Rim1 and Rim2. (A) Short forms of Rims (open arrowhead) bound Rab3A more preferentially than long forms of Rims (closed arrowhead). pEF-T7-Rim-RBD (1 µg of plasmids) and pEF-FLAG-Rab3A (0.5 µg of plasmids) were co-transfected into COS-7 cells, and associations between the T7-Rim-RBD and FLAG-Rab3A were evaluated by co-immunoprecipitation assay as previously described (Fukuda et al. 1999, 2004). Co-immunoprecipitated FLAG-Rab3A and the immunoprecipitated (IP) T7-Rim-RBD were visualized with HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG, IP: anti-T7; middle panel) and HRP-conjugated anti-T7 tag antibody (Blot: anti-T7, IP anti-T7; bottom panel), respectively. Insets in the middle panel showed the results of long X-ray film exposure. Input means 1/80 volume of the reaction mixture used for immunoprecipitation (top panel). (B, C) Competition experiments revealed that short forms of Rims preferentially interact with Rab3A than long forms of Rims. FLAG-Rab3A beads were incubated with solutions containing T7-Rim1-RBD (or T7-Rim2-RBD(+40A) in (C), and T7-Rim2-RBD in the proportions indicated. After washing the beads, proteins bound to the beads were analysed by 12.5% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-T7 tag antibody (Blot: anti-T7, IP: anti-FLAG; middle panels) and HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG, IP: anti-FLAG; bottom panels). Input means 1/80 volume of the reaction mixture used for immunoprecipitation (top panels). Open and closed arrowheads indicate the positions of short and long forms of Rim, respectively. The positions of the molecular mass markers (x 10–3) are shown on the left. (D) T7-Rim2-RBDs were systematically diluted as indicated, and diluted samples were incubated with beads coupled with FLAG-Rab3A protein. Proteins bound to the beads were analysed by 12.5% SDS-PAGE, and visualized with HRP-conjugated anti-T7 tag antibody (top four panels). Bottom panel shows the FLAG-Rab3A protein visualized with HRP-conjugated anti-FLAG tag antibody. The results shown are representative of three independent experiments. (E) Immunoreactive bands of Rims on X-ray films in (D) were captured and quantified as described under ‘Experimental procedures’. The EC50 values of 0.025 for the Rim2·Rab3A interaction () and 0.057 for the Rim2(+4A)·Rab3A interaction (•) were calculated with GraphPad PRISM software (version 4.0). Bars indicate the SE of three independent experiments. (F) Full-length Rim2(+4A) bound Rab3A more preferentially than full-length Rim2(+44A). Co-transfection and co-immunoprecipitation assays were performed as described in (A).

Next, I investigated whether the long form of Rim was recruited to endogenously expressed Rab3A on dense-core vesicles in PC12 cells (Fukuda et al. 2002b). When GFP (green fluorescence protein)-tagged short form of Rims (Rim1-RBD56-105, Rim2-RBD, and Rim2-RBD(+4A)) were transiently expressed in PC12 cells, the vesicular GFP signals at the edge of the cells co-localized well with endogenous Rab3A (Fig. 4C–F, and data not shown). By contrast, the GFP-tagged long form of Rims (Rim1-RBD, Rim2-RBD(+40A), and Rim2-RBD(+44A)) were mostly localized in the cytoplasm, in the nucleus, or near the plasma membrane (Fig. 4A,G and data not shown), although some signals still co-localized with Rab3A. GFP-Rim2-RBD(E36A/R37S), which lacks Rab3A binding activity, was distributed throughout the cytoplasm, and no co-localization of the Rim2 mutant with Rab3A was detected (Fig. 4I,J). I therefore concluded that the Rab3A binding activity of the long form of Rim is insufficient to assess the endogenous expression level of Rab3A in PC12 cells.

View larger version (39K): [in this window] [in a new window]   Figure 4  Rab3A-depedent recruitment of short forms of Rims to dense-core vesicles in PC12 cells. PC12 cells expressing GFP-tagged Rim1-RBD (A, C) or Rim2-RBD (E, G, I) were fixed, permeabilized, and stained with anti-Rab3A mouse monoclonal antibody (B, D, F, H, J). Note that the short forms of Rims (i.e. Rim156-105 and Rim2) overlapped well with Rab3A, whereas the long forms of Rims (i.e. Rim1 and Rim2(+40A)) were mostly found in the cytoplasm as well as in the nucleus (compare A and B, or G and H). The Rab3A-binding-defective mutant of Rim2(E36A/R37S) also showed cytoplasmic localization of the recombinant protein. Scale bar in J indicates 10 µm.

View larger version (25K): [in this window] [in a new window]   Figure 5  Distinct effect of expression of the Rim1-RBD or Rim2-RBD on dense-core vesicle exocytosis in PC12 cells. (A) Expression of the Rim1-RBD (shaded bar) or the Rim1-RBD(E32A/R33S) (open bar) strongly promoted high-KCl-dependent NPY secretion from PC12 cells. (B) Expression of the Rim2-RBD (shaded bar), but not the Rim2-RBD(E36A/R37S) (open bar), promoted high-KCl-dependent NPY secretion from PC12 cells (*P < 0.05, Student's unpaired t-test). The NPY-T7-GST secretion assay was performed as previously described (Fukuda et al. 2002a; Fukuda 2003b). The results are expressed as percent NPY-T7-GST secretion compared to control samples (closed bars) in which no recombinant proteins were expressed. Bars indicate the means  SE of three determinations. The results shown are representative of at least three independent experiments. Bottom panels show recombinant proteins expressed visualized with HRP-conjugated anti-T7 tag antibody.

Since Rim is known to be conserved from C. elegans to humans (Koushika et al. 2001; Wang & Südhof 2003), lastly I attempted to determine whether alternative splicing events in the RBD of Rim had been retained during evolution. However, I did not find any such alternative splicing events in either C. elegans or Drosophila Rims by my RT-PCR analysis (data not shown). Surprisingly, ce-Rim-RBD did not interact with Rab3 from a variety of species (lanes 5-8 in the middle panel of Fig. 6A), whereas mouse Rim2-RBD interacted with Rab3 from C. elegans, Drosophila, squid and the mouse (lanes 1-4). Since ce-rabphilin has recently been shown to interact specifically with ce-Rab27, and not to interact with ce-Rab3 (Fukuda et al. 2004) and mouse Rim1/2 weakly interact with other Rabs (e.g. Rab8A, Rab10, Rab26, or Rab37) in vitro (Fukuda 2003a), I proceeded to investigate the possibility that ce-Rim interacts with other Rabs closely related to the Rab3 subfamily, which belongs to Rab functional group III (e.g. Rab3, Rab27, and Rab37 subfamilies) (see Pereira-Leal & Seabra 2001; Fukuda 2003a; Fukuda et al. 2004). As shown in Fig. 6B, however, ce-Rim did not interact with ce-Rab3, ce-Rab8, ce-Rab27, or ce-Rab37. Similar results were obtained for the dm-Rim-RBD: it did not interact with dm-Rab3, dm-Rab8, or dm-Rab27 (middle panel of Fig. 6C). These results suggest that it is most unlikely that the N-terminal domain of invertebrate Rim functions as a Rab3 effector domain. Consistent with this, investigation of the C. elegans unc-10 mutant (homologue of vertebrate Rim) has revealed little genetic interaction between Rim and Rab3 (Koushika et al. 2001).

View larger version (24K): [in this window] [in a new window]   Figure 6  Rim is not a Rab3-binding protein in invertebrates. (A) Mouse Rim2, but not C. elgans Rim/Unc10, interacts with Rab3 from various species, including C. elegans (ce), Drosophila (dm), squid (s) and mice (m). (B) Interaction between ce-Rim and ce-Rab3, ce-Rab8, ce-Rab27, or ce-Rab37. (C) Interaction between dm-Rim and dm-Rab3, dm-Rab8, or dm-Rab27. Note that neither dm-Rim nor ce-Rim interacted with Rab3. pEF-T7-Rim-RBD and pEF-FLAG-Rab were co-transfected into COS-7 cells, and associations between the T7-Rim-RBD and FLAG-Rab were evaluated by co-immunoprecipitation assay as previously described (Fukuda et al. 1999, 2004). Co-immunoprecipitated FLAG-Rab and the immunoprecipitated (IP) T7-Rim-RBD were visualized with HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG, IP: anti-T7; middle panels) and HRP-conjugated anti-T7 tag antibody (Blot: anti-T7, IP: anti-T7; bottom panels), respectively. Input means 1/80 volume of the reaction mixture used for immunoprecipitation (top panels). The positions of the molecular mass markers (x 10–3) are shown on the left.

View larger version (35K): [in this window] [in a new window]   Figure 7  Interaction between Rim and Munc13-1/Unc13 has been retained during evolution. (A) Mouse Rim1 and Rim2 interact with Munc13-1 regardless of the presence of alternative splicing in the RBD. (B) Interaction between ce-Rim and Unc13. pEF-T7-Rim-RBD and pEF-FLAG-Munc13-1 (or -Unc13-N) were co-transfected into COS-7 cells, and associations between the T7-Rim-RBD and FLAG-Munc13-1/Unc13 were evaluated by co-immunoprecipitation assay as previously described (Fukuda et al. 1999, 2004). Co-immunoprecipitated FLAG-Munc13-1/Unc13 and the immunoprecipitated (IP) T7-Rim-RBD were visualized with HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG, IP: anti-T7; middle panels) and HRP-conjugated anti-T7 tag antibody (Blot: anti-T7, IP: anti-T7; bottom panels), respectively. Input means 1/80 volume of the reaction mixture used for immunoprecipitation (top panels). The positions of the molecular mass markers (x 10–3) are shown on the left.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

Since the Rab3A binding capacity of Rim is regulated by alternative splicing in the RBD, it is important to know which forms of Rim are dominantly expressed in vivo (or the ratio between the long and short form of Rim). To the extent that I tested, by RT-PCR analysis, the longest form of Rim1 and the short forms of Rim2 are dominant in the brain, whereas the long forms of Rim2 are only expressed in the testis (Fukuda 2003a; and Fig. 2B). I therefore speculate that Rim1 and Rim2 have different functions in the brain in terms of Rab3A regulation. Consistent with this notion, Rim2 cannot substitute for Rim1 in Rim1 knockout mice, which lack PKA-dependent presynaptic long-term potentiation at cerebellar parallel fibre synapses (Lonart et al. 2003). It is important to note that the intracellular localization of Rim1 (i.e. longest form) and Rim2 (i.e. shortest form) is completely different in pancreatic ß-cells, where Rim1 is associated with the plasma membrane, possibly through a PDZ domain or C2 domains (Iezzi et al. 2000) and Rim2 is associated with insulin-containing granules through the N-terminal RBD (Shibasaki et al. 2003). Therefore, the long forms of Rim are unlikely to be recruited to Rab3A on secretory vesicles under physiological conditions because of its low Rab3A binding capacity as well as the low endogenous expression level of Rab3A in PC12 cells, and they may instead be targeted to the plasma membrane through other domains or other proteins (e.g. Munc13-1). These observations, together with my own results, strongly suggest that alternative splicing in the RBD1 of Rim regulates intracellular targeting of the Rim molecule (plasma membrane vs. secretory granules) by altering Rab3A binding capacity. In contrast to this notion, Iezzi et al. (2000) have previously shown that enhancement of secretion by Rim1-RBD was reversed by coexpression with Rab3A(Q81L). However, this discrepancy can be explained by the following observations: the Rim1-RBD binds Rab3A(Q81L) more preferentially than the wild-type protein in vitro (Ozaki et al. 2000), and when over-expressed the intracellular concentration of Rab3A(Q81L) should be sufficiently high to bind the Rim1-RBD in intact cells.

During the past decade most of the proteins involved in membrane trafficking (e.g. t- and v-SNAREs, or Rabs) have been shown to be conserved from yeast to humans (Bennett & Scheller 1993; Ferro-Novick & Jahn 1994), and the protein-protein interactions required to tune membrane trafficking (e.g. formation of a SNARE complex, or a Rab-effector interaction) are also believed to have been retained during evolution. In view of this, my discovery that invertebrate (C. elegans and Drosophila) Rim does not interact with invertebrate Rab3 was unexpected and surprising (Fig. 6), because ce-Rim contains a putative Rab3 binding domain that is highly homologous to the short form of the mouse Rim1/2-RBD and the Unc13·ce-Rim interaction is retained across phylogeny (Fig. 7). A similar observation has recently been reported in regard to rabphilin, another putative Rab3 effector protein: invertebrate rabphilin functions as a Rab27 binding protein, rather than a Rab3 binding protein (Fukuda et al. 2004). Since invertebrate Rim and rabphilin do not interact with Rab3, the Rab3 effector(s) in invertebrates have yet been identified (Fukuda 2002b).

In summary, this study is the first to demonstrate that alternative splicing in the RBD1 of mouse Rims regulates Rab3A binding capacity. I also discovered that a putative RBD of invertebrate Rim does not function as a Rab3 binding domain. Further work is necessary to identify the common role of the N-terminal domain of Rim in membrane trafficking during evolution.

	Experimental procedures

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Identification of alternative splicing isoforms of Rim2

In a previous study I identified three alternative splicing isoforms of Rim1 (named Rim1, Rim183-105, and Rim156-105; GenBank accession numbers, AB162895-AB162897) and a single isoform of Rim2 in mouse brain (Fukuda 2003a). To determine whether similar alternative splicing events occur in the Rim2 RBD, PCR was performed on cDNAs from various mouse tissues (mouse MTC panel I; Clontech Laboratories, Inc.; Palo Alto, CA, USA) as previously described (Fukuda et al. 2001a; Fukuda 2003a). Two alternative splicing sites were found in the Rim2 RBD (see Fig. 2A): a 40-amino acid-insertion in the C terminus of the Rim2 RBD1, the same as Rim1, and a 4-amino acid-insertion (i.e. EDKV) in the second zinc finger motif, and these alternative splicing events resulted in the production of four different types of Rim2 RBD (named Rim2, Rim2(+4A), Rim2(+40A), Rim2(+44A); GenBank accession numbers, AB162898-AB162901, respectively). Except for the mouse testis, expression levels of the long forms of Rim2 were very low in all mouse tissues tested by RT-PCR analysis (Fig. 2B). The Rim-RBD fragments were subcloned into the BamHI/SalI site of modified pEF-T7 plasmids (Fukuda et al. 1999) or the BglII/SalI site of pEGFP-C1 plasmids (Clontech Laboratories, Inc.).

Molecular cloning of C. elegans Rim, Unc13, and Rab37, Drosophila Rim, squid Rab3, and mouse Munc13-1 cDNAs

cDNAs encoding the full open reading frame of the C. elegans Rab37 (named ce-Rab37), the N-terminal RBD of C. elegans Rim (named ce-Rim; amino acid residues 1-159), and the N-terminal domain of C. elegans Unc13 (amino acid residues 1-189) were amplified by PCR from the ProQuest C. elegans cDNA library (Invitrogen; Carlsbad, CA, USA) as previously described (Fukuda et al. 2001a; Fukuda et al. 2004). The following primers with a restriction enzyme site (underlined) or a stop codon (in boldface type) were used for amplification: 5'-GGATCCATGTTTTTAAAGGTTATGCT-3' (ce-Rab37 Met primer; sense; GenBank accession number, AB162902), 5'-TCAATTAAACGTGCAACATC-3' (ce-Rab37 stop primer; anti-sense), 5'-CGGATCCATGGACGATCCGTCGATGAT-3' (ce-Rim Met primer; sense; GenBank accession number, AB162903), 5'-CTATTCTGGTACGTTGTCGTCAGT-3' (ce-Rim-RBD stop primer; anti-sense), 5'-GGATCCATGGATGACGTTGGAGATTA-3' (Unc13 Met primer; sense; GenBank accession number, NM_059692 [GenBank] ), and 5'-TTAACTCAAACCCGAATGGTTA-3' (Unc13-3' primer; anti-sense).

No cDNA encoding the N-terminal RBD of Drosophila Rim (named dm-Rim; amino acid residues 1-147) was amplified by PCR from the adult Drosophila body cDNA library constructed in EXlox (Novagen; Madison, WI, USA) using the four different sets of primers, consistent with the fact that expression of dm-Rim-RBD mRNA has not been reported in the fruit fly EST database (Wang & Südhof 2003). I therefore chemically synthesized several pieces of single-strand cDNAs that cover the dm-Rim-RBD and obtained the full-length dm-Rim-RBD by PCR. The details of the methods and the sequences of the oligonucleotides used are available from the author upon request.

cDNA encoding the full open reading frame of squid Rab3 (named s-Rab3) was amplified by PCR with specific primers designed on the basis of the squid sequence in the public database (GenBank accession number, AB162904 and L49400) as previously described (Mikoshiba et al. 1995): 5'-CGGATCCATGATGGCATCAGCAAATGA-3' (s-Rab3-Met primer with a BamHI linker (underlined); sense) and 3'-CTAACACTGACAGCTACCAT-3' (s-Rab3-stop primer with a stop codon (in boldface type); anti-sense).

cDNA encoding the full open reading frame of mouse Munc13-1 was also amplified by PCR with specific primers designed on the basis of the mouse sequence in the public database (GenBank accession number, AB162894 and AF115848) as previously described (Fukuda et al. 1999): 5'-CGGATCCATGTCGCTGCTCTGTGTGCGT-3' (Munc13-1 Met primer with a BamHI linker (underlined); sense), 5'-AGCTCTCTGCTGAGTGAGCTG-3' (Munc3-1 N1 primer; sense), 5'-CAGTATCAGCTGCAGGAACAA-3' (Munc13-1 N2 primer; sense), 5'-AGAATGAATGGAGTCCCTCTC-3' (Munc13-1 C1 primer; anti-sense), 5'-GGGCCAGAAATCCAAGTTCCG-3' (Munc13-1 C2 primer; anti-sense), and 5'-TCAGCTCCCTTCCTCTGTGGA-3' (Munc13-1 stop primer with a stop codon (in boldface type); anti-sense). I identified one amino acid deletion (G1062) and several amino acid differences (E31V, D196S, H250R, R567E, I568V, A792R, M825L, G1267P, V1268L, G1406R, and R1480K) compared with the reported Munc13-1 sequences, and they were unlikely to have been PCR-induced errors, because I found the same differences in at least two independent clones.

The PCR products were purified from an agarose gel, directly inserted into the pGEM-T Easy vector (Promega; Madison, WI, USA), and verified by DNA sequencing as previously described (Fukuda et al. 1999). Construction of pEF-FLAG-ce-Rab37, pEF-FLAG-s-Rab3, pEF-FLAG-Unc13-N, pEF-FLAG-Munc13-1, and pEF-T7-ce/dm-Rim-RBD was essentially performed as previously described (Mizushima & Nagata 1990; Fukuda et al. 1994; Fukuda & Mikoshiba 2000). Other expression constructs (pEF-FLAG-m/ce/dm-Rab3, pEF-FLAG-ce/dm-Rab8, and pEF-FLAG-ce/dm-Rab27) were prepared as previously described (Kuroda et al. 2002; Fukuda 2003a; Fukuda et al. 2004). Plasmid DNA was prepared using Wizard minipreps (Promega) or Qiagen (Chatsworth, CA, USA) maxiprep kits.

Construction of deletion mutants of the Rim2 RBD and site-directed mutagenesis pEF-T7-GST(glutathione S-transferase)-Rim2-RBD1 (amino acid residues 1-70), pEF-T7-Rim2-RBD1 (amino acid residues 71-175), pEF-T7-Rim2-RBD2 (amino acid residues 1-140), and pEF-T7-GST-Rim2-RBD2 (amino acid residues 141-175) were essentially constructed by PCR using the following primers with an appropriate restriction enzyme site (underlined) and/or a stop codon (in boldface type) as previously described (Fukuda 2002a; Fukuda et al. 2004): 5'-GCACTAGTCACTCCTTATACATTTCAAATT-3' (Rim2-RBD1-3' primer; anti-sense), 5'-GGATCCCAAGTCAAGAAGATGGGA-3' (Rim2-RBD1 primer; sense), 5'-TTATTGTTTTCGGCACAAAT-3' (Rim2-RBD2 primer; anti-sense), and 5'-GGATCCCAAGAAATCCTCACTAAATCA-3' (Rim2-RBD2-5' primer; sense). Mutant Rim2 plasmid carrying Glu-to-Ala and Arg-to-Ser substitutions at amino acid positions 36 and 37 (E36A/R37S) was obtained by two-step PCR techniques using the following mutagenic primers with an artificial NheI site (underlined) as previously described (Fukuda et al. 1995): 5'-TTTGCTAGCCTCTTCCGTGAGGTG-3' (Rim2(E36A/R37S)-5' primer; anti-sense) and 5'-GAGGCTAGCAAAATCATCCTGGCT-3' (Rim2(E36A/R37S)-3' primer; sense). pEF-T7-Rim2-RBD(E36A/R37S) and pEGFP-C1-Rim2-RBD(E36A/R37S) were constructed as described above. pEF-T7-Rim1-RBD(E32A/R33S) was similarly constructed by PCR using following primers with an artificial NheI site (underlined) (Fukuda et al. 1995): 5'-GCTAGCCTCCTCGGTCAGGTGGCT-3' (Rim1(E32A/R33S)-5' primer; anti-sense) and 5'-GCTAGCAACATTATCATGGCAGTG-3' (Rim1(E32A/R33S)-3' primer; sense). All constructs were verified by DNA sequencing.

Competition experiments

FLAG-tagged Rab3A proteins were expressed in COS-7 cells and affinity-purified by anti-FLAG tag (M2) antibody-conjugated agarose (Sigma Chemical Co.; St. Louis, MO, USA) (Fukuda et al. 2004). The FLAG-Rab3A beads (wet volume 10 µL) were then incubated with 400 µL of solution (i.e. COS-7 cell lysates) containing T7-Rim1-RBD (or T7-Rim2-RBD(+40A)) and T7-Rim2-RBD in various proportions (indicated in Fig. 3B,C) for 1 h at 4 °C in 50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 1 mM MgCl₂, 1% Triton X-100, and protease inhibitors. After washing the beads three times with 1 mL of 10 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 1 mM MgCl₂, and 0.2% Triton X-100, the proteins bound to the beads were analysed by 10% SDS-PAGE followed by immunoblotting with HRP (horseradish peroxidase)-conjugated anti-T7 tag antibody (1 : 10 000 dilution) and HRP-conjugated anti-FLAG tag antibody (1 : 10 000 dilution) as previously described (Fukuda et al. 2004).

Miscellaneous procedures

An NPY-T7-GST secretion assay of PC12 cells was performed as previously described (Fukuda et al. 2002a; Fukuda 2003b) with a slight modification. Prior to the secretion assay, transfected PC12 cells were incubated with 10 µg/mL brefeldin A for 30 min to eliminate constitutive NPY secretion (Fukuda et al. 2004). Under my experimental conditions, Rim1/2-RBD did not alter the total expression level of NPY (data not shown). NPY cDNA was provided by Dr Wolfhard Almers (Lang et al. 1997). PC12 cell culture, transfection of pEGFP-C1-Rim-RBD into PC12 cells, and immunocytochemical analysis of PC12 cells by confocal fluorescence microscopy (Fluoview; Olympus, Tokyo, Japan) were also performed as previously described (Fukuda et al. 2001b, 2003). T7-tagged Rim (1 µg of plasmid) and FLAG-tagged Rab3 (0.5 µg of plasmid) (or Munc13-1) were coexpressed in COS-7 cells, and association between the two proteins was evaluated by immunoprecipitation in the presence of GTPS as previously described (Fukuda et al. 1999; Kuroda et al. 2002; Fukuda 2003a). The intensity of the bands on X-ray film was quantified with Lane Analyser (version 3.0) (ATTO Corp., Tokyo, Japan) as previously described (Fukuda & Mikoshiba 2000). Statistical analyses and curve fitting were achieved with a GraphPad PRISM computer program (version 4.0). The blots shown in this paper are representative of at least three independent experiments.

	Acknowledgements

 
I would like to thank Eiko Kanno, Yukie Ogata, and Megumi Satoh for technical assistance, Dr Wolfhard Almers (Vollum Institute, Portland, OR) for kindly donating a cDNA of NPY, and Dr Michael L. Nonet (Washington University School of Medicine, St Louis, MO) for sharing unpublished data prior to publication. This work was supported in part by grants from the Ministry of Education, Culture, Sports, and Technology of Japan (15689006 and 16044248 to M.F.).

The accession numbers of the nucleotide sequences are AB162894–AB162904.

	Footnotes

 
Communicated by: Kozo Kaibuchi

^* Correspondence: E-mail: mnfukuda{at}brain.riken.go.jp

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Received: 29 April 2004
Accepted: 18 June 2004

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