Slp4-a/Granuphilin-a Regulates Dense-core Vesicle Exocytosis in PC12 Cells*

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic β-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999)J. Biol. Chem. 274, 28542–28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitroand in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212–9218). How Slp4-a·Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a·Rab8 and Slp4-a·Rab27A complexes, but not Slp4-a·Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.

The SHD consists of two conserved domains, referred to as SHD1 and SHD2 (2). The SHD1 and SHD2 of Slp3-a, Slp4-a, and Slp5 are separated by a sequence containing two zincfinger motifs, whereas Slp1 and Slp2-a lack such zinc-finger motifs, and their SHD1 and SHD2 are linked (2). The SHD has also been found in other proteins, including Slac2-a (Slp homologue lacking C2 domains-a)/melanophilin (2,6,7), Slac2-b/ KIAA0624 (8,9), and Slac2-c/MyRIP (10). 2 We recently discovered that the SHD of the Slp family and Slac2 functions as an effector domain for specific Rab (7,9), a small GTP-binding protein believed to be essential for membrane trafficking in eukaryotic cells (reviewed in Refs. [11][12][13]. All SHDs directly interact with the GTP-bound form of Rab27A (and possibly Rab27B), both in vitro and in intact cells (5, 7, 9, 10, 14 -16), but the Slp4-a SHD is exceptional because it can also interact with Rab8 and Rab3A (9,16), whereas the others only recognize Rab27 isoforms among the Rabs we have tested (more than 20 Rabs) (5,7,9). 2 Since mutations in the rab27A gene cause defects in granule exocytosis in cytotoxic T lymphocytes and melanosome transport in human Griscelli syndrome and ashen mice (17)(18)(19)(20)(21)(22), we hypothesized previously that the Slp family and Slac2 are involved in such membrane trafficking (9). Consistent with this, Slac2-a has recently been identified as a "missing link" between Rab27A in the melanosome and myosin Va, an actinbased motor, indicating that formation of a tripartite protein complex (Rab27A⅐Slac2-a⅐myosin Va) is crucial for melanosome transport (7,16,(23)(24)(25). In contrast, however, functional involvement of the Slp family in Rab27-dependent membrane trafficking (especially in regulated granule exocytosis) remained to be clarified.
In this study, we demonstrated that the Slp4-a⅐Rab27A complex is formed on dense-core vesicles in vivo and controls regulated secretion (high KCl-dependent neuropeptide Y (NPY) secretion) by using PC12 cells in which Slp4-a, Rab3A, Rab8, and Rab27A were endogenously expressed. Based on our findings, we discuss the function of the Slp family as effectors for Rab27A in regulated dense-core vesicle exocytosis.

EXPERIMENTAL PROCEDURES
Antibody Production-cDNA encoding the C2B domain of mouse Slp4-a/granuphilin-a (amino acids 489 -673) (4,9) was amplified by the conventional PCR and subcloned into the pGEX-4T-3 vector (named pGEX-Slp4-a-C2B) (Amersham Biosciences), as described previously (26). GST (glutathione S-transferase) fusion proteins were expressed and purified on glutathione-Sepharose beads by the standard method (27). New Zealand White rabbits were immunized with purified GST-Slp4-a-C2B, and anti-Slp4-a antibody was affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad), as described previously (28). The specificity of the antibody was checked by immunoblotting * This work was supported in part by grants from the Science and Technology Agency to Japan (to M. F.) and Grant 13780624 from the Ministry of Education, Science, and Culture of Japan (to M. F.).
Three days after transfection, PC12 cells (6-cm dish) expressing either T7-GST-Slp4-a or T7-GST, as a control, were harvested and homogenized in 1 ml of the homogenization buffer described above. After solubilization with 1% Triton X-100, insoluble materials were removed by centrifugation, and the expressed GST fusion proteins were affinity-purified on glutathione-Sepharose beads (wet volume, 20 l; Amersham Biosciences) according to the manufacturer's notes. After extensively washing the beads with 10 mM HEPES-KOH, pH 7.2, 150 mM NaCl, and 0.2% Triton X-100, proteins bound to the beads were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-Rab antibodies or HRP-conjugated anti-T7 tag antibody ( 1 ⁄10,000 dilution).

RESULTS
Expression of Rab3A, Rab8, Rab27A, and Slp4-a in PC12 Cells-Our previous in vitro GST pull-down assay showed that the SHD of Slp4-a preferentially binds Rab8 and Rab27A and weakly interacts with Rab3A, whereas other SHDs specifically bind Rab27A (9). To attempt to determine which Rabs are in vivo binding partners of Slp4-a, we used specific antibodies to search for cell line(s) in which these three Rabs and Slp4-a are endogenously expressed. The specificity of each antibody used in this study (anti-Rab3A, anti-Rab8, anti-Rab27A, and anti-Slp4-a antibodies) was confirmed by immunoblotting with recombinant FLAG-Rabs and recombinant T7-Slps, rabphilin, The specificity of anti-Slp4-a antibody was confirmed by using recombinant T7-Slp1-5, -rabphilin, and -Syt I expressed in COS-7 cells (lanes 1-7). The positions of the molecular weight markers (ϫ 10 Ϫ3 ) are shown on the left. and Syt I (Fig. 1A, lanes 1-3 and Fig. 1B, lanes 1-7). The anti-Slp4-a antibody only recognized T7-Slp4-a, not the other Slps, including Slp5, a close homologue of Slp4-a (5). PC12 cells were found to express all four proteins (Fig. 1A, lane 4, and 1B,  lane 8). Quantitative analysis indicated that the ratio of the amounts of the three Rabs in descending order was Rab8: Rab3A:Rab27A ϭ 1:0.4:Ͻ 0.1 (Fig. 1A). The level of expression of Slp4-a was lower than in the pancreas (i.e. pancreatic ␤-cells; data not shown). To investigate whether Slp4-a⅐Rab complex is actually formed in vivo, an immunoprecipitation experiment was performed using anti-Slp4-a-specific antibody (Fig. 2A,  lane 3). Consistent with our previous in vitro binding experiments (9), Rab8 and Rab27A were found to co-immunoprecipitate with Slp4-a, whereas hardly any Rab3A was detected in the anti-Slp4-a IgG immunoprecipitates (Fig. 2A, lane 3). In contrast, none of the Rabs were detected in the control IgG immunoprecipitates (Fig. 2A, lane 2). Since the amounts of bound Rab8 and Rab27A were significantly reduced in the absence of GTP␥S, Slp4-a should recognize the GTP-bound form of Rab8 and Rab27A even under the in vivo conditions (data not shown). Similar results were obtained when exogenously expressed GST-Slp4-a was used (Fig. 2B, lane 2): GST-Slp4-a bound both Rab8 and Rab27A but only a trace amount of Rab3A. Although the relative amount of Rab27A was less than 10% of that of Rab8 in PC12 cells, Slp4-a efficiently co-immunoprecipitated with Rab27A, indicating that Slp4-a prefers Rab27A to Rab8 or Rab3A as an in vivo binding partner.
Slp4-a⅐Rab8 and Slp4-a⅐Rab27A Complexes on Dense-core Vesicles in PC12 Cells-PC12 cells contain two types of secretory vesicles, i.e. dense-core vesicles and synaptic-like microvesicles, and they are often utilized for analysis of regulated secretion. Since Slp4-a/granuphilin-a was first identified as a protein that specifically associated with insulin-containing dense-core vesicles (4), we next investigated whether Slp4-a and three Rabs are localized on the dense-core vesicles of PC12 cells. As shown in Fig. 3, Rab3A (Fig. 3A, green), Rab8 (Fig. 3D, green), Rab27A (Fig. 3G, green), and Slp4-a (Fig. 3, B, E, and H, red) were found to be predominantly localized in the distal portion of neurites, where dense-core vesicles were enriched (Fig. 3, C, F, and I, yellow). Dotted signals for Rab8 were found throughout the cell body in addition to the neurites (Fig. 3D), whereas the others were almost exclusively expressed in the distal portion of the neurites. Similar expression patterns were observed when Slp4-a or Rabs were exogenously expressed in PC12 cells (data not shown).
A subcellular fractionation study was performed to further confirm the presence of Slp4-a and the three Rabs on densecore vesicles (Fig. 4). As expected, Rab3A, Rab27A, and Slp4-a were co-distributed with Syt IX (dense-core vesicle marker (DCV); fractions 5-8) but not with synaptophysin (synapticlike microvesicle marker (SLMV); fractions 2-4) (29,35,36). In contrast, Rab8 was also detected in lighter fractions in addition to the dense-core vesicle-enriched fractions, consistent with the immunocytochemical analysis (Fig. 3D). We therefore concluded that the Slp4-a⅐Rab8 and Slp4-a⅐Rab27A complexes are indeed formed on dense-core vesicles in intact PC12 cells.
Opposite Effects of Slp4-a and Rab27A on Regulated Secretion in PC12 Cells-Finally, we used an NPY-T7-GST secretion assay to investigate whether the Slp4-a⅐Rab8 and/or Slp4-a⅐Rab27A complexes control regulated exocytosis in PC12 cells (see ref. 34 for details). When Rab27A was transiently coexpressed with NPY in PC12 cells, high KCl-dependent NPY secretion was significantly promoted (Fig. 5A, closed bar), but the Rab27A expression had no effect on low KCl-dependent NPY secretion (data not shown). The effect of Rab27A should be GTP-dependent because expression of Rab27A(T23N), a domi-nant negative form that mimics the GDP-bound state, did not promote NPY secretion, consistent with the previous report (14) (data not shown). In contrast, expression of Rab8 had no significant effect on high KCl-dependent NPY secretion (Fig.  5A, open bar). The slight effect of Rab8 on regulated secretion was not due to its expression levels in PC12 cells because

FIG. 2. In vivo formation of Slp4-a⅐Rab complex in PC12 cells.
A, in vivo formation of Slp4-a⅐Rab8 and Slp4-a⅐Rab27A in PC12 cells. Anti-Slp4-a IgG, but not control IgG, immunoprecipitated Rab8 and Rab27A, but no Slp4-a⅐Rab3A complex was detected under our experimental conditions (lane 3). The asterisk indicates the light chain of IgG. B, exogenously expressed T7-GST-Slp4-a, but not T7-GST alone, preferentially interacts with Rab8 and Rab27A but essentially not with Rab3A (lane 2). In both panels, input means 1 ⁄80 volume of the reaction mixture used for immunoprecipitation. The positions of the molecular weight markers (ϫ 10 Ϫ3 ) are shown on the left. similar amounts of FLAG-Rab8 and -Rab27A were detected in PC12 cells by immunoblotting (Fig. 5A, inset). Similarly, expression of Rab3A did not promote NPY secretion, as described previously (14,(37)(38)(39) (data not shown).
When Slp4-a was expressed in PC12 cells, high KCl-dependent NPY secretion was significantly reduced (Fig. 5B, open  bar), but it had no effect on low KCl-dependent secretion (data not shown). Interestingly, expression of Slp3-a, a Rab27A-specific Slp exhibiting Ca 2ϩ -dependent phospholipid binding activity (1,40), in PC12 cells strongly promoted high KCl-dependent NPY secretion (Fig. 5B, closed bar). To further determine whether the effect of Slp4-a (or Slp3-a) expression in high KCl-dependent NPY secretion is indeed Rab27A-dependent, an Slp4-a mutant incapable of Rab27A binding (⌬SHD) was ex-pressed in PC12 cells (Fig. 6A, lane 18). As expected, expression of Slp4-a-⌬SHD completely reversed the inhibitory effect (Fig.  6B, closed bar). A similar result was obtained for Slp3-b (2), an alternatively splicing isoform that lacks an N-terminal SHD (i.e. no significant effect on high KCl-dependent NPY secretion; Fig. 6B, closed bar). We therefore concluded that the observed effect of Slp4-a (or Slp3-a) expression in NPY secretion depends on the presence of the SHD (i.e. Rab27A-binding site).
Since deletion of the SHD of Slp4-a also abrogated Rab3A and Rab8 binding (Fig. 6A, lanes 16 and 17), we could not completely rule out the possibility that the Slp4-a⅐Rab3A or Slp4-a⅐Rab8 complex, but not Slp4-a⅐Rab27A complex, has an inhibitory effect on high KCl-dependent NPY secretion. When Ala-based site-directed mutagenesis was performed to resolve this issue, a single amino acid substitution in the SHD1 was found to alter the Rab binding specificity of Slp4-a (Fig. 6A). Slp4-a(E14A) and Slp4-a(I18A) mutants specifically recognized Rab27A but not Rab3A and Rab8 (Fig. 6B, lanes 4 -9), whereas the Slp4-a(V21A) mutant recognized both Rab8 and Rab27A but not Rab3A (lanes 10 -12). In contrast, a D32A mutation had almost no effect on the Rab binding specificity of Slp4-a (lanes [13][14][15]. If Slp4-a⅐Rab3A or Slp4-a⅐Rab8 complex plays a major role in inhibition of high KCl-dependent NPY secretion, expression of a I18A or V21A mutant should reverse the inhibitory effect. However, when these mutants of Slp4-a were expressed in PC12 cells, no difference in high KCl-dependent NPY secretion was detected between the wild-type and mutant Slp4-a (Fig. 6B, open bars). These results strongly indicated that Slp4-a modulates dense-core vesicle exocytosis via binding to Rab27A in PC12 cells. DISCUSSION The Slp family (Slp1-5) was defined as proteins with an N-terminal SHD and C-terminal tandem C2 domains (1,2). Previously, we showed that the SHD of the Slp family binds the GTP-bound form of Rab27A (5,9), but involvement of the Slp family in Rab27A-dependent membrane trafficking (i.e. melanosome transport in melanocytes and granule exocytosis in cytotoxic T lymphocytes observed in type I human Griscelli syndrome and ashen mice (17-22)) has never been elucidated.  In this study, we demonstrated for the first time that Rab27A and its effector molecules (Slp4-a and Slp3-a) control densecore vesicle exocytosis in PC12 cells.
Although Slp4-a has been found to interact with Rab3A, Rab8, and Rab27A in vitro and in overexpression studies (9,16), endogenous Slp4-a in PC12 cells preferentially interacted with Rab27A on dense-core vesicles (Figs. 2-4). The Slp4-a⅐Rab8 complex was also observed on dense-core vesicles in PC12 cells, but the majority of Rab8 (especially in the cell body; Fig. 3F) was free of Slp4-a, and significant amounts of Slp4-a⅐Rab3A were not detected. In addition, we discovered that expression of Rab27A, but not of Rab8, promoted high KCl-dependent NPY secretion in PC12 cells (Fig. 5A), whereas expression of Slp4-a and Slp3-a had opposite effects on NPY secretion (Fig. 5B). These effects should be Rab27A-dependent because Slp mutants incapable of Rab27A binding had no effect on NPY secretion (Fig. 6B). The opposite effects of Slp4-a and Slp3-a may be attributable to the different biochemical properties of their C2 domains (i.e. the Ca 2ϩ -independent phospholipid binding activity of Slp4-a (4) versus the Ca 2ϩ -dependent phospholipid binding activity of Slp3-a (40)) because Ca 2ϩ -dependent type Syt I and Ca 2ϩ -independent type Syt IV were found to have a positive effect and inhibitory effect, respectively, on transmitter secretion (41)(42)(43). While this manuscript was being reviewed, an inhibitory effect of Slp4-a on dense-core vesi-cle exocytosis has also been reported in pancreatic ␤-cell lines (44), strongly supporting our finding that the Slp4-a⅐Rab27A complex controls dense-core vesicle exocytosis.
Although the function of Slp4-a⅐Rab8 in PC12 cells is unknown, we speculate that Slp4-a⅐Rab8 may have a function different from regulated secretion, in which Slp4-a⅐Rab27A plays a major role. One possible function of Slp4-a⅐Rab8 may be transport of dense-core vesicles (from the trans-Golgi network to the cell periphery) because Rab8 has been shown to regulate polarized membrane traffic (from the trans-Golgi network to the basolateral plasma membrane) in epithelial cells (45). The function of Slp4-a⅐Rab8 complex is now under investigation in our laboratory.
In summary, this study is the first to demonstrate involvement of the Slp family and Rab27A in dense-core vesicle exocytosis in PC12 cells. It will be interesting to determine which Slps function in granule exocytosis in cytotoxic T lymphocytes as well as platelets, where granule exocytosis is known to be defective in Rab27A mutant animals (17,18,20,21).