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Doc2 and Munc13-4 Regulate Ca²⁺-Dependent Secretory Lysosome Exocytosis in Mast Cells¹

Hironori Higashio^*, Noriyuki Nishimura^*, Hiroyoshi Ishizaki, Jun Miyoshi, Satoshi Orita^*,, Ayuko Sakane^* and Takuya Sasaki^2,*

^* Department of Biochemistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan; and Pharmaceutical Research Division, Shionogi and Company, Osaka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The Doc2 family comprises the brain-specific Doc2 and the ubiquitous Doc2β and Doc2. With the exception of Doc2, these proteins exhibit Ca²⁺-dependent phospholipid-binding activity in their Ca²⁺-binding C2A domain and are thought to be important for Ca²⁺-dependent regulated exocytosis. In excitatory neurons, Doc2 interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2-Munc13-1 system is implicated in Ca²⁺-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca²⁺-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2 but not Doc2β was endogenously expressed in the RBL-2H3 mast cell line. Doc2 colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca²⁺-binding C2B domain. In RBL-2H3 cells, Ca²⁺-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2 mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2 also reduced Ca²⁺-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2 but not by its mutant that could not bind to Munc13-4. Moreover, Ca²⁺-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2 knockout mice. These results suggest that the Doc2-Munc13-4 system regulates Ca²⁺-dependent secretory lysosome exocytosis in mast cells.

	Introduction

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Mast cells are immune cells of hematopoietic lineage that play an important role in allergic inflammation. Ag-mediated cross-linking of the high-affinity IgE receptor FcRI induces Ca²⁺-dependent exocytosis of secretory granules, resulting in the release of inflammatory mediators (1, 2). Secretory granules of mast cells contain lysosomal components including β-hexosaminidase and CD63 as well as inflammatory mediators such as histamine (3), and are therefore referred to as secretory lysosomes (4). Upon Ag stimulation, FcRI activation triggers a series of events leading to Ca²⁺ influx, protein kinase C activation, and microtubule-dependent translocation of secretory lysosomes to the plasma membrane (1, 2, 5). Similar to other Ca²⁺-dependent regulated exocytic pathways, the fusion of secretory lysosomes with the plasma membrane is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)³ family proteins in response to Ca²⁺ influx (1, 6, 7).

In addition to SNARE proteins, Rab family small G proteins and Munc13 family proteins are implicated in secretory lysosome exocytosis. Secretory lysosome exocytosis in mast cells, CTLs, and platelets commonly requires Rab27 and Munc13-4 (8, 9, 10, 11, 12, 13, 14, 15). There are >60 Rab proteins in mammalian cells that regulate discrete steps in the vesicular transport pathway (16, 17, 18). The Rab27 subfamily consists of two isoforms, the widely expressed Rab27a and the more restrictedly expressed Rab27b (16, 19), which function in many non-neuronal regulated secretory events (15, 20, 21, 22, 23). The Munc13 family contains three brain-specific isoforms: Munc13-1, Munc13-2, and Munc13-3, and a non-neuronal isoform Munc13-4 expressed in the lung, spleen, and hematopoietic cells (10, 14, 24, 25).

Among Munc13 proteins, Munc13-4 acts as one of the Rab27a target proteins (12, 13, 14). The essential roles of Rab27a and Munc13-4 in secretory lysosome exocytosis in CTLs are manifested in Griscelli syndrome type 2 and familial hemophagocytic lymphohistiocytosis (FHL) type 3 (FHL3), respectively (10, 26). However, the precise mechanism of the Rab27a-Munc13-4 system is still largely unknown.

In contrast to Munc13-4, Munc13-1 has an essential role in priming synaptic vesicles for a fusion competent state in excitatory neurons (27, 28). Munc13-1 interacts with Doc2, a member of the Doc2 family (29), which contains the brain-specific isoform Doc2 (30), and the ubiquitous isoforms, Doc2β (31, 32) and Doc2 (33). Although Doc2 is known to regulate Ca²⁺-dependent synaptic vesicle exocytosis, the cellular roles of ubiquitous Doc2β and Doc2 remain obscure. In PC12 cells, Ca²⁺-dependent regulated exocytosis is enhanced by Doc2 overexpression and reduced by Doc2 depletion (34). In addition, Doc2-deficient mice exhibit reduced postsynaptic long-term potentiation in the hippocampal CA1 region (35). Doc2 interacts with Munc13-1 through the short N-terminal Munc13-1-interacting domain (Mid), and the Doc2-Munc13-1 interaction is crucial for the enhancement of Ca²⁺-dependent regulated exocytosis in PC12 cells and superior cervical ganglion neurons (29, 36).

Doc2 proteins contain an N-terminal Mid domain and tandem Ca²⁺-binding C2A and C2B domains. The C2 domain is found in >50 proteins including the Munc13 family proteins, most of which are involved in signal transduction and Ca²⁺-dependent regulated exocytosis. Many C2 domains exhibit Ca²⁺-dependent phospholipid-binding activity, but some act as protein- or inositol phospholipid-binding modules (37, 38, 39). The C2A domain in Doc2 and Doc2β, but not Doc2, exhibits Ca²⁺-dependent phospholipid-binding activity (30, 32, 33, 40). Although the C2B domain is well-conserved among the Doc2 family proteins (33), its function remains unclear.

In this study, we examined whether Doc2 functions with Munc13-4 in Ca²⁺-dependent secretory lysosome exocytosis in mast cells. To our surprise, Doc2 but not Doc2β was endogenously expressed in mast cells. We found that Doc2 interacts with Munc13-4 and regulates Ca²⁺-dependent secretory lysosome exocytosis in mast cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Plasmid construction

cDNA cloning of human Doc2 (GenBank/EMBL/DDBJ accession no. NM_003586) and construction of pEFBOS-HA-Doc2, an N-terminal influenza hemagglutinin (HA)-tagged Doc2 expression plasmid, were described previously (30, 34). Doc2-F (amino acids 1–400), Doc2-N (amino acids 1–250), Doc2-C (amino acids 82–400), Doc2-Mid (amino acids 1–89), Doc2-C2A (amino acids 82–250), and Doc2-C2B (amino acids 215–400) cDNAs were obtained by PCR using pEFBOS-HA-Doc2 as a template. The mammalian expression vector, pCIneo-HA or pCIneo-3FLAG, was used to express the N-terminal HA-tagged or FLAG-tagged human Doc2 described above. Rat Doc2 cDNA (GenBank/EMBL/DDBJ accession no. NM_022937) was obtained by PCR using the rat brain cDNA library as a template, and the pCIneo-3FLAG vector was used to express the N-terminal FLAG-tagged rat Doc2. cDNA cloning of mouse Munc13-4 (GenBank/EMBL/DDBJ accession no. NM_001009573) and construction of p3FLAG-Munc13-4, an N-terminal FLAG-tagged Munc13-4 expression plasmid, were described previously (12). The mammalian expression vector pCIneo-3HA was also used to express the N-terminal HA-tagged Munc13-4. cDNA cloning of mouse Rab27a (GenBank/EMBL/DDBJ accession no. NM_023635) and generation of its dominant active mutant Rab27aQ78L were described previously (12), and the pCIneo-3FLAG vector was used to express the N-terminal FLAG-tagged Rab27aQ78L. All cDNAs obtained by PCR were sequenced using an ABI Prism 3100 genetic analyzer (Applied Biosystems).

Northern blot analysis

Total RNA from cultured cells and homogenized tissues was isolated using TRIzol (Invitrogen Life Technologies). Total RNA (25 µg) was separated on a 1% formaldehyde/agarose gel and transferred to a Hybond N nylon membrane (GE Healthcare Biosciences). The blot was hybridized at 67°C overnight in hybridization buffer (0.25 M Na₂HPO₄ (pH 7.2), 1 mM EDTA, 7% SDS) (41), with a DNA probe labeled with [-³²P]dCTP using a RediprimeII DNA labeling kit (GE Healthcare Biosciences). After hybridization, the blot was washed at 67°C with 2x SSC containing 0.1% SDS and with 0.2x SSC containing 0.1% SDS. The signals were visualized using a BAS2000 image analyzer (Fuji Photo Film). Probes were obtained by RT-PCR or by PCR and sequenced using an ABI Prism 3100 genetic analyzer (Applied Biosystems). The following probes were used: 1209 bp of full-length rat Doc2 cDNA, 441 bp of rat Doc2β cDNA encoding 147 C-terminal amino acids (GenBank/EMBL/DDBJ accession no. NM_031142), 747 bp of mouse Doc2 cDNA encoding 80 C-terminal amino acids (GenBank/EMBL/DDBJ accession no. BC055768) plus the 3'-untranslated region, and 2028 bp of mouse Munc13-4 cDNA encoding amino acids 239–914.

Cell culture and transfection

COS7 and RBL-2H3 cells were maintained in DMEM (Sigma-Aldrich) containing 10% FBS (Sigma-Aldrich), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies) at 37°C, 5% CO₂ (for RBL-2H3) or 10% CO₂ (for COS7). PC12 cells were maintained in DMEM containing 10% FBS, 5% horse serum (PAA Laboratories), and the antibiotics at 37°C, 5% CO₂. WEHI-3 cells were maintained in RPMI 1640 medium (Sigma-Aldrich) containing 10% FBS, 0.1 mM MEM-nonessential amino acids (Invitrogen Life Technologies), 1 mM sodium pyruvate (Sigma-Aldrich) and the antibiotics (complete RPMI 1640 medium) at 37°C, 5% CO₂, and the cultured supernatant was collected and filtered at every passage. COS7 cells (4 x 10⁵ cells per 60-mm dish) were transiently transfected with 2 µg per plasmid using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies), and RBL-2H3 cells (5 x 10⁶ cells) were transfected with 5 µg per plasmid or with 300 pM of small-interfering RNA (siRNA) duplex by Nucleofector II device (Amaxa) using program T-020 with Amaxa Cell Line Nucleofector kit R. After transfection, the cells were cultured for 36–48 h before use.

RNA interference

The 21-mer siRNA duplex specific for rat Doc2 (5'-GACCAGACGUGGAUAAGAA-3') and a control nonsilencing RNA duplex were purchased from B-Bridge and transfected into RBL-2H3 cells. In rescue experiments, rat Doc2-specific siRNA duplex was cotransfected with knockdown-resistant human Doc2-expression plasmid, pCIneo-3FLAG-Doc2-F or pCIneo-3FLAG-Doc2-C2A.

Quantitative real-time RT-PCR

Total RNA from RBL-2H3 cells transfected with control RNA or rat Doc2 siRNA duplex was isolated by BioRobot EZ1 device (Qiagen) using the EZ1 RNA Universal Tissue kit (Qiagen) and was reverse-transcribed using the Quantitect Reverse Transcription kit (Qiagen). Real-time PCR analysis was then performed with an ABI 7500 Real-Time PCR system (Applied Biosystems) using FastStart Universal SYBR Green Master (Roche Diagnostics). All of these procedures were performed according to the manufacturer’s instructions. Each sample was analyzed in triplicate for each pair of primers. The relative expression of Doc2 to GAPDH was calculated by the relative standard curve method using Sequence Detection Software version 1.4 (Applied Biosystems). Primers for rat Doc2 were 5'-AGCTAGAGCAGGCAGAGCAG-3' (forward) and 5'-AGGTGAGCACAGCGAACAAT-3' (reverse) and rat GAPDH (GenBank/EMBL/DDBJ accession no. BC087743) were 5'-ATGACTCTACCCACGGCAAG-3' (forward) and 5'-CTGGAAGATGGTGATGGGTT-3' (reverse).

Mice

C57BL/6 mice were obtained from Clea Japan. Doc2-deficient mice were on a C57BL/6 background and were established as described previously (35). The mice were maintained under pathogen-free conditions and handled in accordance with the Guidelines for Animal Experimentation of Tokushima University Graduate School.

Preparation of bone marrow-derived mast cells (BMMCs) and flow cytometry

Bone marrow cells were isolated from the femurs and tibias of wild-type (WT) C57BL/6 and Doc2-deficient mice and grown in complete RPMI 1640 medium supplemented with 45% WEHI-3-cultured supernatant as a source of IL-3 (BMMC medium) at 37°C, 5% CO₂. Nonadherent cells were transferred to fresh BMMC medium every 4 days and diluted to a concentration of 2 x 10⁵ cells/ml. Surface IgE receptor expression was assessed by flow cytometry. A total of 5 x 10⁵ cells were washed with analysis buffer (PBS containing 1% BSA and 0.2% sodium azide), resuspended in 100 µl, and incubated with or without 0.05 µg of FITC-conjugated Armenian hamster monoclonal anti-mouse FcRI Ab (MAR-1; eBioscience) on ice for 30 min. The cells were washed twice with analysis buffer and then analyzed using an EPICS XL-MCL flow cytometer (Beckman Coulter). After 5 wk of culture, >90% of the cells were FcRI-positive BMMCs. All experiments were performed with 5- to 9-wk-old BMMCs.

Immunofluorescence microscopy

Transfected RBL-2H3 cells grown on cover slips were fixed with 2% formaldehyde in PBS for 15 min and washed three times with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min, and washed three times with PBS. After blocking with 5% goat serum (Sigma-Aldrich) in PBS, the cells were incubated with primary Abs for 1 h, washed three times with PBS, and then incubated with Alexa 488- or Alexa 594-conjugated secondary Abs (Invitrogen Life Technologies) for 1 h. For staining the nucleus, the cells were incubated with 1 µg/ml 4,6-diamino-2-phenylindole in PBS for 5 min. After washing three times with PBS, the cells were mounted on glass slides. The following primary Abs were used: rat monoclonal anti-HA (3F10; Roche Diagnostics), mouse monoclonal anti-FLAG (M2; Sigma-Aldrich), rabbit polyclonal anti-FLAG (Sigma-Aldrich), and mouse monoclonal anti-rat CD63 (AD1; BD Biosciences). Fluorescent images were acquired using a Radiance 2000 confocal laser-scanning microscope (Bio-Rad) or a C1plus confocal laser-scanning microscope (Nikon).

For Ag stimulation, transfected RBL-2H3 cells on cover slips were sensitized at 37°C, 5% CO₂ for 2 h with 0.5 µg/ml anti-DNP mouse monoclonal IgE (Yamasa) in DMEM containing 10% FBS. After washing twice with PBS containing 10 mM EGTA, the cells were stimulated at 37°C, 5% CO₂ for 10 min with 100 ng/ml DNP-BSA (Calbiochem) in the Ca²⁺-free MEM (Sigma-Aldrich) containing 10 mM EGTA, and subjected to immunofluorescence microscopy as described above.

Transfection efficiency was assessed by counting the number of positive cells vs total number of cells in randomly selected fields. The data were shown as mean ± SD of these ratios in independent transfections.

Immunoprecipitation and Western blotting

Transfected COS7 and RBL-2H3 cells were washed twice with PBS and lysed at 4°C for 15 min with lysis buffer (25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 1 mM MgCl₂, 10 µg/ml (4-amidinophenyl)-methanesulfonyl fluoride, and 10 µg/ml leupeptin) or lysis buffer containing either 2 mM CaCl₂ or 5 mM EGTA. After removing cell debris, the lysates were incubated at 4°C overnight with a mouse anti-HA (12CA5; Roche Diagnostics) Ab bound to protein G-Sepharose beads (GE Healthcare Biosciences). The beads were washed four times with each lysis buffer and boiled in SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 1% SDS, 10% glycerol, and 0.01% bromphenol blue) containing 0.1 M DTT. Lysates and/or immunoprecipitates were subjected to SDS-PAGE and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skim milk in TBST, the membrane was incubated with anti-HA (3F10), anti-FLAG (M2), or mouse monoclonal anti-β-actin (AC-74; Sigma-Aldrich) Ab. After washing the membrane with TBST, the immunoreactive proteins were detected by ECL (ECL-plus kit; GE Healthcare Biosciences) with a HRP-coupled secondary Ab (Jackson ImmunoResearch Laboratories).

Assays for β-hexosaminidase and histamine release

Transfected RBL-2H3 cells (6 x 10⁵ cells) were sensitized with 0.5 µg/ml anti-DNP IgE in DMEM containing 10% FBS at 37°C, 5% CO₂ for 2 h. After washing twice with RPMI 1640 medium without phenol red (Sigma-Aldrich), the cells were stimulated with or without 100 ng/ml DNP-BSA in a final volume of 1.5 ml. During a 37°C, 5% CO₂ incubation, aliquots (30 µl) of the supernatant were harvested at the indicated time periods. At the end of the assay, the residual supernatant was discarded and the cells were lysed with an equal volume of 0.5% Triton X-100 in phenol red-free RPMI 1640 medium. To determine β-hexosaminidase activity, aliquots (30 µl) of the supernatants and the cell lysates were incubated with 50 µl of substrate solution (1.3 mg/ml p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) in 0.1 M citrate (pH 4.5)) at 37°C for 1 h. The reaction was terminated by the addition of stop buffer (0.2 M NaOH, 0.2 M glycine) and absorbance was read at 405 nm (A₄₀₅) using a Beckman DU640 spectrophotometer (Beckman Coulter). The degree of β-hexosaminidase release was calculated by dividing the A₄₀₅ of the supernatant by the sum of the A₄₀₅ in the supernatant and the cell lysate and expressed as a percentage of the total activity. For the BMMC assay, 3 x 10⁵ cells were sensitized with 1 µg/ml anti-DNP IgE in complete RPMI 1640 medium at 37°C, 5% CO₂ for 6 h. After washing with phenol red-free RPMI 1640 medium, the cells were stimulated with the indicated concentrations of DNP-BSA in a final volume of 100 µl. Alternatively, unsensitized BMMCs were washed and directly stimulated with 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich) and the indicated concentrations of A23187 (Calbiochem) in a final volume of 100 µl. The cells were incubated at 37°C, 5% CO₂ for the indicated time periods, then chilled on ice for 5 min to stop β-hexosaminidase release. Supernatants and cell pellets were separated by centrifugation at 4°C, and the cell pellets were lysed with 100 µl of 0.5% Triton X-100 in phenol red-free RPMI 1640 medium. The degree of β-hexosaminidase release was determined as described above. Histamine release was also assessed using a Histamine ELISA kit (Oxford Biomedical Research) according to the manufacturer’s instructions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Doc2 expression in the RBL-2H3 mast cell line

Among Doc2 family proteins, Doc2 and Doc2β show Ca²⁺-dependent phospholipid-binding activity and are thought to be important for Ca²⁺-dependent regulated exocytosis (30, 32, 40, 42, 43, 44). To determine whether Doc2 proteins are involved in secretory lysosome exocytosis in mast cells, we first examined expression of Doc2 and Doc2β in the rat mucosal mast cell line RBL-2H3. Because endogenous Doc2 and Doc2β proteins could not be detected by Western blot or immunofluorescence microscopy using our Abs, Doc2 expression in RBL-2H3 cells was examined by Northern blot analysis using cDNA probes specific for Doc2 or Doc2β. As a control, we also examined Doc2 expression in rat pheochromocytoma PC12 cells. As previously reported, Doc2 and Doc2β were detected as a single transcript with apparent sizes of 2.5 and 5 kb, respectively, in PC12 cells (Fig. 1) (32, 34). However, only the Doc2 transcript was detected in RBL-2H3 cells (Fig. 1), indicating that Doc2 but not Doc2β is endogenously expressed in RBL-2H3 cells.

View larger version (102K): [in this window] [in a new window]   FIGURE 1. Doc2 expression in the RBL-2H3 mast cell line. Total RNA (25 µg) prepared from RBL-2H3 and PC12 cells was analyzed by Northern blot using cDNA probes specific for Doc2, Doc2β, or β-actin. The positions of 28S rRNA (28S), 18S rRNA (18S), and mRNA transcripts (arrowheads) are indicated. The approximate sizes of 28S and 18S rRNAs are 4.5 and 1.9 kb, respectively.

Colocalization of Doc2 and Munc13-4 on secretory lysosomes in RBL-2H3 cells

We next examined the intracellular localization of exogenous HA-Doc2 in RBL-2H3 cells by immunofluorescence microscopy. HA-Doc2 was distributed in the perinuclear region and the nucleus (Fig. 2A, top row). Although the significance of the nuclear distribution of HA-Doc2 is currently unclear (42, 43), the perinuclear HA-Doc2 overlapped with CD63, a secretory lysosome marker in mast cells (Fig. 2A, top row). CD63 distribution also colocalized with the perinuclear cytoplasmic distribution of exogenously expressed FLAG-Munc13-4 (Fig. 2A, middle row), consistent with the previous observation that Munc13-4 colocalizes with multiple secretory lysosome markers (14). Furthermore, exogenous expression of both HA-Doc2 and FLAG-Munc13-4 showed substantial colocalization in the perinuclear region (Fig. 2A, bottom row). Because secretory lysosomes are translocated to the cell periphery upon Ag stimulation in mast cells (5, 45), we next examined whether Doc2 was also translocated to the cell periphery upon Ag stimulation by immunofluorescence microscopy. When the sensitized RBL-2H3 cells expressing HA-Doc2 were stimulated with Ag in the Ca²⁺-free medium to prevent membrane fusion (5), HA-Doc2 as well as CD63 showed a similar relocation to the cell periphery (Fig. 2B). These results collectively suggest that Doc2 localizes to secretory lysosomes together with Munc13-4 in RBL-2H3 cells.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. Intracellular localization of Doc2 in RBL-2H3 cells. A, Intracellular localization of Doc2 in RBL-2H3 cells. RBL-2H3 cells were transfected with pEFBOS-HA-Doc2 (top row), p3FLAG-Munc13-4 (middle row), or both (bottom row). After 48 h, the cells were fixed and coimmunostained with anti-HA (3F10) and anti-CD63 (AD1) (top row), anti-FLAG (polyclonal), and anti-CD63 (AD1) (middle row), or anti-HA (3F10) and anti-FLAG (M2) Abs (bottom row). Merged images are shown on the right of each row with magnified images. Bar, 30 µm. B, Intracellular localization of Doc2 in RBL-2H3 cells upon Ag stimulation. RBL-2H3 cells transfected with pEFBOS-HA-Doc2 were sensitized with 0.5 µg/ml anti-DNP IgE for 2 h and stimulated with 100 ng/ml DNP-BSA for 10 min in the Ca2+-free medium containing 10 mM EGTA. The cells were fixed and coimmunostained with anti-HA (3F10) and anti-CD63 (AD1) Abs. A merged image is shown in the right panel and the arrows indicate the colocalization of Doc2 with CD63 at the cell periphery. Bar, 30 µm.

Physical interaction between Doc2 and Munc13-4

In neurons and PC12 cells, Doc2 functions in Ca²⁺-dependent regulated exocytosis through an interaction with Munc13-1 (29, 36). To gain further insight into the relationship between Doc2 and Munc13-4, we examined their interaction by coimmunoprecipitation analysis using the following Doc2 constructs: Doc2-F (amino acids 1–400, full length), Doc2-Mid containing the Mid domain (amino acids 1–89), Doc2-C containing the C2A and C2B domains (amino acids 82–400), Doc2-C2A containing the C2A domain (amino acids 82–250), Doc2-C2B containing the C2B domain (amino acids 215–400), and Doc2-N containing the Mid and C2A domains (amino acids 1–250) (Fig. 3A). FLAG-Munc13-4 and HA-Doc2-F, HA-Doc2-Mid, or HA-Doc2-C were coexpressed in COS7 cells and the cell lysates were immunoprecipitated with an anti-HA Ab. Immunoprecipitates were then analyzed by Western blot with anti-HA and anti-FLAG Abs. As expected from the interaction between Doc2 and Munc13-1 (29), FLAG-Munc13-4 coimmunoprecipitated with HA-Doc2-F and HA-Doc2-Mid (Fig. 3B, left panels). Furthermore, FLAG-Munc13-4 was also associated with HA-Doc2-C (Fig. 3B, left panels). To define the Munc13-4-binding region in Doc2-C, we then examined the interaction of FLAG-Munc13-4 with HA-Doc2-C2A and HA-Doc2-C2B. FLAG-Munc13-4 specifically coimmunoprecipitated with HA-Doc2-C2B but not with HA-Doc2-C2A (Fig. 3B, right panels). However, HA-Doc2-N, which does not contain the C2B domain, was also associated with FLAG-Munc13-4 (Fig. 3B, right panels). Collectively, these results suggest that both the N-terminal region containing the Mid domain (amino acids 1–89) and the C-terminal region containing the C2B domain (amino acids 215–400) are important for the Doc2-Munc13-4 interaction.

View larger version (54K): [in this window] [in a new window]   FIGURE 3. Interaction between Munc13-4 and Doc2. A, Schematic representation of mutant Doc2 constructs. Doc2 domains and amino acid numbers corresponding to truncation boundaries are indicated for each construct. Mid, Munc13-1-interacting domain; C2A, the first Ca2+-binding C2 domain; C2B, the second Ca2+-binding C2 domain. B, Identification of the Munc13-4-binding regions of Doc2. COS7 cells were cotransfected with p3FLAG-Munc13-4 and the noted pCIneo-HA-based plasmids expressing the HA-Doc2 proteins shown in A. After 48 h, the cells were lysed with lysis buffer and immunoprecipitated with an anti-HA (12CA5) Ab. The lysates (Lysate) and immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-HA (3F10) (Doc2) or anti-FLAG (M2) (Munc13-4) Ab. *, The L chain of the IgG used for immunoprecipitation. C, Ca2+-independent interaction between Munc13-4 and Doc2. COS7 cells were cotransfected with p3FLAG-Munc13-4 and pCIneo-HA-Doc2-F as indicated. After 48 h, the cells were lysed with lysis buffer (Buffer), or lysis buffer containing either 2 mM CaCl2 (2 mM Ca2+) or 5 mM EGTA (5 mM EGTA). Immunoprecipitation and analysis of the lysates (Lysate) and immunoprecipitates (IP) were performed as in B. D, Simultaneous interaction of Munc13-4 with Doc2 and the active form of Rab27a. COS7 cells were cotransfected with p3FLAG-Munc13-4, pCIneo-HA-Doc2-F, and pCIneo-3FLAG-Rab27aQ78L as indicated. Lysate preparation and immunoprecipitation were performed as in B and the analysis of the lysates (Lysate) and immunoprecipitates (IP) were performed by immunoblotting with anti-HA (3F10) (Doc2) or anti-FLAG (M2) (Munc13-4, Rab27aQ78L) Ab.

We further examined the relationship between the Doc2-Munc13-4 and Rab27a-Munc13-4 interactions, because Munc13-4 is a target protein of Rab27a (12, 13, 14). To this end, we performed coimmunoprecipitation analysis with the lysate from COS7 cells coexpressing HA-Doc2-F, FLAG-Munc13-4, and the FLAG-tagged dominant active Rab27a mutant (FLAG-Rab27aQ78L). When HA-Doc2-F was immunoprecipitated, FLAG-Rab27aQ78L was coprecipitated only in the presence of FLAG-Munc13-4 (Fig. 3D). In addition, the extent of FLAG-Munc13-4 coprecipitated with HA-Doc2-F was unaffected by the expression of FLAG-Rab27aQ78L (Fig. 3D). These results suggest that Doc2 and Rab27a can simultaneously bind to Munc13-4 and do not compete for the binding to Munc13-4.

Relationship between Doc2 and Munc13-4 in Ag-induced secretory lysosome exocytosis in RBL-2H3 cells

To examine the involvement of Doc2 in Ag-induced secretory lysosome exocytosis in RBL-2H3 cells, we first examined the exogenous expression of Doc2 and its mutants, FLAG-Doc2-F, FLAG-Doc2-N, and FLAG-Doc2-C, in RBL-2H3 cells by Western blot analysis and confirmed that they were expressed at similar expression levels (Fig. 4A).

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Effects of Doc2 mutants expression on Ag-induced secretory lysosome exocytosis in RBL-2H3 cells. A, Expression of FLAG-Doc2 proteins in RBL-2H3 cells. Cells were transfected with pCIneo-3FLAG (Mock), pCIneo-3FLAG-Doc2-F (Doc2-F), pCIneo-3FLAG-Doc2-N (Doc2-N), or pCIneo-3FLAG-Doc2-C (Doc2-C). After 48 h, the cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted with an anti-FLAG (M2) Ab (IB). A Coomassie brilliant blue staining of PVDF membrane is shown as a loading control (CBB). The arrowheads denote FLAG-Doc2-F, FLAG-Doc2-N, and FLAG-Doc2-C, respectively. B, Time course of β-hexosaminidase release in RBL-2H3 cells. Cells were sensitized with 0.5 µg/ml anti-DNP IgE for 2 h and stimulated with (+Ag) or without (–Ag) 100 ng/ml DNP-BSA for the indicated time periods. β-hexosaminidase activity in the supernatants and the cell lysates were measured and the degree of release was expressed as a percentage of the total activity. C, Effects of expression of Doc2 mutants and Munc13-4 on Ag-induced β-hexosaminidase release. RBL-2H3 cells transfected with pCIneo-3FLAG (Mock), pCIneo-3FLAG-Doc2-F (Doc2-F), pCIneo-3FLAG-Doc2-N (Doc2-N), pCIneo-3FLAG-Doc2-C (Doc2-C), or pCIneo-3HA-Munc13-4 (Munc13-4) were sensitized with 0.5 µg/ml anti-DNP IgE for 2 h and stimulated with 100 ng/ml DNP-BSA for 10 min. Control experiments were done without DNP-BSA. The degree of β-hexosaminidase release was calculated as described above and the unstimulated control values (typically <3% release) were subtracted for each transfectant. Immunofluorescence images of RBL-2H3 cells expressing FLAG-Doc2 protein (red) or HA-Munc13-4 (green) are shown in the lower panels. The nucleus was visualized by 4,6-diamino-2-phenylindole staining. Bar, 30 µm. D, Effects of Munc13-4 expression on reduced β-hexosaminidase release in RBL-2H3 cells expressing a Doc2 mutant. The degree of Ag-induced β-hexosaminidase release in RBL-2H3 cells transfected with pCIneo-3FLAG (Mock), pCIneo-3FLAG-Doc2-N (Doc2-N), or pCIneo-3FLAG-Doc2-C (Doc2-C) alone, or with combinations of mutant Doc2 plasmid and pCIneo-3HA-Munc13-4 (Doc2-N+Munc13-4, Doc2-C+Munc13-4) was examined as in C. Merged immunofluorescence images of RBL-2H3 cells coexpressing FLAG-Doc2 protein (red) and HA-Munc13-4 (green) are shown in the lower panels. Bar, 30 µm. The data of β-hexosaminidase release shown in B–D are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01 compared with the mock-transfectant; ##, p < 0.01 compared with the Doc2 mutant transfectant, as determined by Student’s t test.

We then performed an exocytosis assay using RBL-2H3 cells transfected with FLAG-Doc2-F, FLAG-Doc2-N, FLAG-Doc2-C, or HA-Munc13-4. In our experimental conditions, FLAG-Doc2-F, FLAG-Doc2-N, FLAG-Doc2-C, and HA-Munc13-4 proteins were detected by immunofluorescence microscopy in 66.5 ± 6.5%, 63.0 ± 5.3%, 65.7 ± 4.8%, and 56.6 ± 2.7% of RBL-2H3 cells, respectively (Fig. 4C, lower panels). To precisely compare Ag-induced secretory lysosome exocytosis among transfectants, we measured β-hexosaminidase release for 10 min with or without Ag and subtracted the values of the unstimulated controls for each transfectant. β-hexosaminidase release in FLAG-Doc2-F-transfected cells was higher than in empty vector (mock)-transfected cells (Fig. 4C). In contrast, β-hexosaminidase release was reduced in either FLAG-Doc2-N- or FLAG-Doc2-C-transfected cells (Fig. 4C). Consistent with the previous reports (12, 14), the increased β-hexosaminidase release in HA-Munc13-4-transfected cells was also observed in our assay (Fig. 4C). Next, we examined β-hexosaminidase release in RBL-2H3 cells transfected with HA-Munc13-4 together with FLAG-Doc2-N or FLAG-Doc2-C. In our experimental conditions, HA-Munc13-4 protein was detected by immunofluorescence microscopy in 72.0 ± 4.9% and 71.5 ± 5.7% of FLAG-Doc2-N-positive and FLAG-Doc2-C-positive RBL-2H3 cells, respectively. Conversely, FLAG-Doc2-N and FLAG-Doc2-C proteins were detected in 91.3 ± 2.2% and 91.8 ± 4.0% of HA-Munc13-4-positive cells, respectively (Fig. 4D, lower panels). The reduced β-hexosaminidase release in cells expressing FLAG-Doc2-N and FLAG-Doc2-C was restored upon coexpression of HA-Munc13-4 (Fig. 4D), suggesting that the inhibitory effect of Doc2-N and Doc2-C is potentially suppressed by Munc13-4. Total intracellular β-hexosaminidase was comparable among transfectants and the unstimulated controls were not affected in any transfectant (data not shown).

To gain further insights into the involvement of Doc2 in Ag-induced secretory lysosome exocytosis in RBL-2H3 cells, we designed a rat Doc2-specific siRNA duplex (Doc2 siRNA). When the expression of Doc2 mRNA in RBL-2H3 cells was monitored by quantitative real-time RT-PCR, it was efficiently knockdowned in the Doc2 siRNA-transfected cells compared with the control nonsilencing RNA duplex (control RNA)-transfected cells (Fig. 5A). Doc2 siRNA also efficiently reduced the expression of rat FLAG-Doc2 protein, but not human FLAG-Doc2 protein, in RBL-2H3 cells (Fig. 5B). We then performed an exocytosis assay using Doc2 siRNA- or control RNA-transfected RBL-2H3 cells. Ag-induced β-hexosaminidase release in Doc2 siRNA-transfected cells was lower than in control RNA-transfected cells (Fig. 5C). The reduced β-hexosaminidase release in Doc2 siRNA-transfected cells was rescued by re-expression of human FLAG-Doc2-F but not by human FLAG-Doc2-C2A that lacks the Munc13-4-binding regions (Fig. 5C), suggesting the importance of Munc13-4 binding for the function of Doc2. The expression of human FLAG-Doc2-C2A proteins in RBL-2H3 cells was confirmed by Western blot analysis (Fig. 5D). These results collectively suggest that Doc2 regulates Ag-induced secretory lysosome exocytosis in RBL-2H3 cells through an interaction with Munc13-4.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. Effects of Doc2 knockdown on Ag-induced secretory lysosome exocytosis in RBL-2H3 cells. A, Knockdown of Doc2 mRNA expression in RBL-2H3 cells. Total RNA was prepared from RBL-2H3 cells transfected with a nonsilencing control RNA or a rat Doc2-specific siRNA and the relative mRNA expression of Doc2 to GAPDH was determined by quantitative real-time RT-PCR analysis. The mean expression of Doc2 mRNA in the control RNA (siControl)-transfected cells was set to 1 and the relative expression in the rat Doc2-specific siRNA (siDoc2)-transfected cells was presented as means ± SD of triplicate determinations. B, Knockdown of FLAG-Doc2 protein expression in RBL-2H3 cells. Cells were transfected with the control RNA (siControl) or the rat Doc2-specific siRNA (siDoc2) together with the rat FLAG-Doc2 expression plasmid (rDoc2) or the human FLAG-Doc2 expression plasmid (hDoc2) as indicated. Lysate preparation and immunoblotting with anti-FLAG (M2) Ab (Doc2) were performed as in Fig. 4A. Anti-β-actin immunoblots are shown as loading controls (β-actin). C, Effects of knockdown and re-expression of Doc2 on Ag-induced β-hexosaminidase release in RBL-2H3 cells. Cells were transfected with the control RNA (siControl) or the rat Doc2-specific siRNA (siDoc2) alone, or with combinations of rat Doc2-specific siRNA and either of human Doc2 plasmid, pCIneo-3FLAG-Doc2-F, or pCIneo-3FLAG-Doc2-C2A (siDoc2 + hDoc2-F, siDoc2 + hDoc2-C2A). The degree of β-hexosaminidase release was examined as in Fig. 4C. The data shown are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01 compared with the siControl transfectant; #, p < 0.05 compared with the siDoc2-transfectant, as determined by Student’s t test. D, Expression of human FLAG-Doc2-C2A in RBL-2H3 cells. Cells transfected with either pCIneo-3FLAG (Mock) or pCIneo-3FLAG-Doc2-C2A (hDoc2-C2A) were lysed and subjected to immunoblotting with anti-FLAG (M2) Ab (IB) as in Fig. 4A. A Coomassie brilliant blue staining of PVDF membrane is shown as a loading control (CBB). The arrowhead denotes human FLAG-Doc2-C2A.

Expression of Doc2 and Munc13-4 in BMMCs

To examine whether Doc2 proteins are involved in Ag-induced secretory lysosome exocytosis not only in RBL-2H3 cells but also in primary mast cells, we prepared BMMCs from WT mice and examined expression of Doc2, Doc2β, and Munc13-4 by Northern blot analysis, using mouse brain as a control. In the brain, Doc2 was expressed as two transcripts with apparent sizes of 2.5 and 1.7 kb and Doc2β appeared as a single 5 kb transcript (Fig. 6), consistent with previous analyses on mouse, rat, and human tissues (30, 31, 32, 34, 46). However, only the Doc2 transcripts could be detected in BMMCs (Fig. 6) as in RBL-2H3 cells (Fig. 1). In addition, the Munc13-4-specific probe detected a single 4.5-kb transcript in BMMCs but not in the brain (Fig. 6). Both the transcript size and the absence of Munc13-4 in the brain are consistent with a previous analysis in rat tissues (24). These results indicate that both Doc2 and Munc13-4 are endogenously expressed in BMMCs as in RBL-2H3 cells.

View larger version (107K): [in this window] [in a new window]   FIGURE 6. Expression of Doc2 and Munc13-4 in BMMCs. Total RNA (25 µg) prepared from WT BMMCs (BMMC) and mouse brain (Brain) was analyzed by Northern blot using cDNA probes specific for Doc2, Doc2β, or Munc13-4. The positions of 28S rRNA (28S), 18S rRNA (18S), and mRNA transcripts (arrowheads) are indicated. The approximate sizes of 28S and 18S rRNAs are 4.5 and 1.9 kb, respectively.

Decreased Ag-induced secretory lysosome exocytosis in Doc2-deficient BMMCs

We next prepared BMMCs from previously described Doc2-deficient mice (35) to clearly assess the role of Doc2 in Ag-induced secretory lysosome exocytosis in primary mast cells. Bone marrow cells isolated from WT and Doc2-deficient (Doc2^–/–) mice were cultured for 5 wk in the presence of IL-3, and then surface expression of the IgE receptor FcRI was assessed by flow cytometry. FcRI expression was comparable between WT and Doc2^–/– cells, and >90% of the cells were FcRI-positive BMMCs (Fig. 7A), suggesting that Doc2 expression is dispensable for differentiation and proliferation of BMMCs.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Ag-induced secretory lysosome exocytosis in WT and Doc2-deficient BMMCs. A, IgE receptor expression on BMMCs from WT and Doc2-deficient (Doc2–/–) mice. Cells were stained with (black line) or without (gray line) FITC-conjugated anti-mouse FcRI (MAR-1) Ab and the fluorescence intensity was analyzed by flow cytometry. B, Time course of β-hexosaminidase release in WT BMMCs. Cells were sensitized with 1 µg/ml anti-DNP IgE for 6 h and stimulated with (+Ag) or without (–Ag) 100 ng/ml DNP-BSA for the indicated time periods. β-hexosaminidase activity in the supernatants and the cell lysates was measured and the degree of release was expressed as a percentage of the total activity. C, Dose-dependent effects of Ag on β-hexosaminidase release in WT and Doc2–/– BMMCs. Cells were sensitized with 1 µg/ml anti-DNP IgE for 6 h and stimulated with the indicated concentrations of DNP-BSA for 10 min. Control experiments were done without DNP-BSA. The degree of β-hexosaminidase release was calculated as described above and the unstimulated control values (typically <5% release) were subtracted for each BMMCs. D, Ag-induced histamine release in WT and Doc2–/– BMMCs. Cells were sensitized with 1 µg/ml anti-DNP IgE for 6 h and stimulated with 100 ng/ml DNP-BSA for 10 min. Control experiments were done without DNP-BSA. Histamine content in the supernatants and the cell lysates was determined by ELISA. The degree of histamine release was expressed as a percentage of the total content and the unstimulated control values (typically <10% release) were subtracted for each BMMCs. The data shown in B–D are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01; ns, not significant, as determined by Student’s t test compared with WT BMMCs.

Doc2

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Decreased secretory lysosome exocytosis in Doc2-deficient BMMCs upon stimulation with phorbol ester and Ca²⁺ ionophore

In addition to Ag stimulation, secretory lysosome exocytosis in mast cells can be triggered by Ca²⁺ influx and protein kinase C activation using a Ca²⁺ ionophore and phorbol ester. Therefore, we examined the role of Doc2 in secretory lysosome exocytosis induced by the phorbol ester TPA and the Ca²⁺ ionophore A23187 in BMMCs. In the absence of A23187, neither WT nor Doc2^–/– BMMCs released β-hexosaminidase (Fig. 8A). When WT and Doc2^–/– BMMCs were stimulated with various A23187 concentrations in the presence of TPA, both BMMCs released β-hexosaminidase in an A23187 dose-dependent manner (Fig. 8A). However, β-hexosaminidase release in response to increasing A23187 concentrations in the presence of TPA was significantly reduced in Doc2^–/– BMMCs (Fig. 8A). At 2 µM of A23187, β-hexosaminidase release in Doc2^–/– BMMCs was decreased by 56.5 ± 5.7% compared with that of WT BMMCs. Furthermore, a significant reduction in histamine release was also observed in Doc2^–/– BMMCs (Fig. 8B). These results clearly demonstrate that Doc2 regulates Ca²⁺-dependent secretory lysosome exocytosis in mast cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 8. Secretory lysosome exocytosis in WT and Doc2-deficient BMMCs stimulated with phorbol ester and Ca2+ ionophore. A, β-hexosaminidase release in WT and Doc2-deficient (Doc2–/–) BMMCs stimulated with phorbol ester and Ca2+ ionophore. Cells were stimulated with 50 nM TPA and the indicated concentrations of A23187 for 10 min. Control experiments were done without both TPA and A23187. β-hexosaminidase activity in the supernatants and the cell lysates was determined and the degree of stimulus-induced release was calculated as in Fig. 7C. B, Histamine release in WT and Doc2–/– BMMCs stimulated with phorbol ester and Ca2+ ionophore. Cells were stimulated with 50 nM TPA and 2 µM A23187 for 10 min. Control experiments were done without both TPA and A23187. Histamine content in the supernatants and the cell lysates was determined and the degree of stimulus-induced release was calculated as in Fig. 7D. The data shown are means ± SD of three independent experiments. **, p < 0.01; ***, p < 0.001, as determined by Student’s t test compared with WT BMMCs.

	Discussion

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Our coimmunoprecipitation assay revealed that Doc2 interacts with Munc13-4 through the N-terminal region containing the Mid domain and the C-terminal region containing the C2B domain in a Ca²⁺-independent manner. Munc13-4 has C2 domains separated by a long linker region containing two Munc13-homology domains, a domain alignment that is well-conserved among Munc13 family proteins (24). The linker region of Munc13-1 was previously identified as the Doc2-interacting domain (29). Thus, the Mid-containing region of Doc2 probably interacts with the linker region of Munc13-4. In contrast, the C2B-containing region of Doc2 is likely a specific binding region for Munc13-4, because a Doc2 mutant lacking the Mid domain fails to interact with Munc13-1 (29). Our present study also showed that Doc2 and Rab27a can simultaneously bind to Munc13-4 and do not compete for the binding to Munc13-4. Further studies are required to understand the exact mode of interaction between Doc2, Munc13-4, and Rab27 during secretory lysosome exocytosis in mast cells.

Exogenous expression of a Doc2 mutant lacking either of the Munc13-4-binding regions inhibited secretory lysosome exocytosis in RBL-2H3 cells, which was restored by exogenous expression of Munc13-4. In addition, the decreased secretory lysosome exocytosis in Doc2-knockdowned RBL-2H3 cells was restored by re-expression of human Doc2 but not by Doc2-C2A that lacks the Munc13-4-binding regions. Furthermore, secretory lysosome exocytosis was significantly decreased in Doc2-deficient BMMCs compared with WT BMMCs. Collectively, these results suggest that the Doc2-Munc13-4 system regulates Ca²⁺-dependent secretory lysosome exocytosis in mast cells.

The Munc13 family proteins are suggested to regulate the trans-SNARE complex formation between the vesicle SNARE and the target membrane SNARE that mediates vesicle fusion to the target membrane (47). Munc13-1 is required for priming the plasma membrane-docked synaptic vesicles for a fusion competent state (27, 48). Genetic studies in Caenorhabditis elegans demonstrate that synaptic transmission defects in the unc-13 mutant can be restored by overexpression of an open form of syntaxin (49); therefore, Munc13-1 is suggested to induce the assembly of trans-SNARE complexes by promoting conformational opening of the target membrane SNARE syntaxin1. This priming activity is also suggested for Munc13-4 based on observations that lytic granules correctly dock at the immunological synapse but fail to fuse with the plasma membrane in Munc13-4-deficient CTLs from FHL3 patients (10). Because syntaxin11 is identified as the gene responsible for FHL4 (50) and is also involved in secretory lysosome exocytosis in CTLs and NK cells (51, 52), Munc13-4 is thought to regulate conformational opening of the target membrane SNARE syntaxin 11 in CTLs (53). In mast cells, Doc2 may accelerate Munc13-4-mediated secretory lysosome priming on the plasma membrane after microtubule-dependent movement of secretory lysosomes (5, 54). Recently, the ternary SNARE complex composed of the vesicle soluble N-ethylmaleimide-sensitive fusion protein attachment protein, vesicle-associated membrane protein-2, and the target membrane SNAREs—syntaxin4 and SNAP-23, was identified in lipid rafts during secretory lysosome exocytosis in mast cells (55). As syntaxin4 and SNAP-23 are important for secretory lysosome exocytosis (56, 57, 58), it will be important to determine whether the Doc2-Munc13-4 complex regulates the formation of this SNARE complex.

Secretory lysosome exocytosis in mast cells requires elevated intracellular Ca²⁺ concentrations to execute SNARE-mediated membrane fusion (1, 6, 7), indicating the involvement of Ca²⁺ sensors. Synaptotagmin (Syt) I acts as a Ca²⁺ sensor for synaptic vesicle exocytosis in neuronal cells (59) and SytVII is a Ca²⁺ sensor for lytic granule exocytosis in CTLs (60); therefore, the most likely Ca²⁺ sensor in mast cells is a Syt that is a Ca²⁺-binding transmembrane protein with tandem C2 domains. There are at least 15 Syt proteins in mammalian cells (59). Although SytII, SytIII, SytV, and SytIX are expressed (61, 62), it is still unclear which Syt acts as Ca²⁺ sensor for secretory lysosome exocytosis in mast cells (61, 62, 63). Calmodulin has been shown to act as a Ca²⁺ sensor for secretory lysosome exocytosis in mast cells (64). In this study, we showed that Doc2 is required for Ca²⁺-dependent secretory lysosome exocytosis in response to both increasing Ag concentrations and increasing concentrations of A23187 in the presence of TPA. Like A23187 treatment, Ag-mediated FcRI activation also induces Ca²⁺ influx (1, 2) and secretory lysosome exocytosis in mast cells occurs at submicromolar Ca²⁺ concentrations (1). Both Doc2 and Doc2β are activated at submicromolar Ca²⁺ concentrations and exert their plasma membrane-binding activity in neurons, which is thought to be important for regulating neuronal activity (42, 44). Collectively, our results suggest that Doc2 functions as one of the Ca²⁺ sensors for secretory lysosome exocytosis in mast cells. The residual responsiveness to Ca²⁺ influx in Doc2-deficient BMMCs suggests that other Ca²⁺ sensors also contribute to this process. The Ca²⁺-sensing activity of Doc2 in mast cells will be studied in the future.

In conclusion, we demonstrated that Doc2 and Munc13-4 regulate Ca²⁺-dependent secretory lysosome exocytosis in mast cells. This is the first report that Doc2 functions in Ca²⁺-dependent regulated exocytosis in non-neuronal cells.

	Acknowledgments

 
We are grateful to Dr. M. Matsumoto (Institute for Enzyme Research, University of Tokushima, Tokushima, Japan) for helpful suggestions in preparing BMMCs. We thank Y. Okamura (Support Center for Advanced Medical Sciences, University of Tokushima, Tokushima, Japan) for excellent technical support in flow cytometry.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants-in-Aid for Scientific Research (18790204 to H.H., 18590271 to N.N., and 15079207, 18390089 to T.S.) from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.

² Address correspondence and reprint requests to Dr. Takuya Sasaki, Department of Biochemistry, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail address: sasaki{at}basic.med.tokushima-u.ac.jp

³ Abbreviations used in this paper: SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; FHL, familial hemophagocytic lymphohistiocytosis; Mid, Munc13-1-interacting domain; HA, hemagglutinin; siRNA, small-interfering RNA; BMMC, bone marrow-derived mast cell; PVDF, polyvinylidene difluoride; TPA, 12-O-tetradecanoylphorbol-13-acetate; Syt, synaptotagmin.

Received for publication June 25, 2007. Accepted for publication January 21, 2008.

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