The Doc2 family comprises the brain-specific Doc2α and the ubiquitous Doc2β and Doc2γ. With the exception of Doc2γ, 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, Doc2α interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2α-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 Doc2α but not Doc2β was endogenously expressed in the RBL-2H3 mast cell line. Doc2α 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 Doc2α mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2α also reduced Ca2+-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2α 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 Doc2α knockout mice. These results suggest that the Doc2α-Μunc13-4 system regulates Ca2+-dependent secretory lysosome exocytosis in mast cells.
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 FcεRI 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, FcεRI 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 Doc2α, a member of the Doc2 family (29), which contains the brain-specific isoform Doc2α (30), and the ubiquitous isoforms, Doc2β (31, 32) and Doc2γ (33). Although Doc2α is known to regulate Ca2+-dependent synaptic vesicle exocytosis, the cellular roles of ubiquitous Doc2β and Doc2γ remain obscure. In PC12 cells, Ca2+-dependent regulated exocytosis is enhanced by Doc2α overexpression and reduced by Doc2α depletion (34). In addition, Doc2α-deficient mice exhibit reduced postsynaptic long-term potentiation in the hippocampal CA1 region (35). Doc2α interacts with Munc13-1 through the short N-terminal Munc13-1-interacting domain (Mid), and the Doc2α-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 Doc2α and Doc2β, but not Doc2γ, 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, Doc2α but not Doc2β was endogenously expressed in mast cells. We found that Doc2α interacts with Munc13-4 and regulates Ca2+-dependent secretory lysosome exocytosis in mast cells.
Materials and Methods
cDNA cloning of human Doc2α (GenBank/EMBL/DDBJ accession no. NM_003586) and construction of pEFBOS-HA-Doc2α, an N-terminal influenza hemagglutinin (HA)-tagged Doc2α expression plasmid, were described previously (30, 34). Doc2α-F (amino acids 1–400), Doc2α-N (amino acids 1–250), Doc2α-C (amino acids 82–400), Doc2α-Mid (amino acids 1–89), Doc2α-C2A (amino acids 82–250), and Doc2α-C2B (amino acids 215–400) cDNAs were obtained by PCR using pEFBOS-HA-Doc2α 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 Doc2α described above. Rat Doc2α 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 Doc2α. 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 [α-32P]dCTP using a RediprimeII DNA labeling kit (GE Healthcare Biosciences). After hybridization, the blot was washed at 67°C with 2× SSC containing 0.1% SDS and with 0.2× 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 Doc2α cDNA, 441 bp of rat Doc2β cDNA encoding 147 C-terminal amino acids (GenBank/EMBL/DDBJ accession no. NM_031142), 747 bp of mouse Doc2α 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 × 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 × 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.
The 21-mer siRNA duplex specific for rat Doc2α (5′-GACCAGACGUGGAUAAGAA-3′) and a control nonsilencing RNA duplex were purchased from B-Bridge and transfected into RBL-2H3 cells. In rescue experiments, rat Doc2α-specific siRNA duplex was cotransfected with knockdown-resistant human Doc2α-expression plasmid, pCIneo-3FLAG-Doc2α-F or pCIneo-3FLAG-Doc2α-C2A.
Quantitative real-time RT-PCR
Total RNA from RBL-2H3 cells transfected with control RNA or rat Doc2α 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 Doc2α to GAPDH was calculated by the relative standard curve method using Sequence Detection Software version 1.4 (Applied Biosystems). Primers for rat Doc2α 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).
C57BL/6 mice were obtained from Clea Japan. Doc2α-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 Doc2α-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 × 105 cells/ml. Surface IgE receptor expression was assessed by flow cytometry. A total of 5 × 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 FcεRIα 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 FcεRI-positive BMMCs. All experiments were performed with 5- to 9-wk-old BMMCs.
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 × 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 × 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.
Doc2α expression in the RBL-2H3 mast cell line
Among Doc2 family proteins, Doc2α 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 Doc2α and Doc2β in the rat mucosal mast cell line RBL-2H3. Because endogenous Doc2α 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 Doc2α or Doc2β. As a control, we also examined Doc2 expression in rat pheochromocytoma PC12 cells. As previously reported, Doc2α 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 Doc2α transcript was detected in RBL-2H3 cells (Fig. 1), indicating that Doc2α but not Doc2β is endogenously expressed in RBL-2H3 cells.
Colocalization of Doc2α and Munc13-4 on secretory lysosomes in RBL-2H3 cells
We next examined the intracellular localization of exogenous HA-Doc2α in RBL-2H3 cells by immunofluorescence microscopy. HA-Doc2α was distributed in the perinuclear region and the nucleus (Fig. 2,A, top row). Although the significance of the nuclear distribution of HA-Doc2α is currently unclear (42, 43), the perinuclear HA-Doc2α overlapped with CD63, a secretory lysosome marker in mast cells (Fig. 2,A, top row). CD63 distribution also colocalized with the perinuclear cytoplasmic distribution of exogenously expressed FLAG-Munc13-4 (Fig. 2,A, middle row), consistent with the previous observation that Munc13-4 colocalizes with multiple secretory lysosome markers (14). Furthermore, exogenous expression of both HA-Doc2α and FLAG-Munc13-4 showed substantial colocalization in the perinuclear region (Fig. 2,A, bottom row). Because secretory lysosomes are translocated to the cell periphery upon Ag stimulation in mast cells (5, 45), we next examined whether Doc2α was also translocated to the cell periphery upon Ag stimulation by immunofluorescence microscopy. When the sensitized RBL-2H3 cells expressing HA-Doc2α were stimulated with Ag in the Ca2+-free medium to prevent membrane fusion (5), HA-Doc2α as well as CD63 showed a similar relocation to the cell periphery (Fig. 2 B). These results collectively suggest that Doc2α localizes to secretory lysosomes together with Munc13-4 in RBL-2H3 cells.
Physical interaction between Doc2α and Munc13-4
In neurons and PC12 cells, Doc2α functions in Ca2+-dependent regulated exocytosis through an interaction with Munc13-1 (29, 36). To gain further insight into the relationship between Doc2α and Munc13-4, we examined their interaction by coimmunoprecipitation analysis using the following Doc2α constructs: Doc2α-F (amino acids 1–400, full length), Doc2α-Mid containing the Mid domain (amino acids 1–89), Doc2α-C containing the C2A and C2B domains (amino acids 82–400), Doc2α-C2A containing the C2A domain (amino acids 82–250), Doc2α-C2B containing the C2B domain (amino acids 215–400), and Doc2α-N containing the Mid and C2A domains (amino acids 1–250) (Fig. 3,A). FLAG-Munc13-4 and HA-Doc2α-F, HA-Doc2α-Mid, or HA-Doc2α-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 Doc2α and Munc13-1 (29), FLAG-Munc13-4 coimmunoprecipitated with HA-Doc2α-F and HA-Doc2α-Mid (Fig. 3,B, left panels). Furthermore, FLAG-Munc13-4 was also associated with HA-Doc2α-C (Fig. 3,B, left panels). To define the Munc13-4-binding region in Doc2α-C, we then examined the interaction of FLAG-Munc13-4 with HA-Doc2α-C2A and HA-Doc2α-C2B. FLAG-Munc13-4 specifically coimmunoprecipitated with HA-Doc2α-C2B but not with HA-Doc2α-C2A (Fig. 3,B, right panels). However, HA-Doc2α-N, which does not contain the C2B domain, was also associated with FLAG-Munc13-4 (Fig. 3 B, 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 Doc2α-Munc13-4 interaction.
Because Doc2α is a Ca2+-binding protein, we next investigated whether the Doc2α-Munc13-4 interaction was affected by Ca2+ concentration. COS7 cells coexpressing HA-Doc2α-F and FLAG-Munc13-4 were lysed in the presence of 2 mM Ca2+ or 5 mM EGTA, and subjected to coimmunoprecipitation analysis. In either case, FLAG-Munc13-4 was efficiently coprecipitated with HA-Doc2α-F (Fig. 3 C), implying that the Doc2α-Munc13-4 interaction is not affected by intracellular Ca2+ concentration.
We further examined the relationship between the Doc2α-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-Doc2α-F, FLAG-Munc13-4, and the FLAG-tagged dominant active Rab27a mutant (FLAG-Rab27aQ78L). When HA-Doc2α-F was immunoprecipitated, FLAG-Rab27aQ78L was coprecipitated only in the presence of FLAG-Munc13-4 (Fig. 3,D). In addition, the extent of FLAG-Munc13-4 coprecipitated with HA-Doc2α-F was unaffected by the expression of FLAG-Rab27aQ78L (Fig. 3 D). These results suggest that Doc2α and Rab27a can simultaneously bind to Munc13-4 and do not compete for the binding to Munc13-4.
Relationship between Doc2α and Munc13-4 in Ag-induced secretory lysosome exocytosis in RBL-2H3 cells
To examine the involvement of Doc2α in Ag-induced secretory lysosome exocytosis in RBL-2H3 cells, we first examined the exogenous expression of Doc2α and its mutants, FLAG-Doc2α-F, FLAG-Doc2α-N, and FLAG-Doc2α-C, in RBL-2H3 cells by Western blot analysis and confirmed that they were expressed at similar expression levels (Fig. 4 A).
We next monitored the time course of Ag-induced release of β-hexosaminidase, a secretory lysosome-associated enzyme, in RBL-2H3 cells. The cells were sensitized with anti-DNP IgE, stimulated with or without DNP-BSA, and then examined for β-hexosaminidase release into the medium at various time periods. Upon Ag stimulation, β-hexosaminidase was mainly released within the first 10 min followed by a slow residual release in the next 10 min, finally reaching ∼30% of the total (Fig. 4,B, +Ag). In a control experiment without stimulus, the initial β-hexosaminidase content in the medium (typically 3% of the total) increased slightly during incubation, representing the background of the assay due to spontaneous release (Fig. 4 B, −Ag).
We then performed an exocytosis assay using RBL-2H3 cells transfected with FLAG-Doc2α-F, FLAG-Doc2α-N, FLAG-Doc2α-C, or HA-Munc13-4. In our experimental conditions, FLAG-Doc2α-F, FLAG-Doc2α-N, FLAG-Doc2α-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. 4,C, 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-Doc2α-F-transfected cells was higher than in empty vector (mock)-transfected cells (Fig. 4,C). In contrast, β-hexosaminidase release was reduced in either FLAG-Doc2α-N- or FLAG-Doc2α-C-transfected cells (Fig. 4,C). 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. 4,C). Next, we examined β-hexosaminidase release in RBL-2H3 cells transfected with HA-Munc13-4 together with FLAG-Doc2α-N or FLAG-Doc2α-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-Doc2α-N-positive and FLAG-Doc2α-C-positive RBL-2H3 cells, respectively. Conversely, FLAG-Doc2α-N and FLAG-Doc2α-C proteins were detected in 91.3 ± 2.2% and 91.8 ± 4.0% of HA-Munc13-4-positive cells, respectively (Fig. 4,D, lower panels). The reduced β-hexosaminidase release in cells expressing FLAG-Doc2α-N and FLAG-Doc2α-C was restored upon coexpression of HA-Munc13-4 (Fig. 4 D), suggesting that the inhibitory effect of Doc2α-N and Doc2α-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 Doc2α in Ag-induced secretory lysosome exocytosis in RBL-2H3 cells, we designed a rat Doc2α-specific siRNA duplex (Doc2α siRNA). When the expression of Doc2α mRNA in RBL-2H3 cells was monitored by quantitative real-time RT-PCR, it was efficiently knockdowned in the Doc2α siRNA-transfected cells compared with the control nonsilencing RNA duplex (control RNA)-transfected cells (Fig. 5,A). Doc2α siRNA also efficiently reduced the expression of rat FLAG-Doc2α protein, but not human FLAG-Doc2α protein, in RBL-2H3 cells (Fig. 5,B). We then performed an exocytosis assay using Doc2α siRNA- or control RNA-transfected RBL-2H3 cells. Ag-induced β-hexosaminidase release in Doc2α siRNA-transfected cells was lower than in control RNA-transfected cells (Fig. 5,C). The reduced β-hexosaminidase release in Doc2α siRNA-transfected cells was rescued by re-expression of human FLAG-Doc2α-F but not by human FLAG-Doc2α-C2A that lacks the Munc13-4-binding regions (Fig. 5,C), suggesting the importance of Munc13-4 binding for the function of Doc2α. The expression of human FLAG-Doc2α-C2A proteins in RBL-2H3 cells was confirmed by Western blot analysis (Fig. 5 D). These results collectively suggest that Doc2α regulates Ag-induced secretory lysosome exocytosis in RBL-2H3 cells through an interaction with Munc13-4.
Expression of Doc2α 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 Doc2α, Doc2β, and Munc13-4 by Northern blot analysis, using mouse brain as a control. In the brain, Doc2α 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 Doc2α 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 Doc2α and Munc13-4 are endogenously expressed in BMMCs as in RBL-2H3 cells.
Decreased Ag-induced secretory lysosome exocytosis in Doc2α-deficient BMMCs
We next prepared BMMCs from previously described Doc2α-deficient mice (35) to clearly assess the role of Doc2α in Ag-induced secretory lysosome exocytosis in primary mast cells. Bone marrow cells isolated from WT and Doc2α-deficient (Doc2α−/−) mice were cultured for 5 wk in the presence of IL-3, and then surface expression of the IgE receptor FcεRI was assessed by flow cytometry. FcεRI expression was comparable between WT and Doc2α−/− cells, and >90% of the cells were FcεRI-positive BMMCs (Fig. 7 A), suggesting that Doc2α expression is dispensable for differentiation and proliferation of BMMCs.
We then monitored the time course of Ag-induced β-hexosaminidase release in WT BMMCs. As in RBL-2H3 cells (Fig. 4,B), β-hexosaminidase release in WT BMMCs was almost complete within the first 10 min of stimulation (Fig. 7,B, +Ag), and the initial β-hexosaminidase content in the medium (typically <5% of total) did not increase in unstimulated control cells (Fig. 7,B, −Ag). Therefore, to compare Ag-induced secretory lysosome exocytosis between WT and Doc2α−/− BMMCs, we measured β-hexosaminidase release for 10 min after stimulation with various Ag concentrations (0–100 ng/ml) and subtracted the unstimulated control values for each BMMCs. WT BMMCs showed a dose-dependent β-hexosaminidase release upon Ag stimulation (Fig. 7,C). At low Ag concentrations (12.5 ng/ml), β-hexosaminidase release was comparable between WT and Doc2α−/− BMMCs (Fig. 7,C). However, Doc2α−/− BMMCs were significantly less responsive to increasing Ag concentrations (25–100 ng/ml). At 100 ng/ml Ag, β-hexosaminidase release in Doc2α−/− BMMCs was decreased by 67 ± 4.4% compared with that of WT BMMCs (Fig. 7,C). Total intracellular β-hexosaminidase was comparable between WT and Doc2α−/− BMMCs and the unstimulated controls were not affected by the loss of Doc2α (data not shown). Furthermore, a significant reduction in Ag-induced histamine release was also observed in Doc2α−/− BMMCs (Fig. 7 D). These results suggest that Doc2α also regulates Ag-induced secretory lysosome exocytosis in primary mast cells.
Decreased secretory lysosome exocytosis in Doc2α-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 Doc2α 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 Doc2α−/− BMMCs released β-hexosaminidase (Fig. 8,A). When WT and Doc2α−/− BMMCs were stimulated with various A23187 concentrations in the presence of TPA, both BMMCs released β-hexosaminidase in an A23187 dose-dependent manner (Fig. 8,A). However, β-hexosaminidase release in response to increasing A23187 concentrations in the presence of TPA was significantly reduced in Doc2α−/− BMMCs (Fig. 8,A). At 2 μM of A23187, β-hexosaminidase release in Doc2α−/− 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 Doc2α−/− BMMCs (Fig. 8 B). These results clearly demonstrate that Doc2α regulates Ca2+-dependent secretory lysosome exocytosis in mast cells.
In this study, we investigated the role of Doc2 in secretory lysosome exocytosis in mast cells. Northern blot analysis revealed that Doc2α but not Doc2β is expressed in both RBL-2H3 cells and BMMCs. Although this result was somewhat surprising, previous analyses defining brain-specific Doc2α and ubiquitous Doc2β isoforms were performed with tissues but not individual cell types. Then, we showed that Doc2α and Munc13-4 are colocalized on secretory lysosomes in RBL-2H3 cells and that Doc2α can interact with Munc13-4. We further demonstrated that Doc2α regulates Ca2+-dependent secretory lysosome exocytosis in mast cells likely through the interaction with Munc13-4.
Our coimmunoprecipitation assay revealed that Doc2α 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 Doc2α probably interacts with the linker region of Munc13-4. In contrast, the C2B-containing region of Doc2α is likely a specific binding region for Munc13-4, because a Doc2α mutant lacking the Mid domain fails to interact with Munc13-1 (29). Our present study also showed that Doc2α 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 Doc2α, Munc13-4, and Rab27 during secretory lysosome exocytosis in mast cells.
Exogenous expression of a Doc2α 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 Doc2α-knockdowned RBL-2H3 cells was restored by re-expression of human Doc2α but not by Doc2α-C2A that lacks the Munc13-4-binding regions. Furthermore, secretory lysosome exocytosis was significantly decreased in Doc2α-deficient BMMCs compared with WT BMMCs. Collectively, these results suggest that the Doc2α-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, Doc2α 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 Doc2α-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 Doc2α 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 FcεRI activation also induces Ca2+ influx (1, 2) and secretory lysosome exocytosis in mast cells occurs at submicromolar Ca2+ concentrations (1). Both Doc2α 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 Doc2α functions as one of the Ca2+ sensors for secretory lysosome exocytosis in mast cells. The residual responsiveness to Ca2+ influx in Doc2α-deficient BMMCs suggests that other Ca2+ sensors also contribute to this process. The Ca2+-sensing activity of Doc2α in mast cells will be studied in the future.
In conclusion, we demonstrated that Doc2α 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.
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.
The authors have no financial conflict of interest.
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.
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.
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.