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Protein and Vesicle Trafficking, Cytoskeleton
1Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California; and 2Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan
Submitted 15 December 2006 ; accepted in final form 19 December 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-thio)-triphosphate, GTP, or guanosine-5'-O-(2-thiodiphosphate), binding of Rab3DWT to pIgR was inhibited. In blot overlays, recombinant Rab3DWT bound to immunoprecipitated pIgR, suggesting that Rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant Rab3DT36N in LGACs inhibited CCH-stimulated SC release, and, in CCH-stimulated LGACs, pull down of pIgR with Rab3DWT and colocalization of pIgR with endogenous Rab3D were decreased relative to resting cells, suggesting that the pIgR-Rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGACs and its secretion therefrom may be affected by its novel interaction with Rab3D. secretory component; acinar cells; transcytosis; guanine nucleotide exchange factor
Tear fluid is relatively rich in sIgA and SC, and the SC concentration in rat tear fluid is 10 times higher than its concentration in saliva (22). The lacrimal gland is the primary source of the aqueous portion of tear film that contains water, electrolytes, and proteins and is necessary for the health and maintenance of the ocular surface (25). Proteins in lacrimal gland fluid are secreted predominantly by acinar cells. We and others have previously reported the presence of pIgR and SC in lacrimar gland acinar cells (LGACs) from rabbits (28, 47), rats (57, 58), and humans (1). LGACs may therefore represent a good, physiologically relevant, organ-based model system to characterize the mechanism of regulation of pIgR trafficking and SC secretion. Thus far, the pIgR-transfected Madin-Darby canine kidney (MDCK) cell line has served as the predominant cellular model for the characterization of the molecular mechanisms that regulate the transcytosis of pIgR (52).
Key regulators of vesicular traffic are Rab proteins, members of the Ras superfamily of small-molecular-weight GTPases (19, 21, 45, 53, 67, 77). Rab proteins are known to regulate cargo selection into nascent vesicles, vesicle budding and motility, and tethering, docking, and fusion of vesicles to target organelles. More than 60 Rab proteins have been identified, and each is associated with a specific membrane compartment. Four highly homologous Rab3 isoforms (Rab3A, Rab3B, Rab3C, and Rab3D) are expressed in cells with regulated secretory pathways, and these isoforms have been shown to have both positive and negative regulatory functions in a number of steps in regulated secretion (18, 54). Of interest here, Rab3D is predominantly localized to secretory vesicles of various exocrine secretory cells, such as acinar cells of the pancreas (41, 63), parotid (41, 48), lacrimal glands (16, 41, 54, 73), and chief cells of the stomach (49, 59). However, it has also been found in other cell types, usually those associated with secretory function, such as neuroendocrine cells (5), osteoclasts (43), endothelial cells (30), alveolar type II cells (68), adipocytes (6), and mast cells (51, 61), and it has been localized to the Golgi apparatus in enterocytes and in acinar cells of Brunner's glands (64).
Functionally, Rab3D has been shown to regulate amylase secretion by pancreatic acinar cells. Overexpression of wild-type (WT) Rab3D (Rab3DWT) in transgenic mice stimulates amylase release (42), and expression of dominant negative Rab3D in isolated acini inhibits amylase release (12). On the other hand, Rab3DWT appears to block pepsinogen release from gastric chief cells (40). In addition, in Rab3D knockout mice, functional changes in secretion from exocrine cells were not detected, but these mice had twofold larger secretory granules in pancreatic and parotid acinar cells compared with those in WT cells (50), suggesting that Rab3D may play a role in secretory granule biogenesis. Thus, the role of Rab3D in regulating secretion from exocrine cells is still not well defined. The lacrimal gland, with concentrations of Rab3D twice that of Rab3A in the brain, where Rab3A is the most abundant Rab isoform (54), may represent a good model system to characterize the role of Rab3D in exocrine secretion.
A number of Rab proteins have been implicated as possible transcytotic regulators (10, 26, 29, 66, 72). Many of these Rabs are colocalized with pIgR and dIgA and regulate their trafficking through particular membrane compartments, such as the apical endosomal compartment. With respect to Rab3 isoforms, in pIgR-transfected MDCK cells, endogenous and heterologously expressed Rab3B have been shown to interact directly with pIgR and regulate the transcytosis of pIgR in a GTP-dependent fashion (66). In addition, Rab3B binding to pIgR is inhibited when dIgA is added to these transfected MDCK cells, suggesting that the Rab3B-pIgR interaction can be regulated by ligand binding and signaling pathways. In rat hepatocytes, Rab3D copurifies with pIgR in vesicles that are putative transcytotic vesicles (32, 33). Thus, depending on the cell type, Rab3 GTPases may also regulate transcytosis, and, due to the relatively high expression of both Rab3D and pIgR in lacrimal gland, these cells may also represent a good model system to characterize the role of Rab3D in the trafficking of pIgR, a marker of the transcytotic pathway.
In the work reported here, evidence is presented to support the novel finding that pIgR is a cargo protein of the regulated secretory pathway in rabbit LGACs and that its trafficking to, or secretion from, this pathway may be regulated by a direct interaction between pIgR and Rab3D. pIgR is localized by immunofluorescence to Rab3D-positive vesicles, and pIgR binds to Rab3D in pull-down assays and blot overlays. In addition, the interaction of pIgR and Rab3D is regulated by carbachol (CCH) stimulation of LGACs and by the GTP-binding state of Rab3D, suggesting that the interaction between pIgR and Rab3D may be physiologically regulated. Rab3D-dependent trafficking of pIgR into the regulated secretory pathway may occur directly from the trans-Golgi network or indirectly via the transcytotic pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-thio)-triphosphate (GTPS), GTP, guanosine-5'-O-(2-thiodiphosphate) (GDPβS), His-Select Nickel Affinity gel, donkey anti-sheep secondary antibody conjugated to FITC, rhodamine phalloidin, and other chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO). Ni2+-nitriloacetate (Ni-NTA) beads were purchased from Qiagen (Valencia, CA). Protein G-Sepharose was from Pharmacia Biotech (Alameda, CA). The PCR kit and Taq PCRx DNA polymerase (recombinant) were purchased from Invitrogen (Carlsbad, CA). The total RNA Purification kit for cells and tissues was from Gentra Systems (Valencia, CA). High Capacity cDNA Reverse Transcription kits were obtained from Applied Biosystems (Foster City, CA). Sheep anti-rabbit SC polyclonal antiserum was generated by a commercial vendor (Capralogics, Hardwick, MA) against SC purified from rabbit bile (Pel-Freeze, Rogers, AR) by preparative gel electrophoresis. The antiserum was of sufficient titer to use diluted for Western blots, immunoprecipitation, and immunofluorescence. Plasmids encoding (His)6 epitope-tagged forms of Rab3DWT, the constitutively active mutant Q81L (Rab3DQ81L), and the dominant negative mutant T36N (Rab3DT36N) were gifts from Dr. John A Williams (University of Michigan, Ann Arbor, MI). They were expressed in Escherichia coli and purified on Ni-NTA beads. Anti-Rab3D polyclonal antibodies were generated in rabbits against recombinant (His)6 epitope-tagged Rab3DWT expressed in E. coli and purified by chromatography over protein A/G agarose (Antibodies, Davis, CA).
ProLong antifade mounting kit, goat anti-rabbit secondary antibody conjugated to Alexa fluor 568 and Alexa fluor-647-phalloidin were from Molecular Probes (Eugene, OR). Goat anti-rabbit IRDye800- and donkey anti-sheep IRDye700-conjugated secondary antibodies were purchased from Rockland (Gilbertsville, PA). Cell culture reagents were from Invitrogen.
Acinar cell isolation and primary culture. Isolation of lacrimal acini from female New Zealand White rabbits (1.8–2.2 kg) obtained from Irish Farms (Norco, CA) was in accordance with the Guiding Principles for the Use of Animals in Research and approval from the institution's Institutional Animal Care and Use Committee (IACUC). Lacrimal acini were isolated as previously described (15) and cultured for 2–3 days. Cells prepared in this way aggregate into acinus-like structures while individual cells within these structures display distinct apical and basolateral domains and maintain a robust secretory response (15, 16, 73). CCH was used at a concentration of 100 µM to stimulate LGACs.
Isolation of mouse lacrimal glands and olfactory bulbs. Isolation of tissues from Rab3D knockout mice (a generous gift from Dr. Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) and C57BL/6 mice (Harland, Indianapolis, IN) was in accordance with the Guiding Principles for Use of Animals in Research and approval from the institution's IACUC. The lacrimal gland and olfactory bulb were removed from euthanized C57BL/6 mice and prepared for RT-PCR. Some fragments of the lacrimal gland from C57BL/6 and Rab3D knockout mice were fixed in 4% paraformaldehyde-4% sucrose in Dulbecco's PBS (DPBS) at room temperature for 3–4 h and then transferred to 30% sucrose in DPBS at 4°C overnight. The next morning, the fragments were dried, immersed in OCT solution (Sakura Finetek USA, Torrance, CA), rapidly frozen with liquid nitrogen, and cryosectioned at 5 µm with a Mikrom cryostat. Cryosections placed on glass coverslips were processed for confocal fluorescence microscopy.
Confocal fluorescence microscopy. Reconstituted rabbit lacrimal acini cultured on Matrigel-coated coverslips were fixed and processed as previously described (15, 16, 73). Acini were incubated with the appropriate primary and fluorophore-conjugated secondary antibodies or Alexa fluor-647-phalloidin or FITC-phalloidin.
Cryosections of mouse lacrimal glands placed on glass coverslips were sequentially incubated with 50 mM NH4Cl, 0.1% Triton X-100, and 1% SDS (each in DPBS) with washes in DPBS between each incubation. Samples were blocked with 1% BSA in DPBS and incubated with the appropriate primary and fluorophore-conjugated secondary antibodies and rhodamine phalloidin.
Most confocal images were obtained with a Zeiss LSM 510 Meta NLO imaging system equipped with argon and red and green HeNe lasers mounted on a vibration-free table and attached to an incubation chamber controlling temperature, humidity, and CO2. The ability of this system to acquire fluorescence emission signals resolved within narrow ranges in multitrack mode as well as the use of singly labeled control samples ensured the validity of colocalization experiments by assessing lack of signal bleedthrough. Analysis of the extent of colocalization between Rab3D and SC/pIgR was determined using similar to the methods described in Ref. 28. With the "Enhanced Colocalization" tool available with Zeiss LSM510 software, the thresholding function was used to establish background intensity, and a region of interest was then selected around the luminal area that was <2.0 µm beneath the apical actin. The channel representing SC/pIgR was used to calculate the colocalization coefficient (c), which was calculated as follows: c = colocalizing pixels/total pixels. The coefficient represents the ratio of the sum of intensities of colocalizing pixels with the overall sum of pixel intensities above threshold. The values ranged from 0 to 1, with 0 representing no colocalization and 1 representing complete colocalization. Panels of images were compiled in Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).
RT-PCR for Rab3 proteins. Olfactory bulbs and lacrimal glands collected from C57BL/6 mice were homogenized with a Polytron PT-2100 tissue homogenizer (Kinematica, Newark, NJ) in the lysis buffer provided in the Total RNA Purification Kit for Cells and Tissues from Gentra Systems. The protocol provided by Gentra Systems was used to prepare total RNA. RT was carried out using a protocol provided by ABI in the High Capacity cDNA RT Kit. RT was conducted with total RNA at a concentration of 1 µg/10 µl reaction at 25°C for 10 min, 37°C for 2 h, 85°C for 5 s, and 4°C overnight. The PCR was performed with a PCR kit from Invitrogen. Sequences of the primer pairs used were as follows: Rab3A, 5'-ATGAGCGAGTGGTGTCCTCA-3' and 5'-GGAGCAGCAGTGACCACAAT-3'; Rab3B, 5'-GGAAAGAGTGGTCCCAACTG-3' and 5'-TAATGGAGAGAAGCGGAGGA-3'; Rab3C, 5'-TTGGGATAATGCCCAGGTTA-3' and 5'-GGGACATTAGCAGCCACAGT-3'; and Rab3D, 5'-ACGAACGGGTCGTACCTGCT-3' and 5'-GCCCTGAGCTGAGAGACAGT-3'. PCR conditions were programmed in a GeneAmp PCR System 9700 from ABI in the order of four cycles as follows: cycle 1 (1x), 94°C for 5 min; cycle 2 (35x), 94°C for 45 s, 58°C for 45 s, and 72°C for 1 min; cycle 3 (1x), 72°C for 6 min; and cycle 4 (1x), 4°C overnight. These PCR fragments were analyzed by 1.2% agarose gel electrophoresis.
Adenoviral constructs and transduction.
Replication-defective adenoviral (Ad) constructs used in these experiments included Ad encoding (His)6-tagged Rab3DWT and green fluorescent protein (GFP) separately (Ad-Rab3dWT), Ad encoding (His)6-tagged dominant negative Rab3DT36N (Rab3DT36N) and GFP separately (Ad-Rab3DT36N), Ad encoding (His)6-tagged constitutively active Rab3DQ81L (Rab3DQ81L) and GFP separately (Ad-Rab3DQ81L), Ad encoding GFP alone (Ad-GFP) (73), and Ad encoding a syncollin-GFP fusion protein (Ad-syncollin-GFP) (a kind gift of Dr. Chris Rhodes, University of Chicago, Chicago, IL). All Ad-Rab3D constructs were generous gifts from Dr. John A. Williams (University of Michigan, Ann Arbor, MI). Reconstituted rabbit LGACs cultured for 2 days were exposed to Ad constructs for 1–2 h at a multiplicity of infection of 5, washed twice with DPBS, and then incubated in fresh culture medium for 18–20 h at 37°C and 5% CO2. On day 3 of culture, the transduction efficiency was determined by observing the GFP fluorescence. Lacrimal acinar cultures with at least an 
80% efficiency of cellular transduction, as determined by GFP expression, were used for analysis.
SC secretion analysis. Rabbit lacrimal gland acini seeded on Matrigel-coated 12-well plates were transduced on day 2 with Ad-syncollin-GFP, Ad-Rab3DWT, Ad-Rab3DT36N, Ad-Rab3DQ81L, or Ad-GFP. On day 3 of culture, untransduced or transduced lacrimal acini were stimulated with CCH (100 µM), and media were collected at time points up to 30 min. Cell pellets were dissolved in 0.5 N NaOH. Equal volumes of media were concentrated in YM-10 Microcons (Millipore, Bedford, MA) and resolved by SDS-PAGE. Proteins of interest were detected by Western blot analysis. Signal intensities were quantitated and normalized to pellet protein for each sample.
Pull down of Rab3D and pIgR.
(His)6 epitope-tagged forms of Rab3DWT, constitutively active mutant Rab3DQ81L, and dominant negative mutant Rab3DT36N were expressed in E. coli and purified on Ni-NTA bead columns. Resting or CCH-stimulated LGACs (3.6 x 107 cells each) or 5.0 x 107 MDCK cells were solubilized in a buffer containing 1% Triton X-100, 20 mM Na-HEPES (pH 7.4), and 50 mM KCl and incubated overnight at 4°C with 40 µg of recombinant Rab3DWT or mutant Rab3D. In some cases, the lysate was supplemented with 10 µM nonhydrolyzable GTPS, 10 µM nonhydrolyzable GDPβS, or 0.5 mM GTP. Rab3D was recovered from lysates by an incubation with His-Select Nickel Affinity Gel beads for 1 h at room temperature and washed. Rab3D and any interacting proteins were eluted from the beads with SDS-PAGE sample buffer and analyzed on Western blots.
Immunoprecipitation and overlay. Resting LGACs (7.2 x 107 cells) were solubilized in a buffer containing 2.5% Triton X-100, 100 mM triethanolamine (pH 8.6), 100 mM NaCl, 5 mM EDTA, and 0.02% NaN3, and pIgR was immunoprecipitated at 4°C overnight with anti-SC polyclonal antibodies covalently coupled to Protein G-Sepharose (6 µl of packed beads). After being washed, the immunoprecipitate was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was soaked in a solution containing 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.3% Tween 20, 3% BSA, and 0.05% NaN3 for 6–7 h at room temperature and then incubated with 10 µg recombinant Rab3DWT in 10 ml of a solution containing 10 mM Tris·HCl (pH 7.5), 20 mM NaCl, 1 mM DTT, and 0.5 mM EGTA at 4°C overnight. After being washed, the membrane was analyzed by Western blot analysis.
Stripping of nitrocellulose membranes. The nitrocellulose membrane was soaked in a buffer containing 100 mM 2-mercaptoethanol, 62.5 mM Tris·HCl (pH 6.8), and 2% SDS at 50°C for 30 min to strip previously bound primary and secondary antibodies. After being washed and blocked, the membrane was reprobed with the appropriate new primary and secondary antibodies and analyzed by Western blot analysis.
Western blot analysis. Western blots were processed using the appropriate primary antibodies and secondary antibodies conjugated to either IRDye-800 or IRDye-700. Blots were quantified using the Li-Cor Odyssey Scanning Infrared Fluorescence Imaging System (Lincoln, NE). For display, fluorescent signals were converted digitally to black and white images.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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pIgR and Rab3D are colocalized in resting LGACs.
Intracellular distributions of pIgR/SC and Rab3D were analyzed by confocal fluorescence microscopy of primary cultures of reconstituted lacrimal acini. The polyclonal anti-SC antiserum recognizes both full-length pIgR and SC. Analysis of the immunofluorescence associated with pIgR/SC (Fig. 2, left, green) revealed that pIgR/SC immunoreactivity was distributed on the basolateral membrane, and additional pIgR/SC immunoreactivity was detected in large apparent vesicles or organelles localized beneath the apical plasma membrane (Fig. 2, arrows) surrounding the luminal regions (Fig. 2, asterisk). Rab3D, a regulated secretory vesicle marker (Fig. 2, middle, red), was also localized to this subapical region in similar structures. In the merged image (Fig. 2, right), there appeared to be significant colocalization of pIgR/SC and Rab3D signals near the apical membrane. Quantitation of the colocalization between the signals from the two proteins revealed that 40% of total cellular Rab3D pixels were colocalized with those of pIgR/SC (Fig. 2, right, yellow). These results are the first to show the colocalization of pIgR/SC with a marker protein of regulated secretory vesicles (Rab3D) and suggest that pIgR/SC is a cargo protein packaged into regulated secretory vesicles in LGACs.
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S or 0.5 mM GTP was added to the lysates used in the pull-down assays with Rab3DWT, binding of pIgR was also significantly inhibited, with only 9 ± 5% (mean ± SE; n = 7 independent experiments) and 25 ± 8% (mean ± SE; n = 9 independent experiments), respectively, of the amount of pIgR bound to Rab3DWT. Interestingly, incubation of Rab3DWT with GDPβS in the pull-down assay also inhibited pIgR binding (Fig. 5, C and D). These data suggest that the interaction of pIgR with Rab3D is sensitive to the GTP-bound state of Rab3D. The GTP-bound form of Rab3D, whether induced by a mutation or by the addition of GTP or GTPS, appears to be incapable of interacting with pIgR. The interaction of pIgR with Rab3D is more complex with the GDP-bound form of Rab3D, since the T36N mutation allows mutated Rab3D to bind effectively to pIgR, whereas the addition of GDPβS to Rab3DWT inhibits the binding of pIgR.
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S or GTP included in the pull-down assays. The results shown in Fig. 5E demonstrate that the binding of Rab3D and pIgR can be replicated in pIgR-transfected MDCK cells. In addition, Rab3DWT did not bind to pIgR when GTPS or GTP was added in pull-down assays. Thus, the binding characteristics of Rab3D and pIgR from LGACs can be reproduced with pIgR-transfected MDCK cells, indicating that the interaction of Rab3D with pIgR may be independent of the source of pIgR-containing cell lysates.
Direct interaction between Rab3D and pIgR.
The pull-down assays did not distinguish whether the interaction between Rab3D and pIgR was a direct or an indirect one. Thus, blot overlay assays (far Western blots) were performed to test whether Rab3D and pIgR can interact directly. pIgR was immunoprecipitated from LGACs, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. The blot was then incubated with recombinant Rab3DWT and probed with anti-Rab3D antibody to detect where Rab3D had bound to the blot. As shown in Fig. 6, left lane, two proteins, one migrating with an Mr of 120 kDa and the other at 130 kDa, showed significant binding to Rab3D. To confirm whether the lower Mr band was pIgR, the blot was stripped of Rab3DWT and reprobed with anti-SC antibody to detect pIgR. As shown in Fig. 6, right lane, anti-SC antibodies reacted with the protein migrating at 120 kDa. Upon comparison of both blots, the 120-kDa protein binding to Rab3D and anti-SC immunoreactivity coincided precisely. Immunoprecipitated SC, migrating around 80 kDa, did not bind to Rab3D. These results are consistent with a direct interaction between Rab3D and pIgR, and this interaction appears to be dependent on the presence of the cytoplasmic and/or membrane-spanning domain of pIgR.
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Acute treatment of cholinergic agonist abolishes Rab3D-pIgR binding. We (28) have previously reported that 100 µM CCH stimulated the acute (30 min) release of SC from LGACs. CCH may then regulate the interaction of pIgR with Rab3D, ultimately to regulate SC release from LGACs. Stimulation of LGAC secretion with 100 µM CCH for 30 or 60 min before lysis, followed by an incubation of lysates with recombinant Rab3DWT in pull-down assays, resulted in a loss of pIgR binding to Rab3DWT from 100% (0 min) to 43 ± 15% (30 min) and 26 ± 13% (60 min) (means ± SE; n = 5 independent preparations; Fig. 8, A and B). Although there was a decrease in pIgR content in acini after stimulation by CCH (Fig. 8, C and D), the magnitude and time course of this decrease differed from those observed in pull-down assays, suggesting that the overall decrease in pIgR content in acini appears to be unlikely to account for the decrease in pIgR binding to Rab3D in pull-down assays from CCH-stimulated cells. Thus, the CCH-dependent physiological signaling pathway in LGACs modulates the interaction between Rab3D and pIgR. The CCH-dependent loss of interaction between Rab3D and pIgR could provide a mechanism for the observed stimulation of SC secretion in LGACs, particularly if the Rab3D-pIgR interaction negatively regulates the terminal steps in SC release.
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DISCUSSION |
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2 (69). In studies with mutants of Rab3A, Rab3AT36N has been shown to bind to GDP but exhibits a very high dissociation rate for GDP, up to 60 times higher than that for WT Rab3A (8). In addition, this particular mutant does not bind GTP (8), nor does the Rab3DT36N mutant (X. Chen, unpublished observations), suggesting that it may spend a significant amount of time in the nucleotide-free state in vitro and in vivo. Finally, Rab3AT36N has a 10-fold higher affinity for the Rab3A GEF (Rab3A-guanine nucleotide-releasing factor) compared with WT Rab3A (8). Thus, if pIgR is a GEF for Rab3D, and if the Rab3DT36N mutant behaves similarly to the identical mutant of Rab3A, then, in the pull-down assays, 1) binding of pIgR to Rab3DWT should be inhibited in the presence of either GTP or GDP and 2) pIgR would bind better to the T36N mutant compared with nucleotide-free or nucleotide-bound Rab3DWT. Both of these predicted results were observed here. In addition, in vivo, the observed inhibition of SC secretion by Ad-mediated expression of Rab3DT36N is consistent with this mutant interacting better with pIgR to form unproductive interactions. These data raise the intriguing possibility that pIgR may act as an exchange factor for Rab3D. Such an effector function would have two important functional outcomes: 1) Rab3D would be targeted to membranes containing pIgR, such as mature secretory granules and the trans-Golgi network; and 2) Rab3D would be activated by exchanging GDP for GTP at the site of action. Testing whether pIgR is a GEF for Rab3D is currently underway. Once in the GTP-bound form, Rab3D may then recruit effectors for vesicle translocation, vesicle fusion, or sorting of nascent pIgR to new regulated secretory granules. In other cell types, there is significant evidence to support a role for Rab3D GTPases in vesicle docking or fusion (5, 11, 35, 40, 54) and/or secretory granule biogenesis (43, 64).
Alternatively, in previous studies of the inhibitory effect of the expression of the Rab3DT36N mutant on the early phase of amylase secretion by pancreatic acinar cells, it was shown that the expression of this mutant reduces the level of GTP-bound endogenous Rab3D (12), suggesting that it inhibits GDP/GTP exchange on endogenous Rab3D. Expression of WT or constitutively active Rab3DQ81L did not alter levels of GTP-bound endogenous Rab3D and did not affect amylase secretion. Thus, the effects of expression of these proteins in pancreatic acinar cells may be due to their effects on GDP/GTP exchange on endogenous Rab3D, and their similar effects on SC secretion by LGAC may be due to similar mechanisms.
On the other hand, the variable effects of Rab3D overexpression in LGACs may reflect the functional redundancy of other Rab3 isoforms expressed in LGACs. We detected both Rab3D and Rab3A by RT-PCR analysis of mouse LGAC mRNA; however, at the protein level, another study (54) could only detect Rab3D in lacrimal glands. We could not independently determine protein levels of Rab3A in rabbit LGACs. Nonetheless, the issue of functional redundancy has been well documented for Rab3 isoforms (54) as well as the differing and sometimes opposite effects of overexpression of Rab3D and its mutants in regulating secretion from other exocrine cells (11, 40, 42). Finally, the variable effects of overexpression may also be due to the regulation of more than one step of pIgR trafficking by Rab3D along the merocrine secretory pathway.
The findings reported here clearly differ in several fundamental aspects from the study of van Ijzendoorn et al. (66), in which Rab3B was shown to bind to pIgR in a manner sensitive to the GTP-bound state of Rab3B and also to the binding of pIgR ligand dIgA. In contrast to our results with Rab3D, they reported that the GTP-bound form of Rab3B interacted with pIgR. This difference may reflect the different functional roles of Rab3B and Rab3D in cell types in which these Rabs and pIgR are expressed. Another difference is that while they could reconstitute Rab3B binding to the cytoplasmic domain of pIgR in vitro, in those same experiments, they did not detect an interaction between Rab3D and pIgR. Since Rab3B does not appear to be expressed in lacrimal acini, we have not independently verified that Rab3B binds to pIgR in our system; however, we did verify that recombinant Rab3D could bind to pIgR from transfected MDCK cells in pull-down assays. Thus, we interpret these data to suggest that, since both Rab3B and Rab3D can bind to pIgR directly, there may be a common motif shared by Rab3B and Rab3D that mediates binding to pIgR. Indeed, van Ijzendoorn et al. have shown that the membrane-proximal 14 amino acids of the cytoplasmic domain of pIgR are necessary for the Rab3B-pIgR interaction; interestingly, this region also contains the basolateral targeting motif of pIgR (4). Alternatively, we might also speculate that both Rab3B and Rab3D bind to pIgR, but to different motifs on the cytoplasmic domain of the pIgR, and the nature of these interactions are sensitive to the types of fusion proteins used with the in vitro binding assays. Specifically, the previous study used glutathione-S-transferase fusions of Rab3B and the pIgR cytoplasmic domain, whereas here (His)6-tagged versions of Rab3D and either solubilized pIgR from cell lysates or immunoprecipitated pIgR were used.
Having acknowledged the differences between these two studies, it is probably more instructive to focus on their similarities. The first is the identification and characterization of a direct interaction between a Rab protein and its putative cargo. There are still only a few other cargo proteins that have been shown to interact with Rab proteins directly, such as Rab5A with the angiotensin-1 receptor (55), Rab11A with the transient receptor potential (TRP)V5 and TRPV6 Ca2+ channels (65), and Rab21 with -integrin (44).
The second similarity between the van Ijzendoorn et al. study and ours is that the interaction between the Rab3 isoform and pIgR is sensitive to the GTP-binding state of the Rab3 isoform, suggesting that both interactions are functional ones. Third, the interaction between the Rab3 isoform and pIgR is also sensitive to physiological stimuli. In the case of Rab3B, it is dIgA, and, with Rab3D in lacrimal acinar cells, it is CCH. In both cases, these stimuli either prevent binding or stimulate the dissociation of the Rab3 isoform and pIgR. Interestingly, both dIgA and CCH stimulate the elevation of intracellular Ca2+ in their respective cell types (9, 78), and, therefore, intracellular Ca2+ may be an important regulator of Rab3-dependent pIgR trafficking. It will be of significant interest in future studies to characterize the signaling pathway stimulated by dIgA binding to pIgR and to characterize the effect of dIgA on Rab3D-pIgR interactions in LGACs.
The localization of pIgR to mature secretory granules in LGACs suggests that pIgR has a motif that targets it to the regulated secretory pathway. Given the heterogeneity of the few motifs that have been characterized in proteins that are targeted to regulated secretory granules (31, 37, 60, 70, 74), scanning the pIgR sequence for such motifs has not yielded any useful information. Thus, the secretory granule-targeting motif in pIgR will need to be independently characterized. The assays used here to characterize the interaction of Rab3D with pIgR may be valuable in characterizing this motif, and the LGAC system may be a good model system in which to pursue this line of investigation. In addition, the intracellular route taken by pIgR on its way to regulated secretory vesicles has yet to be characterized. Is pIgR delivered to the regulated secretory pathway directly from the trans-Golgi network, similar to other tear proteins (76), or is there a novel route to regulated secretory vesicles from the transcytotic pathway (Fig. 10) ? Since LGACs are not cultured in the presence of dIgA, dIgA would apparently not be necessary for the trafficking of pIgR to secretory vesicles. What would be the purpose of a storage pool for pIgR in secretory granules? Perhaps it is to secure a source for the generation of free SC in the presence of a relative abundance of dIgA. In addition to sIgA, free SC is highly abundant in the tear film, and most of the SC and sIgA in tears is produced by the lacrimal gland. It has been proposed that the free SC may play a significant role in mucosal immunity (46) by providing a scavenger function by binding to microorganisms in mucosal fluids (17, 20), binding to IL-8 to regulate neutrophil function (34), or regulating eosinophil function (39). All of these functions are certainly relevant to the secretion of SC into tears by LGACs to effect its protection of ocular surfaces.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* E. Evans and W. Zhang contributed equally to this work (listed alphabetically). 
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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(CCH-CON). B: comparison of SC released into culture medium in LGACs transduced with either Ad-GFP or dominant negative Ad-Rab3DT36N. SC release was analyzed as in A. C: comparison of SC release into culture medium in LGACs transduced with Ad-GFP or constitutively active Ad-Rab3DQ81L. SC release was analyzed as in A. Results are from 6–8 independent experiments and are means ± SE. *Significant at P 
0.05.
