Secretogranin III Directs Secretory Vesicle Biogenesis in Mast Cells in a Manner Dependent upon Interaction with This information is current as of October 4, 2021. Prerna Prasad, Angel A. Yanagihara, Andrea L. Small-Howard, Helen Turner and Alexander J. Stokes J Immunol 2008; 181:5024-5034; ; doi: 10.4049/jimmunol.181.7.5024

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Secretogranin III Directs Secretory Vesicle Biogenesis in Mast Cells in a Manner Dependent upon Interaction with Chromogranin A1

Prerna Prasad,2*§ Angel A. Yanagihara,2‡ Andrea L. Small-Howard,2* Helen Turner,2,3*† and Alexander J. Stokes2*†¶

Mast cells are granular immunocytes that reside in the body’s barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results Downloaded from show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1–210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis http://www.jimmunol.org/ suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1. The Journal of Immunology, 2008, 181: 5024–5034.

he granin family (Chromogranin A (CgA),4 Chromo- pacity, low affinity, calcium binding proteins that are closely as- granin B (CgB), Secretogranin (Sg) II, and the less well sociated with calcium-regulated intracellular ion channels T studied Secretogranins III-VII) comprises a group of (10–13). Moreover, emerging evidence suggests that these pro- acidic proteins that are present in the secretory granules of a wide teins play an active role in directing the biosynthesis of secretory variety of endocrine and neuro-endocrine cells (1–4). The molec- granules. This biological role for granins has been proposed on the by guest on October 4, 2021 ular functions of the granins are poorly understood. There is evi- basis of work in neuroendocrine cells, where several granin pro- dence that granins may be the precursors of biologically active teins are expressed and have been shown to cooperatively contrib- peptides (3, 5–9). However, granins cannot be considered simply ute to secretory vesicle biogenesis (14, 15). This cooperation is as precursors of granule cargo. They may act as helper proteins in exemplified by the interaction between SgIII and CgA (15, 16). In the packaging of peptide and neuro-peptides (3, 5–9). this study, an intermolecular interaction between the two granins is The EF-hand containing CgA and CgB may also act as high ca- needed for their targeting to secretory granules. Courel and coau- thors (15) have described a “granulogenic domain” in CgA that promotes the biogenesis of secretory granules probably through *Center for Biomedical Research at The Queen’s Medical Center, Honolulu, HI 96813; †Department of Cell and Molecular Biology, John A. Burns School of Med- exerting control over cholesterol sequestration and the dynamics of icine, University of Hawaii, Honolulu, HI 96813; ‡Pacific Bioscience Research Cen- intracellular vesicle budding/fusion. The potential contribution of ter, University of Hawaii, Honolulu, HI 96922; §Graduate Program in Microbiology, University of Hawaii, Honolulu, HI 96822; and ¶Program in Molecular Biosciences granins to the granularity of secretory immunocytes has not been and Bioengineering, University of Hawaii, Honolulu, HI 96822 addressed. Received for publication April 26, 2007. Accepted for publication July 23, 2008. Mast cells are densely granular, secretory leukocytes that reside The costs of publication of this article were defrayed in part by the payment of page in tissues (17, 18). In response to a variety of stimuli, including charges. This article must therefore be hereby marked advertisement in accordance immunological challenge, exposure to secretagogues and exposure with 18 U.S.C. Section 1734 solely to indicate this fact. to physical stimuli, mast cells release preformed, and de novo syn- 1 This work was supported by the Leahi Fund for Pulmonary Research (awards to thesized, inflammatory mediators into their surrounding tissues. H.T. and A.J.S.), McKee Fund (to A.J.S.), NIH RO1 GM 070634 (to H.T.) and NCRR INBRE P20RR016467 (sub-award to H.T.). The preformed mediators are housed within membrane-delimited 2 Current affiliations: H.T., Chaminade University of Honolulu; A.J.S., Center for secretory granules. The contents of mast cell secretory granules Cardiovascular Research, John. A. Burns School of Medicine, University of Hawaii; include inflammatory mediators such as histamine, serotonin, P.P., Institute for Biogenesis Research, John. A. Burns School of Medicine, Univer- ␣ sity of Hawaii; A. S.-H., AMDL, Tustin, CA. platelet activating factor, some cytokines (TNF- ), and matrix- 3 Address correspondence and reprint requests to Dr. Helen Turner, Castle Science active proteases of the chymase and tryptase families (17, 18). Center 116, Chaminade University, 3140 Waialae Avenue, Honolulu HI 96816. Together, these mediators exert diverse proinflammatory effects E-mail address: [email protected] or [email protected] upon tissue. These include the promotion of vasodilation, induc- 4 Abbreviations used in this paper: CgA, chromogranin A; CgB, chromogranin B; Sg, tion of inflammatory pain, and recruitment of other immunocytes secretogranin; w/v, weight-to-volume ratio; RT, room temperature. to the inflammatory site. The central role of these granules in in- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 flammation has led to an intense interest in the signaling www.jimmunol.org The Journal of Immunology 5025

mechanisms that control granule exocytosis, because targets for anti- For production of TRex HEK293 cells with inducible expression of inflammatory therapeutics could lie within this system. Moreover, the SgIII constructs, parental cells were electroporated with 15 ␮g of plasmid ␮ mechanisms that control the biogenesis and abundance of these gran- DNA in a 500 l volume of DMEM/10% FBS/2 mM glutamine. Electro- porations were performed at 280 V/950 ␮F in a 4-mm path length cuvette ules are also of therapeutic interest, but remain poorly studied. (BioRad). Clonal cell lines were selected by limiting dilution in 400 ␮g/ml Mast cell granules are structurally, and functionally, related to zeocin and 5 ␮g/ml blasticidin, and screened by immunoprecipitation and the dense core vesicles of neuro-endocrine cells and neurons. Western blot. Transfected gene expression was induced using 1 ␮g/ml There has been no published analysis of the expression, or func- tetracycline for 16 h at 37°C. Whenever stable cell lines were produced, steps were taken to avoid the tion, of granins in the mast cell system, although their involvement possibility that experiments would be performed on progeny cells derived in secretory vesicle biogenesis in other neuroendocrine cells and from an original parental clone. After electroporation, a brief (12–14 h) neurons has been established (6, 7, 14, 19–23). In the current re- recovery period for the cells was used such that no discernable cell division port, we tested the idea that SgIII drives the production of mast cell took place. Recovered cells were trypsinized to a single cell suspension to secretory granules. Moreover, we propose that its interaction with disperse any potential daughter cell pairs. The trypsinized cell suspension was subjected to a limiting dilution (plated in a 96-well plate at less than CgA is critical to this biological role. Although SgIII and CgA one cell per well, (i.e., 0.5 cells per 250 ␮l medium)). Each well is there- proteins are abundant in various mast cells, we show that SgIII is fore unlikely to have more than one cell, but of course across the 960 wells depleted from the cytosol after stimulation of mast cells via either plated there will occasionally be wells where more than one cell is dis- pharmacological or immunological methods. Moreover, SgIII lev- pensed. After 3 wk, typically 5–15 wells on a given 96-well plate had visible colonies (i.e., colonies at the 100–200 cell level). Before these els in mast cells recover during the poststimulation refractory colonies were harvested and transferred to a larger culture vessel, the wells phase during which mast cells replenish their granule population. were inspected microscopically and any wells in which more than one Over-expression of SgIII in mast cells causes an increase in the colony appears were discarded. After harvesting, great care was taken to number of mast cell granules, and thus when SgIII-expressing mast avoid cross-contamination of the clonal cell lines. Depending on the effi- Downloaded from cells are stimulated we note an increase in the release of proin- ciency of the transfection, we observed between five to fifteen clonal col- onies per 96-well plate. Between five and ten individual clones were se- flammatory mediators. Our results also confirm that CgA interacts lected at random, expanded, and cryopreserved. These clones were with SgIII via a specific granulogenic domain and that the granu- screened for expression of the transfected gene by immunoprecipitation logenic activity of SgIII in mast cells depends upon its ability to and Western blot. Experiments were performed on three independent, ran- form a functional interaction with CgA. Although the enigmatic domly selected clones, but results from one clonal cell line are presented

throughout this article. http://www.jimmunol.org/ granin proteins should therefore be considered as components of the secretory apparatus in mast cells, our data also suggest poten- Antibodies and stimuli tially wider functions as regulators of organellar ion channels. Affinity-purified goat polyclonal anti-Secretogranin III (Santa Cruz Bio- technology) was used to study SgIII protein expression. Rabbit polyclonal Materials and Methods anti-Secretogranin I (Abcam) and affinity purified goat polyclonal anti- Cell cultures Secretogranin II (Santa Cruz Biotechnology) were used to perform com- parative studies. Rabbit monoclonal anti-serotonin (Sigma-Aldrich) and RBL2H3, HEK293, and PC12 cells were maintained in DMEM (Mediat- mouse monoclonal anti-V5 (Serotec) were used for immunostaining and ech), 10% FBS (Mediatech), and 2 mM glutamine (Invitrogen) in humid- immunoblotting. Mouse anti-CgA (Spring Bioscience) was used to study

ified 5% CO2 at 37°C. LAD2 cells were a generous gift from Dr. A. Kir- the interaction of SgIII and CgA. Anti-FLAG M2 was from Sigma-Aldrich. by guest on October 4, 2021 shenbaum (National Institute for Allergic and Infections Diseases, The anti-TRPA1 has been described previously (26). IgE anti-DNP, DNP- Bethesda, MD; Ref. 24). LAD2 cells were maintained in DMEM with 2% BSA, PMA, and ionomycin were all from Sigma-Aldrich. FBS, 2 mM Gln and rSCF at 100 ng/ml (PeproTech). Growth medium for stably transfected cells were supplemented with appropriate antibiotics; Immunoprecipitation and Western blotting 200 ␮g/ml zeocin (Invivogen), 5 ␮g/ml blasticidin (Invitrogen), 200 ␮g/ml hygromycin (Invivogen). Cells were pelleted (2000 ϫ g, 2 min) and washed once in PBS at room temperature. Approximately 107 cells were lysed on ice for 20 min in 500 cDNA constructs, transient transfection, and stable cell lines ␮l of lysis buffer (100 mM HEPES (pH 7.4), 150 mM NaCl, 80 mM NaF, 20 mM iodoacetamide, 50 mM PMSF (phenyl methyl sulfonyl fluoride), Full-length human Secretogranin III cDNA (Open Biosystems) was ob- 500 mg/ml aprotinin, 1.0 mg/ml leupeptin, and 2.0 mg/ml chymostatin, 1% tained by PCR and cloned into the pcDNA4/TO and pcDNA5/TO GFP (A. (weight-to-volume ratio, w/v) IGEPAL) dissolved in distilled water. Ly- Stokes, unpublished observations) vectors using NotI/KpnIorEcoRV/NotI sates were then clarified (10000 ϫ g, 5 min at 4°C) to remove nuclei. For digestion respectively. A V5 epitope tag was incorporated into the carboxyl preparation of total protein, lysates were acetone precipitated and again terminus of SgIII during this PCR. A truncated SgIII cDNA (residues centrifuged at 10000 ϫ g for 5 min at 4°C. For immunoprecipitation, 1–210) was obtained by PCR and subcloned into pcDNA5/TO empty vec- supernatants were tumbled (4°C/2 h) with the indicated Ab, followed by tor and pcDNA5/TO GFP vector (A. Stokes, unpublished observations). capture of immunocomplexes using ϳ50 ␮g Protein A conjugated to aga- All constructs were verified by sequencing. The pcDNA5/TO TRPA1 con- rose beads (Sigma-Aldrich). Excess liquid was aspirated with a Hamilton struct was generated as described previously (25). syringe. Samples were then boiled (8 min) in reducing sample buffer (20% For transient transfection, HEK293 cells were seeded and grown until (v/v) glycerol, 62.5 mM Tris-HCl (pH 6.8), 0.05% (w/v) bromophenol 50% confluent. All cDNA was purified using a QIAfilter Plasmid Maxi Kit blue, 2 mM 2-mercapto-ethanol) and then resolved by 10% SDS-PAGE for (Qiagen). The cDNA quality was evaluated using a spectrophotometer and 16 h. Ͼ was used in transfection only if O.D. 260/280 1.7. HEK293 cells were After being resolved on SDS-PAGE, the proteins were electro-trans- transiently transfected using reagent LT1 (Mirus). Serum-free DMEM (100 ferred to Polyvinylidene fluoride membrane (1.4 A for 180 min in 25 mM ␮ ␮ l) and LT1 reagent (10 l) were added together and vortexed for 2 min. Tris base-HCl, 192 mM glycine (pH 8.63)). Membranes were blocked us- The mixture was then left at room temperature for 15 min. After 15 min, ing 5% (w/v) nonfat milk (1 h at RT). Membranes were incubated with 7.5 ␮g of V5-SgIII cDNA was added to the mixture and mixed gently. primary Abs (Ab dissolved in TBS, 0.05% Tween, 0.05% NaN3, and 0.5% After 15 min at room temperature (RT), the mixture was added to the cells w/v BSA) for 16 h at 4°C. Membranes were washed with TTBS (4 ϫ 5 in a drop-wise manner while swirling the plate. Cells were incubated for min) and incubated with secondary Abs (HRP-conjugated IgG) for 1 h. The 48–72 h and then harvested and lysed for Western blots. membranes were washed with TTBS (4 ϫ 5 min) and then incubated with For stable transfection, the RBL2H3 and PC12 cell lines were electro- ECL Plus Western Blotting Detection Reagent (Amersham Biosciences). ⌬ ⌬ porated with V5-SgIII, V5-SgIII-GFP, V5- SgIII, and V5- SgIII-GFP Emitted light was then detected using autoradiography film (Kodak Sci- ␮ ␮ ϫ constructs. Cells were electroporated with 15 g of DNA in 500 lof1 entific Imaging). DMEM, 10% FBS, and 2 mM glutamine at 280 V and 950 ␮Fina4mm path length cuvette (BioRad). Clonal cell lines were selected by limiting Protein determination assay dilution in 96-well plates. Selection was conducted in zeocin (V5-SgIII construct) or hygromycin (V5-SgIII-GFP, V5-⌬SgIII, and V5-⌬SgIII-GFP Protein samples were matched for total protein levels on the basis of a

constructs). colorimetric protein determination assay, the Dc Protein Determination Kit 5026 SgIII AND CgA DIRECT SECRETORY GRANULE BIOGENESIS IN MAST CELLS

(BioRad) according to the manufacturer’s instructions. Color development Analysis was read at 710 nm in a Benchmark Plus (BioRad) plate reader. Experiments are n ϭ 3 unless otherwise indicated. Results are shown as the Ϯ Tritiated serotonin release assay mean SD. Statistical significance was determined based on a two-way ANOVA (Student’s t test). Adjacent to data points in the respective graphs, ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء :Adherent RBL2H3 and RBL-SgIII (1.25 ϫ 105 cells/cm2) were incubated significant differences were recorded as follows .p Ͻ 0.001; no symbol, p Ͼ 0.05 ,ءءء .with 1 ␮g/ml IgE–anti DNP and 0.5 ␮Ci/ml [3H]serotonin for 16 h at 37°C Monolayers were then washed with normal medium at 37°C (2ϫ) and cells were incubated with 10 ␮l of indicated stimuli (1 ␮M PMA, 10 ␮M iono- mycin, or 10% Triton X-100) or vehicle (PBS) in 190 ␮l/cm2 medium for Results 1 h at 37°C. Reactions were stopped on ice. The supernatant was centri- fuged (10,000 ϫ g for 30 s) and 100 ␮l of supernatant were counted in Secretogranin III and its partner Chromogranin A are liquid scintillation mixture (Scintiverse BD mixture, Fisher Scientific). Re- expressed in mast cells lease in response to PMA/ionomycin was between 60 and 80% of the Triton X-100 releasable signal. Signals from Fc␧RI stimulation approached SgIII and CgA are considered to be components of the secretory 30–50% of the Triton X-100 releasable [3H] serotonin. vesicle biogenesis system in neuroendocrine cells. Because the secretory vesicle system in mast cells shares multiple features with Immunofluorescence the neuroendocrine system we asked whether these proteins are Cells were seeded onto glass coverslips. The cells were fixed with pure expressed in mast cells. We assayed the presence of SgIII and CgA methanol at –20°C for 5 min. They were then washed with PBS, and then by Western blot in rodent and human mast cells. The data in Figs. blocked with 0.7% (w/v). Fish skin gelatin (prepared in PBS) for 20 min. 1, A and B show an expression survey for Secretogranin III and The cells were incubated with primary Ab diluted in the blocking reagent CgA. IGEPAL lysates were prepared from the following mast cell (0.7% (w/v) fish skin gelatin) for 1 h. The cells were washed four times lines RBL2H3 (rat basophilic leukemia), P815 (mouse mastocy- Downloaded from with PBS and then incubated with Alexa fluorophore conjugated Abs (In- vitrogen) prepared in the blocking reagent for 20 min. Cells were washed toma), mouse bone marrow derived mast cell, and LAD2 (human four times with PBS and then finally the cover-slips were dipped in water mast cell leukemia). LAD2 cells exhibit a high MW (Ͼ75 kDa) and mounted on microscope slides using Crystal mount (Biomedia). Im- form of SgIII. This variation in apparent MW of SgIII may reflect aging was performed with an Olympus IX70 fluorescence inverted micro- an, as yet undefined, posttranslational modification. The size vari- scope with quadruple dichroic filter block and excitation filter set 88000 (Chroma), connected to an F-view monochrome CCD camera. ance is unlikely to be attributatble to species differences because HEK are derived from humans, as are the LAD2. The neuroendo- http://www.jimmunol.org/ Toluidine blue staining crine PC12 cells were used as positive controls for SgIII expres- sion (Fig. 1C). SgIII protein is present in each of the mast cell lines Cells were seeded onto glass cover-slips. The cells were first fixed with pure methanol at Ϫ20°C for 5 min. They were then washed with PBS, tested. SGIII was also detected in CAD catecholaminergic neu- followed by incubation for 30 min with 0.5% (w/v) Toluidine blue (J. T. rons, RIN5MF pancreatic ␤ cells and the Jurkat T lymphocyte line Baker, Phillipsburg, NJ) dissolved in distilled water. Coverslips were (data not shown). We also noted that in some cell lines SgIII mi- washed 4 times with PBS and mounted using Crystal mount (Biomedia). grated as a single species on SDS-PAGE (Fig. 1A), supporting the data of Ottiger and coauthors (25) who suggested that SgIII, unlike Macroarray analysis other granin family members, is not inevitably proteolysed to form by guest on October 4, 2021 Total RNA was extracted from adherent RBL2H3 cells that were either left bioactive peptides. A high MW (75–80 kDa), uncleaved, version ␮ untreated or exposed to IgE (1 g/ml for 16 h followed by two washes) of SgIII that binds to CgA has recently been reported by the Ho- followed by 250 ng/ml DNP-BSA for the time indicated. Total RNA was purified using a Nucleospin RNA II kit according to the manufacturer’s saka laboratory (27). instructions (BD Biosciences). Before hybridization on the BD Atlas Rat 1.2 Arrays, total RNA samples were labeled with [␣-33P]dATP using the Atlas Pure Total RNA Labeling System (BD Biosciences). A set of Secretogranin III is depleted from mast cells following matched BD Atlas Rat 1.2 Array blots were hybridized (68°C, 16 h). Mem- stimulation branes were washed four times in 2 ϫ SSC/1% SDS (68°C, 15 min), then once in 0.1 ϫ SSC/0.5% SDS (68°C, 15 min) and once in 2 ϫ SSC (RT, We hypothesized that SgIII is a component of the mast cell secre- 5 min). Blots were exposed to storage phosphor screens (Packard Bio- tory granule. We reasoned that if so, then SgIII levels in the mast sciences) for 8 days. Data were captured using a Cyclone System (Packard cells may be altered after stimulation of mast cells using agents Biosciences). BD Atlas Image 2.7 (BD Biosciences) software was used to assign genes to specific coordinates on the array membranes, to correct for that evoke secretory responses. We assayed the levels of SgIII background counts, to establish the threshold for positive gene expression protein in mast cells treated with secretion-evoking pharmacolog- (2 ϫ background level counts ϩ 10%), and to normalize between-blot ical stimuli or antigenic stimuli. The presence of SgIII was as- hybridization intensity differences based on the median-signal intensity. sessed by Western blot of matched cytosolic extracts from un- Per gene differences in mRNA levels were expressed as a fold-change stimulated and stimulated RBL2H3. Our results show that resting relative to the corresponding unstimulated controls. cells express SgIII protein, which is markedly depleted at 1–2 h Single cell calcium assay after stimulation with PMA/Ionomycin (Fig. 2A) or via antigenic cross-linking of the Fc␧RI (Fig. 2B). Control experiments suggest Fura-2 AM ester (Molecular Probes) loading of intact cells was performed by incubating cells for 45 min in a standard modified Ringer’s solution of that this is not a protein degradation event as secreted SgIII can be detected by Western blot in the extra-cellular milieu of resting the following composition (in mM): NaCl 145, KCl 2.8, CsCl 10, CaCl210, ⅐ MgCl22, glucose 10, HEPES NaOH 10 (pH 7.4), supplemented with 5 mast cells (Fig. 2C). We have also detected full-length endogenous ␮M fura-2-AM (Molecular Probes). Cytosolic calcium concentration was SgIII in the supernatants of resting RBL2H3 but at very low levels monitored at a rate of 5 Hz with a photomultiplier-based system using a (data not shown). SgIII protein starts to reappear at 4 h after stim- monochromatic light source that excited fura-2 at 360 and 390 nm for 20 ms each. Emission was detected at 450–550 nm with a photomultiplier ulation and this expression stabilizes by 24 h after stimulation, and whose analog signals were sampled and processed by the X-Chart software re-attains resting levels (Fig. 2, A and B). The depletion in SgIII package (HEKA Electronics). Fluorescence ratios were translated into free levels is consistent with the idea that this protein is a component of intracellular calcium concentration based on calibration parameters derived the mast cell secretory granule. Other granule components such as from patch-clamp experiments with calibrated calcium concentrations. Os- molarity was maintained at 300 mOsm. Local perfusion of individual cells chymase and tryptase show a distinctive pattern of depletion and was achieved through a wide-tipped, pressure-controlled application pi- then replenishment in activated mast cell populations (28). SgIII pette (3-␮m diameter). appears to behave in a similar fashion. Moreover, the recovery in The Journal of Immunology 5027

SgIII protein is apparently due to a transient transcriptional up- regulation of the SGIII gene (Fig. 2D) following mast cell stimulation.

Secretogranin III over-expression causes expansion of the secretory vesicle compartment in mast cells The granulogenic properties of granins have been established in neuroendocrine cells on the basis that over-expression of these proteins is necessary and sufficient to cause the appearance of a novel secretory vesicle compartment. We evaluated the secretory compartment of RBL2H3 mast cells with stable over-expression of SgIII using several methods. First, we used an immunofluores- cence approach in RBL2H3 mast cells, HEK cells, and PC12 neu- roendocrine cells. We compared each of these cell types with a matched clonal cell line in which V5-SgIII is over-expressed (Fig. 3A). Interestingly, we observed that V5-SgIII over-expression in each cell type was accompanied by the appearance of a prominent vesicle population that could be immunodecorated with anti-V5 (Fig. 3A). This vesicular organelle was most prominent in the se- Downloaded from cretory RBL2H3 and PC12 cells, while the SgIII-associated struc- tures in HEK293 were relatively smaller and less numerous. The vesicular structures associated with SgIII over-expression in RBL2H3 cells did not immunodecorate with Abs toward mark- ers of the Golgi apparatus (anti-Golgi-58K), lysosomes (anti- LAMP2), endoplasmic reticulum (anti-calreticulin), or peroxi- somes (anti-PMP70) (data not shown). We therefore hypothesized http://www.jimmunol.org/ that these structures may represent secretory granules in mast cells. Because serotonin is a marker for secretory granules (29, 30) we asked whether SgIII-associated vesicles could be counterstained with an anti-serotonin Ab. We noted that these vesicles contained significant serotonin, which colocalized with V5-SgIII, indicating that they may be bona fide granules capable of packaging inflam- matory mediators in mast cells (Fig. 3B). We also detected some

subplasma membrane staining with anti-serotonin, which may re- by guest on October 4, 2021 flect its presence in a subpopulation of small, endogenous, exocy- totic vesicles. Further, we performed staining on mast cells with toluidine blue, a metachromatic dye that detects the numerous sul- fated groups in mast cell granules. RBL2H3 with over-expression of SgIII displayed a large number of toluidine blue-positive gran- ules that were not observed in control cells (Fig. 3C), again sug- gesting that SgIII over-expression leads to an increase in the se- cretory vesicle load of mast cells.

SgIII over-expression confers a functional increase in the intensity of mast cell proinflammatory responses With the idea that V5-SgIII over-expressing granules could pack- age inflammatory mediators such as serotonin, we hypothesized that these V5-SgIII over-expressing mast cells may exhibit en- hanced secretion of proinflammatory mediators. In V5-SgIII over- expressing RBL2H3 resting cells, there is a significant increase in basal efflux of [3H]serotonin (Fig. 3D). Together with the fact that the detergent-releasable amount of [3H]serotonin is also enhanced in V5-SgIII over-expressing cells (Fig. 3E), these data suggest that there may be an expansion of the compartment storing [3H]sero- FIGURE 1. SgIII and CgA protein levels in mast cells. A, SgIII protein tonin in these cells. Indeed, the V5-SgIII cells exhibit enhanced in mast cells. IGEPAL lysates were prepared from RBL2H3 (rat mast cell), secretory responses to pharmacological stimulation (Fig. 3F), sug- P815 (mouse mast cell), BMMC (mouse bone marrow derived mast cell), gesting that the vesicles arising from V5-SgIII over-expression HEK (human endothelial cells), and LAD2 (human mast cells). Total pro- tein was recovered by acetone precipitation and was resolved by 10% SDS- may be functionally equivalent to mast cell granules. The potential PAGE. After electrotransfer and blocking, Western blotting was performed for V5-SgIII over-expression to induce apparent granulogenesis in with 0.26 ␮g/ml anti-SgIII. Molecular mass shown in kDa. Predicted mo- lecular mass of SgIII is 68 kDa. B, CgA protein levels in mast cells. IGE- PAL lysates were prepared as described above and Western blotting was blots for CgA and SgIII in the line PC12. IGEPAL performed with 0.4 ␮g/ml anti-CgA. Molecular mass shown in kDa. Pre- lysates were prepared as described above and Western blotting was per- dicted molecular mass of CgA is 68–75 kDa. C, Positive control Western formed with 0.26 ␮g/ml anti-SgIII or 0.4 ␮g/ml anti-CgA. 5028 SgIII AND CgA DIRECT SECRETORY GRANULE BIOGENESIS IN MAST CELLS Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 2. SgIII protein levels are depleted and subsequently restored following pharmacological or antigenic activation of mast cells. A, Western blot analysis of SgIII levels in stimulated mast cells. RBL2H3 mast cells were stimulated with PMA and ionomycin (1 ␮M each) (A) or IgE (0.1 ␮g/ml 16h) (B) followed by DNP-BSA (200 ng/ml) for 0, 1, 2, 4, and 24 h. After stimulation, cells were harvested and IGEPAL postnuclear lysates were prepared. Total protein was recovered by acetone precipitation and resolved by 10% SDS-PAGE. After electro-transfer and blocking, Western blotting was performed with 0.26 ␮g/ml anti-SgIII. C, SgIII is present in the culture supernatant of RBL2H3 cells. RBL2H3 culture supernatant was harvested by acetone precipitation, then Western blotted for the presence of SgIII. Left lanes present culture supernatant or cell lysates prepared from untransfected cells, while right lanes present supernatants or lysates harvested from RBL2H3 stably transfected with V5-SgIII. Positive control blots for the cytosolic proteins pyruvate dehydrogenase (PDH) and Grb2 are presented. D, Transcript levels for granin family members in stimulated mast cells. Rat 1.2 macroarrays (BD Clontech) were hybridized with cDNA probes derived from RBL2H3 cells left unstimulated or stimulated for 0.5, 3, or 9 h, as described in Materials and Methods. Each of the arrayed genes is represented by a column whose height is defined by the fold change in the hybridization intensities of the gene at that respective time point relative to a matched control. mast cells may speak to a role for endogenous SgIII in directing crine cells and mast cells. In neuroendocrine cells, over-expres- this process physiologically. sion of CgA has been shown to be granulogenic, as is over- expression of SgIII. Moreover, in neuroendocrine cells the CgA is a functional partner of SgIII in mast cells granulogenic properties of CgA are critically dependent upon Taken together, our data indicate similarities in the role of SgIII its interaction with SgIII. Hosaka and coauthors (16) showed in two major physiological secretory systems: the neuroendo- that the SgIII (214–373) and CgA (48–111) domains mediate The Journal of Immunology 5029 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 3. A, Immunofluorescence analysis of SgIII over-expressing cells and wild-type cells. i, RBL2H3 cells were grown on cover slips and fixed with methanol. After blocking, the cells were stained with anti-V5 (0.2 ␮g/ml) and an Alexa 568 nm-conjugated secondary Ab (pseudo-colored red). Nuclei were stained using Hoechst 33342 (100 ␮g/ml, pseudo-colored blue). ii, Overexpression of V5-SgIII in RBL cells. iii, HEK-SgIII cells without induction and iv with tetracycline induction. v, Represents PC12 cells and vi, PC12-SgIII. Cells in panels iv were induced with tetracycline (1 ␮g/ml) overnight. The left panels are the nuclei, middle panels represent the V5 stained vesicles, and the right panels are the overlaid images. B, SgIII over-expression results in vesicle formation in mast cells. Control RBL2H3 (top panel) and RBL2H3 with constitutive overexpression of V5-SgIII (bottom panel) were grown on glass cover slips and fixed in methanol. After blocking, V5-tagged proteins were visualized using anti-V5 (0.2 ␮g/ml) and an Alexa 568 nm-conjugated secondary Ab. Serotonin was labeled using anti-serotonin (84 ␮g/ml) and an Alexa 488 nm-conjugated secondary Ab. Nuclei were stained using Hoechst 33342 (100 ␮g/ml). Merged image with anti-V5 (pseudo-colored red), anti-serotonin (pseudo-colored green), and Hoechst 33342 (pseudo-colored blue). C, SgIII overexpression results in an increased number of toluidine blue-positive granules in mast cells. Left panels, Control RBL2H3 (a), RBL2H3 with constitutive over-expression of V5-SgIII (b). Right panels, Control HEK (a) and HEK with constitutive over-expression of V5-SgIII (b). Cells were grown on glass cover slips and fixed in methanol. After washing, the cells were flooded with 0.5% toluidine blue (pH 3.14) for 30 min. The cells grown on coverslips were washed and mounted to be observed under the microscope. The RBL2H3- V5-SgIII cells show the presence of an increased number of granules as compared with control RBL2H3 cells. D, Basal secretion of [3H]serotonin from V5-SgIII overexpressing mast cells. The y-axis represents the average scintillation counts per min in 100 ␮l of supernatant from a culture of 1 ϫ 105 mast cells and SgIII over-expressing cells. Cells were not stimulated. Counts from triplicate wells were averaged. Error bars represent SD around the mean. A Student’s t test was done p Ͻ 0.001. E, Enhanced [3H]serotonin content in V5-SgIII overexpressing mast cells. The y-axis represents ,ءءء ;p Ͻ 0.01 ,ءء ;to calculate the p values. ϩ, p Ͼ 0.5 the average scintillation counts per min in 100 ␮l of supernatant from a culture of 1 ϫ 105 mast cells and SgIII over-expressing cells. The x-axis represents the presence 10% v/v Triton. Counts from triplicate wells were averaged. Error bars represent SD around the mean. A Student’s t test was done to calculate the p values. p Ͻ 0.001. F, Enhanced degranulation of [3H]serotonin content in V5-SgIII over-expressing mast cells. The cells were stimulated ,ءءء ;p Ͻ 0.01 ,ءء ;ϩ, p Ͼ 0.5 with PMA (1 ␮M, 1 h) and iono (ionomycin, 10 ␮M, 1 h). The y-axis represents the average scintillation counts per min in 100 ␮l of supernatant from a culture of 1 ϫ 105 mast cells and SgIII over-expressing cells. Counts from triplicate wells were averaged. Error bars represent SD around the mean. A Student’s t test .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;was performed to calculate the p values. ϩ, p Ͼ 0.5 5030 SgIII AND CgA DIRECT SECRETORY GRANULE BIOGENESIS IN MAST CELLS

FIGURE 4. A, Expression of full-length and truncated SgIII constructs in

RBL2H3 mast cells. RBL2H3 were stably transfected with the indicated con- Downloaded from structs. IGEPAL lysates were prepared and immunoprecipitated using 2 ␮gof the anti-V5 Ab. Immunocomplexes were resolved by 10% SDS-PAGE and Western blotted using anti-V5 (0.5 ␮g/ml). Arrows indicate position of Ig heavy (55 kDa) and light (26 kDa) chains. B, Coimmunoprecipitation of CgA, full-length SgIII and truncated mutant of SgIII. IGEPAL lysates were prepared from wild-type RBL2H3 cells (lanes 1 and 4), RBL2H3 stably overexpressing

SgIII (lanes 2 and 5), and RBL2H3 stably overexpressing the truncated mutant http://www.jimmunol.org/ of SgIII (lanes 3 and 6). Cellular lysates were immunoprecipitated using 0.4 FIGURE 5. Lack of prominent vesicular structures in mast cells over- ␮g/ml anti-CgA, and total protein was recovered by acetone precipitation expressing truncated compared with full-length SgIII. RBL2H3-V5-SgIII (lanes 1–3). Although in this exposure we do not see significant differences (left panel) and RBL2H3-V5-⌬SgIII (right panel) were grown on glass in SgIII content when measuring cellular lysate, SgIII transfectants clearly cover slips and fixed in methanol. After blocking, V5-tagged proteins were contain an enhanced level of V5-SgIII when recovered by coimmunoprecipi- visualized using anti-V5 (0.2 ␮g/ml) and an Alexa 568 nm-conjugated tation. The total protein and the immunocomplexes were then resolved by 10% secondary Ab. Nuclei were stained using Hoechst 33342 (100 ␮g/ml). SDS-PAGE. After electrotransfer and blocking, Western blotting was per- Images are separated into anti-V5 (upper panels) and Hoechst nuclear formed with 0.26 ␮g/ml anti-SgIII. MW shown in kDa. stains (lower panels). by guest on October 4, 2021 the functional interaction between CgA and SgIII in mouse cor- complex plays an, as yet poorly understood, role in directing ticotrope-derived AtT-20 cells. We asked whether SgIII and granule biosynthesis. However, there are precedents for the in- CgA interact functionally in the mast cell system, and we pro- teractions of granin proteins with nongranin binding partners, duced a truncated form of SgIII that lacks the putative CgA- including the inositol (1, 4, 5) trisphosphate receptor, and in- interacting (and granulogenic) domain (Fig. 4A). We were able tracellular calcium release channel (10–13, 31). We performed to coimmunoprecipitate CgA and SgIII in mast cells (Fig. 4B). an interaction trap analysis (a Sos-Recruitment Screen yeast Our data indicate that removal of residues 211–468 of SgIII two-hybrid (Stratagene) performed using the amino-terminal (Fig. 4A) resulted in a loss of interaction with CgA (Fig. 4B). cytoplasmic tail of rat TRPA1 as bait and a rat brain cDNA We asked whether full-length and the SgIII (1–210) trunca- library a target, data not shown), which suggested that the ion tion mutant were equivalent in their ability to induce the for- channel TRPA1 may form a protein complex with SgIII. Be- mation of a novel granule population in mast cells. RBL2H3 cause the work of several groups does suggest that ion channels were transfected with either V5-SgIII or V5-SgIII (1–210). The in intracellular membranes may interact with certain granins, presence of SgIII-associated vesicle population in the cytosol of we asked whether this is a physiological interaction in mast these cells was assessed by anti-V5 immunofluorescence. These cells. Fig. 6, A and B show that TRPA1 and SgIII can be co- data (Fig. 5) show that the over-expression of the truncated immunoprecipitated in both an HEK over-expression system V5-SgIII (1–210) did not result in the appearance of a promi- and in RBL2H3 mast cells. It is clear that a small proportion of nent vesicular compartment. Diffuse, faint staining of the trun- the over-expressed, or endogenous, TRPA1 can be immunopre- cated V5-SgIII was observed, which quenched rapidly. More- cipitated in a complex with SgIII. Similarly, we find that of the over, a GFP-tagged version of SgIII (1–210) with truncation of multiple mobility forms of SgIII that are present in cells over- CgA binding domain showed a lack of vesicular organization expressing V5-SgIII, only the lowest apparent m.w. (perhaps (data not shown). Thus, the granulogenic potential of SgIII in corresponding to the least processed, immature form of SgIII) mast cells appears to be dependent upon an intact SgIII C ter- species forms a complex with TRPA1. The functional signifi- minus (residues 211–468), which is the region that mediates cance of this remains unclear, but we note that TRPA1 predom- interaction between SgIII and CgA. inantly localizes to vesicular structures in resting mast cells (Fig. 6C), although these immuno-decorate only weakly with SgIII binding partners in mast cells include the ion channel anti-SgIII (data not shown). The interaction between TRPA1 TRPA1 and SgIII clearly has functional implications for the channel The data presented above suggest that SgIII in mast cells par- however, because intracellular calcium release responses ticipates in an interaction with CgA, and that the CgA-SgIII evoked by the TRPA1 ligand icilin are compromised in cells The Journal of Immunology 5031 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 6. Secretogranin III is a binding partner for TRPA1 in mast cells. A, Coimmunoprecipitation of V5-SgIII and FLAG-TRPA1. HEK cells stably expressing FLAG-tagged TRPA1 were transiently retransfected with V5-SgIII. Lysates were immunoprecipitated with 1 ␮g of the indicated Ab and duplicated membranes were Western-blotted for the presence of FLAG- and V5-tagged proteins. B, Coimmunoprecipitation of TRPA1 and SgIII. RBL2H3 mast cells were lysed and immunoprecipitated with 5 ␮g of the indicated Abs. Immunocomplexes were resolved by SDS-PAGE and Western blotted with anti-TRPA1 (left panel) or anti-SgIII (right panel). C, TRPA1 subcellular localization in resting mast cells. RBL2H3 were grown on glass coverslips and fixed in methanol, before immunofluorescence analysis using anti-TRPA1 (decorated with Alexa-488 nm conjugated goat ant-rabbit IgG) and anti-Golgi 58K mAb (decorated with rabbit anti-mouse Alexa 568 conjugated IgG). Nuclear material was stained (blue pseudo-color) using Hoechst 33342. Scale bar, 10 microns. D, TRPA1-mediated calcium responses in the absence and presence of over-expressed SgIII. HEK cells (lower gray trace), HEK stably expressing TRPA1 (black trace), and TRexTRPA1 cells transiently retransfected with V5SgIII (upper gray trace, offset by ϩ0.1 ␮M for clarity) were loaded with fura-2 and a single cell calcium assay was performed as described. After establishing a baseline (white bar), individual cells were perfused with nominally calcium-free external buffer containing 12 ␮M icilin (gray bar). 5032 SgIII AND CgA DIRECT SECRETORY GRANULE BIOGENESIS IN MAST CELLS over-expressing SgIII (Fig. 6D). These data present the first hint minus, which in turn is the domain of SgIII that interacts with of a wider role for SgIII in mast cells, and their implications the amino terminus of CgA. Thus, in mast cells, as in sympa- will be discussed below. thoadrenal AtT-20 cells (16, 35), a functional interaction be- tween SgIII and CgA seems to be required for their granulo- Discussion genic capacity. Hosaka and coauthors (34, 35) have suggested Granin family members are present in the secretory granules of that SgIII binds to the amino terminus of CgA thereby initiating a wide variety of endocrine and neuroendocrine cells (1, 3, 4, the secretory granule biogenesis in the AtT-20 cells. This SgIII- 32). Granins are involved in granulogenesis in neuroendocrine CgA complex binds to the cholesterol moieties within the se- cells (6, 7) but there has been no published analysis of ex- cretory granule membrane, but the exact mechanism by which pression or function of granins in the mast cell system. Our this binding promotes granulogenesis is not yet fully under- data demonstrate the expression of granins in mast cells, and stood. Several groups have determined that cholesterol deple- imply an important role for these proteins in this secretory tion blocks the formation of constitutive secretory vesicles from immunocyte. the Golgi apparatus (30), and the formation of microdomains of The biological roles of granin proteins are not well under- the “cone-shaped” cholesterol molecules may contribute to the stood. Several ideas, which are not mutually exclusive, have negative curvature of the lumenal leaflet of the vesicle mem- been proposed for the molecular functions of granins. It is clear brane. One key open question regards the localization of en- that CgA and CgB act as cargo precursors that are proteolyti- dogenous SgIII in untransfected mast cells. We have not been cally cleaved to form bioactive peptides. In addition to being able to produce any convincing immunofluorescence analysis sources of granule content in the form of proteolytically derived for endogenous SgIII in mast cells, because the available anti- peptides, Chromogranins have also been described to chaperone SgIII gives only weak, diffuse vesicular staining that may either Downloaded from secretory granule components (1, 3, 9, 33, 34). Chromogranins indicate a vesicular localization or may represent the type of have also been described as cholesterol binding proteins, and a background staining that is typically associated with low affin- model for their role in the cholesterol sequestration events that ity Abs in this application. participate in granulogenesis has been derived from several The data from our SgIII over-expression experiments in mast studies (35). One common observation for several granins sug- cells suggest that SgIII drives granule biogenesis. The data con- gests that their over-expression result in expansion of the gran- cerning an expansion of the granule content that can be released http://www.jimmunol.org/ ule compartment (6, 15, 20, 22). Both their proposed roles as in response to antigenic or pharmacological treatment of mast precursors of granule cargo, and as regulators of granule mem- cells could also reflect an alternate explanation, such as an ac- brane dynamics, would be consistent with these observations of tion of SgIII in promoting expression, or activity, of the trans- granulogenic capacity. In a further, intriguing, set of publica- porters that load serotonin into preexisting granules. Similarly, tions, the CgA and CgB proteins have been described as low we cannot exclude that SgIII over-expression enhances the ac- affinity, high capacity, calcium buffering proteins that associate tivity of serotonin efflux transporters, a caveat to the secretion with intracellular calcium channels in the ER and Golgi appa- assays presented in this study. However, our microscopy data ratus. In this context, the calcium reservoir that is provided by are more consistent with the fact that SgIII over-expression by guest on October 4, 2021 CgA appears to play a feedback role in regulating the open simply results in the presence of more mast cell granules. status of these calcium permeant channels (10–13, 31). Knockdown or knockout of SgIII would reveal whether this Secretogranin III has not been extensively investigated, and protein is needed for the biosynthesis of endogenous vesicles in is only weakly similar to CgA/CgB at the sequence level. Our a native expression system. We have found SgIII knockdown in data, and that from other laboratories, allow us to make a num- mast cells to be associated with suppression in secretory capac- ber of statements about SgIII. First, it is heavily sulfated, which ity, but these experiments are difficult to interpret in the light of is consistent with it being a component of the mast cell secre- the confounding effects of decreased viability, possibly because tory granule (Ref. 36 and Prerna Prasad, unpublished observa- this protein may plays central role in membrane trafficking (data tion). Second, SgIII is not extensively proteolyzed to form pep- not shown). A SgIII knockout mouse has been described by tides in resting mast cells (this article and Ref. 25). These data Dopazo and coauthors (Ref. 40; unpublished observations) but suggest that its major physiological role in this system is not to no endocrine or immunological phenotype was described and act as a precursor for bioactive peptides. Although Holthuis and the mice are no longer extant. However, a recent paper by coauthors (36) have reported proteolytic fragments that derive Hendy and coauthors (23) suggests that knockout of granins from SgIII, no biological activity has been assigned to any of may not be very informative, due to a compensatory up-regu- the resulting peptides, in contrast with the situation for the CgA lation in the expression levels of other granin genes. and CgB (37–39). It remains to be seen whether any SgIII- The data summarized above suggest that the function of SgIII derived fragments act as signaling peptides in neuroendocrine in mast cells closely parallels that described in the neuroendo- cells, and we have not seen any evidence that pharmacological crine secretory system, where SgIII is involved in regulating the or immunological activation of mast cells can induce proteol- membrane dynamics of secretory vesicles via an interaction ysis of SgIII. In the mast cell system at least, we can argue that with CgA. Intriguingly, our interaction trap and coimmunopre- the granulogenic capacity of transfected SgIII does not simply cipitation data suggest that CgA is not the only binding partner reflect an adaptive response to the sudden need to package large for SgIII in mast cells. SgIII clearly interacts with the ligand- quantities of SgIII-derived bioactive peptides. gated cation channel TRPA1. These data are strongly reminis- Our third observation concerning SgIII is that transfection of cent of the observations of the Ehrlich and Yoo laboratories (10, SgIII cDNA is necessary and sufficient to drive expansion of a 11, 13, 31), where CgA and CgB bind to and regulate the ino- vesicular compartment in mast cells, neuroendocrine cells, and sitol (1, 4, 5) trisphosphate receptors, which are intracellular even in the nonsecretory HEK cell line. This expansion is ac- calcium channels. We had initially considered the possibility companied by a concomitant increase in the proinflammatory that interaction between SgIII and TRPA1 might reflect the fact capacity of the transfected mast cells. The granulogenic poten- that an SgIII-derived peptide could be a ligand for this channel. tial of SgIII depends on the presence of an intact carboxyl ter- However, the clear interaction between TRPA1 and full-length The Journal of Immunology 5033

SgIII, and the data from studies of the granin/InsP3R interaction 8. Chanat, E., U. Weiss, W. B. Huttner, and S. A. Tooze. 1993. Reduction of the would seem to argue against this model. TRPA1 seems, like disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways. EMBO J. 12: TRPV1, to function as an intracellular calcium release channel 2159–2168. when supplied with an appropriate membrane-permeant (li- 9. Natori, S., and W. B. Huttner. 1996. Chromogranin B (secretogranin I) pro- pophilic) ligand (41), and it is clearly localized to vesicular motes sorting to the regulated secretory pathway of processing intermediates derived from a peptide precursor. Proc. Natl. Acad. Sci. USA 93: intracellular structures. Moreover, channels of the TRP family 4431–4436. have been reported to be associated with cholesterol-rich mi- 10. Choe, C. U., K. D. Harrison, W. Grant, and B. E. Ehrlich. 2004. Functional crodomains, at least in the plasma membrane (42). Thus, the coupling of chromogranin with the inositol 1,4,5-trisphosphate receptor shapes calcium signaling. J. Biol. Chem. 279: 35551–35556. association between TRPA1 and SgIII may simply reflect the 11. Thrower, E. C., C. U. Choe, S. H. So, S. H. Jeon, B. E. Ehrlich, and S. H. Yoo. localization of the latter to lipid membrane microdomains. 2003. A functional interaction between chromogranin B and the inositol 1,4,5- ϩ However, there is also a clear precedent for a regulatory role of trisphosphate receptor/Ca2 channel. J. Biol. Chem. 278: 49699–49706. 12. Thrower, E. C., H. Y. Park, S. H. So, S. H. Yoo, and B. E. Ehrlich. 2002. granins that associate with certain intracellular calcium release Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage channels, the InsP3R. protein chromogranin A. J. Biol. Chem. 277: 15801–15806. Choe and coauthors (10) demonstrated that CgA and CgB 13. Yoo, S. H., S. H. So, Y. H. Huh, and H. Y. Park. 2002. Inositol 1,4,5-trisphos- phate receptor/Ca2ϩ channel modulatory role of chromogranins A and B. Ann. associate with the InsP3R, and that provision of competing con- NY Acad. Sci. 971: 300–310. centrations of a peptide derived from the interacting portion of 14. Loh, Y. P., T. Kim, Y. M. Rodriguez, and N. X. Cawley. 2004. Secretory granule the granin had a dominant inhibitory effect upon Ins(1,4,5)P3- biogenesis and sorting to the regulated secretory pathway in neu- evoked calcium release. Similarly, our over-expression of SgIII roendocrine cells. J. Mol. Neurosci. 22: 63–71. 15. Maite Courel, C. R., S. T. Nguyen, A. Pance, A. P. Jackson, D. T. O’ Connor, and seems to suppress calcium release via TRPA1. In the case of the L. Taupenot. 2006. Secretory granule biogenesis in sympathoadrenal cells: iden- Downloaded from Chromogranin/InsP3R interaction, the calcium-binding activity tification of a granulogenic determinant in the secretory prohormone chromo- of the granin, through conserved EF-hand motifs, has been pro- granin A. J. Biol. Chem. 281: 38038–38051. 16. Hosaka, M., T. Watanabe, Y. Sakai, Y. Uchiyama, and T. Takeuchi. 2002. Iden- posed to play a role in shaping the channel’s open probability tification of a chromogranin A domain that mediates binding to secretogranin III (10, 43). In the case of SgIII, there is no obvious EF-hand motif and targeting to secretory granules in pituitary cells and pancreatic ␤-cells. Mol. (CgA/CgB and SgIII are only weakly similar at the sequence Biol. Cell 13: 3388–3399. 17. Krishnaswamy, G., O. Ajitawi, and D. S. Chi. 2006. The human mast cell: an level; Ref. 4) and no calcium-buffering activity has been as- overview. Methods Mol. Biol. 315: 13–34. cribed to this protein. At this time, it seems likely that the loss 18. Metcalfe, D. D., D. Baram, and Y. A. Mekori. 1997. Mast cells. Physiol Rev. 77: http://www.jimmunol.org/ of function in TRPA1 that is associated with SgIII over-expres- 1033–1079. 19. Beuret, N., H. Stettler, A. Renold, J. Rutishauser, and M. Spiess. 2004. Expres- sion is likely to reflect a disruption of its localization to cho- sion of regulated secretory proteins is sufficient to generate granule-like structures lesterol-rich microdomains. Future work will determine in constitutively secreting cells. J. Biol. Chem. 279: 20242–20249. whether the activity of TRPA1 in intracellular membranes ab- 20. Chanat, E., S. W. Pimplikar, J. C. Stinchcombe, and W. B. Huttner. 1991. What solutely depends upon correct localization to SgIII-containing the granins tell us about the formation of secretory granules in neuroendocrine cells. Cell Biophys. 19: 85–91. lipid rafts. 21. Rong, Y. P., F. Liu, L. C. Zeng, W. J. Ma, D. Z. Wei, and Z. G. Han. 2002. In summary, the present report suggests that granins in mast Cloning and characterization of a novel human secretory protein: secretogranin cells recapitulate the varied functional contributions made by these III. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao 34: 411–417. 22. Huh, Y. H., S. H. Jeon, and S. H. Yoo. 2003. Chromogranin B-induced secretory by guest on October 4, 2021 proteins to the neuroendocrine secretory system. Granins such as granule biogenesis: comparison with the similar role of chromogranin A. J. Biol. SgIII can obviously contribute to multiple facets of cell biology, Chem. 278: 40581–40589. and the reaching of a consensus as to their molecular roles is 23. Hendy, G. N., T. Li, M. Girard, R. C. Feldstein, S. Mulay, R. Desjardins, R. Day, A. C. Karaplis, M. L. Tremblay, and L. Canaff. 2006. Targeted ab- clearly a high priority. 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