Disruption of SLP-76 Interaction with Gads Inhibits DynamicClustering of SLP-76 and FcεRI Signaling in Mast Cells†
Michael A. Silverman, Jonathan Shoag, Jennifer Wu, and Gary A. Koretzky*Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, Department of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received 18 August 2005/Returned for modification 19 September 2005/Accepted 16 December 2005
We developed a confocal real-time imaging approach that allows direct observation of the subcellularlocalization pattern of proteins involved in proximal Fc�RI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of76 kDa (SLP-76) is critical for Fc�RI-induced calcium flux, degranulation, and cytokine secretion. In thisstudy, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon Fc�RI cross-linking, SLP-76translocates to the cell membrane, forming clusters that colocalize with the Fc�RI, the tyrosine kinase Syk, theadaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive bindingpartner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76,inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads iscritical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of theGads-binding region of SLP-76 in bone marrow-derived mast cells inhibits Fc�RI-induced calcium flux,degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signalingclusters following Fc�RI stimulation and demonstrated that the Gads-binding region of SLP-76 regulatesclustering of SLP-76 and Fc�RI-induced mast cell responses.
Mast cells are tissue resident hematopoietic cells that expressthe high-affinity immunoglobulin E (IgE) receptor FcεRI. Thisreceptor contains immunoreceptor tyrosine-based activation mo-tifs (ITAMs) that become phosphorylated by Src family kinasesfollowing cross-linking by IgE and cognate antigen. Syk kinase,which binds the phosphorylated ITAMs, then becomes activatedand phosphorylates a number of critical signaling proteins, includ-ing the transmembrane adaptor linker for the activation of T cells(LAT), the guanine nucleotide exchange factor Vav, and theadaptor molecule Src homology 2 (SH2) domain-containingleukocyte phosphoprotein of 76 kDa (SLP-76). These proximalevents lead to the activation of phospholipase C�1 (PLC�1)and phospholipase C�2 (PLC�2), which cleaves the membranephospholipid phosphatidylinositol 4,5-bisphosphate into inosi-tol 1,4,5-trisphosphate and diacylglycerol, inducing calciumflux and protein kinase C activation, respectively. This signal-ing cascade regulates diverse mast cell responses, includinggranule release, cytokine secretion, cytoskeletal rearrange-ment, and gene transcription (for reviews, see references 13,26, and 31).
SLP-76 is a hematopoietic-cell-specific adaptor protein thatfunctions downstream of both ITAM-containing receptors andintegrins (44). Loss-of-function studies have demonstrated acritical role for SLP-76 in FcεRI signaling. SLP-76-deficientbone marrow-derived mast cells (BMMCs) fail to flux calcium,degranulate, or secrete interleukin-6 (IL-6) in response toFcεRI stimulation. In addition, SLP-76-deficient mice are re-
sistant to FcεRI-induced passive systemic anaphylaxis (PSA)(16, 30, 43).
SLP-76 contains three N-terminal tyrosines that induciblybind the guanine nucleotide exchange factor Vav (16, 27, 36,42), the adaptor Nck (45), and the Tec family kinase Btk (16);a P1 region that binds the phospholipase PLC� (46); a C-terminal SH2 domain that inducibly binds the adaptor adhe-sion- and degranulation-promoting adaptor protein (10, 24)and the hematopoietic progenitor kinase 1 (33); and a proline-rich region that constitutively binds the adaptor molecule Gads(1, 21). Mutational analysis has demonstrated that the N-ter-minal tyrosines and the proline-rich region are absolutely re-quired, while the SH2 domain is partially required for FcεRI-induced mast cell responses (16, 43).
These findings suggest an important role for a number of sig-naling proteins, including the cytosolic adaptor Gads, a memberof the Grb2 family of proteins. Gads is expressed specifically inhematopoietic cells and contains a central SH3 domain flanked bytwo SH2 domains (20). Gads constitutively binds through its SH3domain to the RxxK motif in the proline-rich region of SLP-76,and Gads inducibly associates through its SH2 domain with phos-phorylated LAT (3, 21). Mice deficient in SLP-76 or LAT haveprofound defects in thymocyte development (8, 48), while Gads-deficient mice have a partial block in thymocyte development thatphenocopies the defects seen in mice expressing a Gads-bindingmutant of SLP-76 (18, 25, 47). These studies suggest that Gadsmediates the formation of a signaling complex between SLP-76and LAT that is critical for T-cell-receptor signaling and thymo-cyte development.
The formation of multimolecular signaling complexes hasbeen proposed to regulate FcεRI signaling, leading to diversemast cell responses. Previous studies of RBL cells using im-munoelectron microscopy indicated that FcεRI cross-linking
* Corresponding author. Mailing address: University of PennsylvaniaSchool of Medicine, 415 BRBII/III, 421 Curie Blvd., Philadelphia, PA19104. Phone: (215) 746-5522. Fax: (215) 746-5525. E-mail: [email protected].
† Supplemental material for this article may be found at http://mcb.asm.org/.
induces the formation of a “primary signaling domain” thatincludes FcεRI, Syk, PLC�2, Gab2, and the p85 regulatorysubunit of phosphatidylinositol-3 kinase and a “second signal-ing domain” that includes LAT, PLC�2, and p85 (39–41).These studies offered high-resolution “snapshots” of signalingcomplexes but did not allow direct observation of the kineticsof complex formation. While these experiments have describedthe spatial relationships of a number of key FcεRI signalingmolecules, the localization of SLP-76 was not addressed. Wehave therefore developed a real-time confocal imaging ap-proach that facilitates investigation of the temporal as well asspatial regulation of proximal FcεRI signaling.
The mechanisms that regulate the formation of these mul-timolecular signaling complexes remain poorly understood.Adaptor molecules have been proposed to play a critical role inthe formation of signaling complexes in numerous cell types.We therefore investigated the subcellular localization of theadaptor protein SLP-76 in real time in the RBL cell line and inprimary mast cells. Our results demonstrated that SLP-76translocates from the cytosol to the cell membrane, where itforms signaling clusters that colocalize with the FcεRI, LAT,Syk, and phosphotyrosine-containing proteins. Further, weshowed that formation of the dynamic signaling clusters re-quires interaction between SLP-76 and Gads. Disruption ofthis interaction blocks SLP-76 translocation to the membrane,abrogates formation of signaling clusters, and inhibits FcεRI-induced mast cell responses.
MATERIALS AND METHODS
Cell culture. RBL cells were cultured in RPMI medium, 15% fetal calf serum,100 U/ml penicillin, 100 �g/ml streptomycin, and 2.92 mg/ml glutamine. Bonemarrow cells were cultured in RPMI medium, 20% fetal calf serum, 100 U/mlpenicillin, 100 �g/ml streptomycin, 2.92 mg/ml glutamine, 25 mM HEPES, 0.1mM nonessential amino acids, 1 mM sodium pyruvate, and 50 �g/ml gentamicin(complete RPMI medium) supplemented with 20 ng/ml of recombinant IL-3 and20 ng/ml of recombinant stem cell factor (SCF) (Peprotech). After 4 weeks ofculture, �90% of cells were mast cells, as determined by flow cytometric analysisfor the expression of FcεRI. Functional assays were performed on cells that hadbeen in culture for 4 to 8 weeks.
cDNA constructs and production of retrovirus. Syk-green fluorescent protein(GFP) chimera was kindly provided by Robert Geahlen (Purdue University) andhas been described previously (22). LAT-GFP chimera was kindly provided byLarry Samelson (NIH) and has been described previously (5). The monomericred fluorescent protein (mRFP) vector was kindly provided by Roger Tsien(UCSD). It was PCR amplified from pRSET-mRFP1 (7) by using the primers5�-CCT TAA GCC ACC ATG GCC TCC TCC GAG GAC GTC AT-3� and5�-CCC AAG CTT GGC GCC GGT GGA GTG GCG GC-3�. The PCR productwas digested with EcoRI, blunted with Klenow fragment, and then digested withHindIII. The digested product was placed upstream of the multiple cloning siteof pEGFP-C1 (Clontech) after the removal of enhanced GFP by digestion withAgeI, blunting, and digestion of HindIII. RFP-SLP-76 chimera was generated byPCR amplifying SLP-76 from GFP-SLP-76 (35) and adding EcoRI and Xmalinkers with primers 5�-GGA ATT CCC ATG GCC TTG AAG AAT G-3� and5�-TTT CCC GGG CTA CAG ACA GCC TGC AGC-3�. SLP-76 was thendigested with EcoRI and XmaI and was cloned in frame with mRFP. Thisconstruct was confirmed by sequencing. The SLP-76 G2 mutant and the GBF-DsRed2 and DsRed2 murine stem-cell virus (MSCV)-based vectors have beendescribed previously (35). High-titer retroviral supernatants were produced viacotransfection of 293-T cells with retroviral construct and helper virus packagingconstruct (Imgenex).
Retroviral infection of BMMCs. Freshly isolated bone marrow from B6/129mice was cultured overnight in complete RPMI medium supplemented with 10ng/ml IL-3, 10 ng/ml IL-6 (R&D Systems), and 50 ng/ml SCF. Retroviral super-natant was then added, and cells were spin-infected at 2,500 rpm for 90 min atroom temperature in the presence of 8 �g/ml of Polybrene (Sigma). Cells wereagain incubated overnight at 37°C with 5% CO2, and the retroviral spin infection
was repeated the following day. Following overnight incubation, cells were cul-tured to generate mast cells. After 3 to 4 weeks, DsRed2-expressing cells weresorted using a FACSVantage SE flow cytometer (Beckton Dickinson).
Flow cytometric analysis. Cells were stained according to standard protocols byusing the following labeled antibodies: mouse anti-2,4-dinitrophenol (DNP) IgE(Sigma), anti-mouse IgE-biotin (Pharmingen), and streptavidin-APC (Pharmingen).Two-color flow cytometry was performed with a FACSCalibur (Becton Dickinson).
�-Hexosaminidase release assay. A total of 5 � 105 RBL cells per well weresensitized overnight at 37°C with 1 �g/ml anti-DNP IgE in a 96-well, flat-bottomtissue culture plate. Cells were then washed twice with Tyrode’s buffer (130 mMNaCl, 10 mM HEPES, 1 mM MgCl2, 5 mM KCl, 1.4 mM CaCl2, 5.6 mM glucose,1 mg/ml bovine serum albumin, pH 7.4) and then stimulated with DNP-humanserum albumin (HSA) or phorbol myristate acetate (PMA) and ionomycin for 60min at 37°C. Total cellular granule contents were released by lysing cells withTyrode’s buffer with 1% Triton X-100. A total of 30 �l of supernatant wascollected and transferred to a 96-well, flat-bottom plate. A total of 30 �l of 1 mMp-nitrophenyl-N-acetyl-�-D-glucosamide was then added to each supernatant andmixed before incubation for 1 h at 37°C. The reaction was terminated by theaddition of 200 �l of 0.1 M Na2CO3-NaHCO3 buffer, and the optical density wasread on a plate reader at a wavelength of 405 nm. BMMCs (1 � 106/ml) werestarved of SCF overnight and then sensitized at 1 � 107/ml in complete RPMImedium without cytokines and with 1 �g/ml anti-DNP IgE (clone SPE-7; Sigma)for 4 h at 37°C with 5% CO2. Cells were then washed once in Tyrode’s buffer andresuspended at 2 � 106/ml in the buffer. A total of 200 �l of cells was thenstimulated with various amounts of DNP (0 to 1,000 ng/ml) for 1 h at 37°C. Cellswere spun down, and 30 �l of supernatant was transferred to a 96-well, flat-bottom plate. A total of 30 �l of 1 mM p-nitrophenyl-N-acetyl-�-D-glucosamidewas then added to each supernatant and mixed before incubation for 1 h at 37°C.The reaction was terminated by the addition of 200 �l of 0.1 M Na2CO3-NaHCO3 buffer, and the optical density was read on a plate reader at a wave-length of 405 nm.
IL-6 production assay. BMMCs (1 � 106/ml) were starved of SCF overnightand then sensitized at 1 � 107/ml in complete RPMI medium without cytokinesand with 1 �g/ml anti-DNP IgE (clone SPE-7, Sigma) for 4 h at 37°C with 5%CO2. Cells were then washed once and resuspended at 1 � 106/ml in completeRPMI medium. A total of 5 � 104 cells in complete RPMI medium was thenincubated with various concentrations of DNP overnight at 37°C plus 5% CO2 ina total volume of 100 �l in a 96-well, flat-bottom plate. Each sample was assayedin triplicate. The following day, the plate was removed from the incubator andfrozen at �20°C. An enzyme-linked immunosorbent assay (ELISA) was per-formed on thawed supernatants by using a murine IL-6 ELISA kit (Pierce/Endogen).
Lysate preparation and immunoblotting. RBL cells and BMMCs werewashed, pelleted, and lysed in ice-cold 1% NP-40 containing proteinase (50�g/ml aprotinin, 10 �g/ml leupeptin, 50 �g/ml pepstatin A, and 1 mM Pefablock)inhibitors. Western blotting was performed using anti-DsRed2 (Clontech).
Calcium flux assay. RBL cells were sensitized overnight with 1 �g/ml anti-DNP IgE. RBL cells were then incubated with Versene (sodium chloride, 8g/liter; potassium chloride, 0.2 g/liter; dibasic sodium phosphate, 1.15 g/liter;EDTA, 0.2 g/liter; and phenol red, 0.1 g/liter; at pH 7.34) for 2 min and detachedfrom tissue culture dishes and washed once in RBL medium. BMMCs weresensitized with 1 �g/ml anti-DNP IgE for 4 h at 37°C. Cells were then washedonce in Tyrode’s buffer and resuspended at 1 � 107 cells/ml in Tyrode’s buffercontaining 6.25 mM Probenecid (Sigma) and 2 mg/ml Indo-1 (MolecularProbes). Cells were protected from light and incubated at 37°C for 30 min.Indo-1-loaded cells were washed twice and resuspended in warm Tyrode’s buffer.Data were collected using an LSR flow cytometer (Becton Dickinson). BaselineCa2� levels were measured for 30 s prior to the addition of DNP (100 ng/ml).The sample was collected for a total of 260 s with approximately 500 to 700events collected per second. Ionomycin was added 35 s prior to the end of theassay. FL5 represents Indo-1 bound by Ca2�, and FL4 represents Indo-1 notbound by Ca2�. Data were analyzed using FlowJo software (TreeStar), and Ca2�
flux is defined by the mean ratio of FL5/FL4 over time.RBL transfection and generation of stable cell lines. RBL cells were detached
with Versene and washed once in RBL medium. The cells were then incubatedwith 10 �g of plasmid DNA for 10 min and electroporated (900 �F/ 250 V) inGene Pulser cuvettes (Bio-Rad). Cells were then rested for 10 min before platingin RBL medium. Transiently transfected cells were assayed 18 to 72 h aftertransfection. Stable cell lines were generated by selection in G418 (0.1 mg/ml;Mediatech) followed by fluorescence-activated cell sorting (FACS) for equallevels of GFP of DsRed2.
Cellular imaging. The dynamics of signaling complexes in mast cells weremonitored using an adaptation of a T-cell spreading assay (6). Delta T micro-
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scope dishes (Bioptechs) were incubated overnight with 1 ml 0.01% poly-L-lysine(Sigma) at 37°C. Poly-L-lysine was aspirated, and plates were dried at 37°C for 10min. The microscope dishes were then incubated with 1 ml of 1 �g/ml DNP-HSAin carbonate-binding buffer (35 mM sodium bicarbonate and 75 mM sodiumcarbonate) for 1 h at 37°C. DNP-HSA was then aspirated, and the microscopedishes were dried for 10 min at 37°C. RBL cells were prepared by detaching themfrom tissue culture plates with Versene, washing them in RBL medium, andsensitizing them with 1 �g/ml anti-DNP IgE for 1 h in RBL medium at 37°C.BMMCs were prepared by sensitizing them in cytokine-free mast cell media with1 �g/ml anti-DNP IgE for 1 h at 37°C. Cells were then washed and resuspendedin RBL medium or cytokine-free mast cell medium. To initiate an assay, RBLcells or BMMCs were dropped onto a microscope dish coated with poly-L-lysinealone or poly-L-lysine and DNP-HSA. As the cells initiated contact with theantigen-coated coverslip, multiple z-axis images were collected starting from justbelow the plane of interaction of the cell and the coverslip. Images were collectedfor the indicated times. Live-cell imaging was performed on a Perkin-Elmer 5wavelength laser UltraVIEW LCI spinning disk confocal microscope(Yokogawa) that was attached to a Nikon TE-300 inverted microscope equippedwith a 60� objective, a z-axis servo controller (Physik Instrumente), a Delta T4culture dish controller (Bioptechs), and an objective heater (Bioptechs) to main-
tain the sample temperature at 37°C. Samples were excited, and emission wave-lengths were collected using an argon ion laser emitting 488- and 514-nm linesand an argon-krypton laser emitting 568- and 647-nm lines in conjunction with a488/568 RGB dichroic mirror. A Hamamatsu Orca-ER charge-coupled-devicecamera (Hamamatsu) was used to collect images. Image analysis was performedusing IP Labs version 3.9.3 r4.
For fixed images, the RBL cells, BMMCs, and delta T microscope disheswere prepared as described for the live images. Cells were added to themicroscope dishes and incubated at 37°C for the indicated times. The disheswere then aspirated and washed in phosphate-buffered saline to removemedium and nonadherent cells. The cells were fixed in 4% paraformaldehydefor 20 min, quenched in 50 mM ammonium chloride for 3 min, permeabilizedin 0.5% Triton X-100 for 1 min, and blocked (phosphate-buffered saline,0.01% saponin, 0.25% gelatin, and 0.02% sodium azide) overnight. Rhoda-mine-conjugated PY20 antiphosphotyrosine antibody was used for immuno-fluorescence staining. Multiple z-axis images were acquired and then analyzedusing IP Labs software to enhance contrast and to merge color channels.Representative images were chosen for the figures.
Clustering analysis. Cells were counted as containing GFP-SLP-76 clusters ifdistinct areas of GFP fluorescence above the background were observed specif-
FIG. 1. FcεRI cross-linking induces dynamic SLP-76 clustering at the membrane. (A and B) Anti-DNP IgE-sensitized RBL cells expressingGFP-SLP-76 were dropped onto a coverslip coated with either PLL or DNP-HSA. Upon contact with the coverslip, multiple z-stack images of livecells were acquired using the UltraVIEW spinning disk confocal system. Images shown are from the plane of interaction between the cell and thecoverslip.
ically at the plane of interaction between the cell and the coverslip. More than 75cells from at least 10 different high-powered fields were analyzed from eachgroup. Cells were scored for clustering by a blinded observer. The percentages ofcells with GFP-SLP-76 clustering were compared between GBF-DsRed2 andDsRed2 vector by Student’s t test.
GBF peptide. GBF-antennapedia peptide (RQIKIFQNRRMKKKNHSPLSPPHPNHEEPSRSGNNKTAKLPAPSIDRSTKPPLDRSLAPLDREPF) wassynthesized by the W. M. Keck Biotechnology Resource Center (New Haven, CT).The peptide was initially dissolved in dimethyl sulfoxide and then diluted to theappropriate concentration in water.
RESULTS
Fc�RI stimulation induces SLP-76 clustering and translo-cation to the membrane. To investigate SLP-76 subcellular lo-calization following FcεRI cross-linking, stable RBL cell linesexpressing GFP alone or GFP fused to SLP-76 were generated bytransfection followed by FACS for equal expression of GFP. RBLcells were preincubated with IgE specific for DNP-HSA and
FIG. 2. SLP-76 colocalizes with FcεRI, Syk, LAT, and phosphotyrosine. (A) RBL cells expressing GFP-SLP-76 (green) were sensitized withAlexa 568-conjugated anti-DNP IgE (red) and then dropped onto a coverslip coated with either PLL or DNP-HSA. Live cells were imaged from0 to 30 min using the UltraVIEW spinning disk confocal system. Multiple vertical z-stack images were acquired. Images shown are from the planeof interaction between the cell and the coverslip. (B and C) RBL cells were cotransfected with mRFP-SLP-76 and Syk-GFP (B) or mRFP-SLP-76and LAT-GFP (C), sensitized with anti-DNP IgE, and then dropped onto coverslips with either PLL or PLL with DNP-HSA. Cells were then fixedwith 4% paraformaldehyde, and multiple confocal z-stack images were acquired in the GFP and RFP channels that were merged using IP Labssoftware. Images shown are from the plane of interaction between the cell and the coverslip. Inset images are from the same cell 1 �m away fromthe plane of interface between the cell and the coverslip, allowing visualization of the cell membrane and cytosol. (D) Anti-DNP IgE-sensitizedRBL cells expressing GFP-SLP-76 were dropped onto a coverslip coated with either PLL or DNP-HSA. Cells were then fixed, permeabilized, andstained with rhodamine-conjugated PY20 antiphosphotyrosine (red). Multiple confocal z-stack images were obtained in the GFP and rhodaminechannels that were merged using IP Labs software. Images shown are from the plane of interaction between the cell and the coverslip. pY,phosphotyrosine.
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then were dropped onto a coverslip coated with poly-L-lysine(PLL) as a negative control or with DNP-HSA. As the cellscontacted the coverslip, the antigen DNP-HSA cross-linkedthe FcεRI receptor and initiated signaling. Live confocal visu-alization at the plane of interaction between the cell and thestimulatory coverslip allowed direct observation of proximalFcεRI signaling events in real time. These images were col-lected and analyzed with high temporal and spatial resolution.
To determine the pattern of SLP-76 localization in response toFcεRI activation, we compared the subcellular localization ofGFP-SLP-76 on PLL-coated versus that on DNP-HSA-coatedcoverslips. In RBL cells interacting with the PLL surface, GFPfluorescence remained diffusely cytoplasmic (Fig. 1A) (see MovieS1 in the supplemental material), whereas a dramatic clusteringof GFP fluorescence was observed when the cells came in contactwith DNP-HSA-coated coverslips (Fig. 1B) (see Movie S2 in thesupplemental material). GFP-SLP-76 formed numerous clusters,which remained for 15 to 30 min, at the cell membrane as early as15 s after contacting the stimulatory surface (data not shown) (seeMovie S2 in the supplemental material). Analysis of multipleconfocal images demonstrated that these clusters were found atonly the interface between the cell and the coverslip and thatSLP-76 clustering was not observed in the interior of the cell or atthe plasma membrane in areas not interacting with the coverslip(data not shown). In contrast, fluorescence in RBL cells express-ing GFP not fused to SLP-76 remained diffusely cytoplasmic andnuclear when cells contacted PLL- or DNP-HSA-coated cover-
slips (data not shown). These results demonstrated that FcεRIcross-linking leads to rapid and sustained clustering of SLP-76 atthe antigen contact site.
SLP-76 clusters colocalize with Fc�RI, Syk, LAT, and phos-photyrosine. Cross-linking of the FcεRI is also known to in-duce clustering of the receptor (39). To investigate whether theSLP-76 clusters colocalized with FcεRI, GFP-SLP-76 RBLcells were incubated with Alexa 568-conjugated IgE and thenwere dropped onto PLL- or DNP-HSA-coated coverslips.FcεRI formed distinct clusters at the cell contact sites with theDNP-HSA-coated coverslip within seconds, whereas FcεRI re-mained diffusely distributed as the cells spread upon PLL-coated coverslips. In the stimulated RBL cells, some smallerclusters of IgE could also be seen inside the cells, which likelyrepresented endocytosis of the receptor and bound IgE (29);however, the majority of the FcεRI clusters were seen at theplane of interface between the cell and the coverslip. TheseFcεRI clusters colocalized with the SLP-76 clusters as shownby the merged fluorescence images (Fig. 2A). These experi-ments indicated that FcεRI stimulation induces SLP-76 trans-location from the cytoplasm to the cell membrane, formingclusters that colocalize with the FcεRI.
Following FcεRI cross-linking, Syk kinase becomes activatedand phosphorylates SLP-76. We therefore investigated whetherSyk colocalized with SLP-76 clusters. RBL cells stably expressingmRFP fused to SLP-76 were generated and transiently trans-fected with Syk-GFP. Both mRFP-SLP-76 and Syk-GFP formed
FIG. 3. Anti-DNP-IgE-sensitized GFP-SLP-76 expressing BMMCs were dropped onto a coverslip coated with either PLL or DNP-HSA. Cellswere then fixed, permeabilized, and stained with rhodamine-conjugated PY20 antiphosphotyrosine (red). Multiple confocal z-stack images wereobtained from the GFP and rhodamine channels and were merged with IP labs software. Images shown are from the plane of interaction betweenthe cell and the coverslip. pY, phosphotyrosine.
clusters when the anti-DNP-HSA IgE-sensitized RBL cells con-tacted the DNP-HSA-coated surface (Fig. 2B). mRFP-SLP-76formed clusters with kinetics and distributions similar to those ofGFP-SLP-76 (data not shown). In contrast, mRFP and GFP flu-orescence remained diffusely cytoplasmic when the RBL cellsinteracted with the PLL-coated coverslip. Merging of the mRFPand GFP images demonstrated that SLP-76 clusters and Sykclusters colocalize (Fig. 2B).
We next investigated whether SLP-76 clusters also colocalizewith the membrane adaptor LAT. mRFP-SLP-76-expressingRBL cells were transiently transfected with LAT-GFP andwere sensitized with DNP-HSA-specific IgE. When the RBLcells interacted with a DNP-HSA-coated coverslip, bothSLP-76 and LAT formed distinct clusters at the cell contactsites (Fig. 2C). These clusters were found specifically at theinterface between the cell and the coverslip, whereas clusteringwas not observed in the cytoplasm or on other cell membranes,suggesting specificity for this interaction. In unstimulated cells,LAT was evenly distributed at the periphery of the cells, asexpected for a transmembrane protein, while SLP-76 was dif-fusely distributed in the cytoplasm. Merging of the RFP andGFP images demonstrated colocalization of the SLP-76 andLAT clusters (Fig. 2C).
We hypothesize that SLP-76 functions by regulating the for-mation of multimolecular signaling complexes that contain ac-tivated signaling proteins. Since tyrosine phosphorylation ofmultiple signaling intermediates is required for a productivesignal, we investigated whether SLP-76 clusters would colocal-ize with phosphotyrosine-containing proteins at the mem-brane. RBL GFP-SLP-76 cells were dropped onto PLL- orDNP-HSA-coated coverslips, fixed, and stained with rhoda-
mine-conjugated antiphosphotyrosine. RBL cells adhered toand spread upon the PLL-coated coverslips but did not exhibitphosphotyrosine staining, while cells adhering and spreadingon DNP-HSA-coated coverslips exhibited dramatic phospho-tyrosine clusters (Fig. 2D). These clusters were seen within 2min (the earliest time point analyzed) of dropping the cells onthe coverslip and remained for �30 min (data not shown).Importantly, phosphotyrosine staining was seen primarily inthe plane of interaction between the cell and the stimulatorysurface. Very little phosphotyrosine staining was seen in otherregions of the cell. SLP-76 colocalized with phosphotyrosine-containing proteins, as seen with the merged fluorescence im-ages (Fig. 2D).
While the RBL cell line is a powerful model system forinvestigating FcεRI signaling, it is important, whenever possi-ble, to also examine events in primary cells. Accordingly, weinvestigated the subcellular localization pattern of SLP-76 inBMMCs. SLP-76-deficient BMMCs expressing GFP-SLP-76were generated by infecting SLP-76-deficient bone marrowwith the MSCV-based retrovirus containing GFP fused toSLP-76, followed by growth in IL-3- and SCF-containing mediafor 4 weeks to allow for differentiation and proliferation. Anti-DNP IgE-sensitized BMMCs were dropped on to coverslipscoated with PLL or DNP-HSA. BMMCs adhered to PLL-coated coverslips in the absence of stimulation, but DNP-HSAstimulation increased the adhesion and spreading of BMMCson the coverslips, likely via inside-out up-regulation of integrinfunction. SLP-76 was diffusely cytoplasmic when BMMCs weredropped on PLL alone but formed distinct clusters upon con-tact with DNP-HSA-coated coverslips. In addition, these
FIG. 4. FcεRI-induced calcium flux and SLP-76 clustering requires an interaction between Gads and SLP-76. (A) Anti-DNP-IgE-sensitizedSLP-76-deficient BMMCs reconstituted with GFP-SLP-76, GFP-SLP-76 G2, or GFP alone were loaded with the calcium-sensitive dye Indo-1.Calcium flux was assessed by flow cytometry with DNP-HSA added at 30 s and ionomycin added at 225 s. (B) Anti-DNP IgE-sensitized RBL cellsexpressing GFP-SLP-76 or GFP-SLP-76 G2 were dropped onto a coverslip coated with either PLL or DNP-HSA. Cells were then fixed followinga 10-min incubation. Multiple confocal z-stack images were obtained in the GFP channel. Images shown are from the plane of interaction betweenthe cell and the coverslip.
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SLP-76 clusters colocalized with phosphotyrosine staining, sug-gesting that they represent complexes enriched for proteinsimportant for signaling (Fig. 3).
A SLP-76 Gads-binding mutant does not cluster at the cellmembrane. The cytosolic adaptor protein Gads constitutivelybinds SLP-76, and this interaction is mediated by the SH3region of Gads and the RxxK motif found in the proline-richregion of SLP-76. Previous studies of BMMCs by our groupand others demonstrated that the SLP-76/Gads interaction isrequired for FcεRI-induced calcium flux, degranulation, andcytokine production (16, 43). An arginine-to-alanine substitu-tion at amino acids 237 and 240 (G2) abrogates SLP-76 bindingwith Gads (3). Expression of this minimal mutation of GFP-SLP-76 was unable to rescue FcεRI-induced calcium flux inSLP-76-deficent BMMCs (Fig. 4A). To correlate SLP-76 traf-ficking and competence of FcεRI-induced signaling, we inves-tigated whether the Gads-binding region of SLP-76 is requiredfor SLP-76 to translocate and cluster at the cell membrane. Wegenerated a stable RBL cell line expressing GFP fused toSLP-76 containing the G2 mutation. Confocal imaging of RBLGFP-SLP-76 G2 cells dropped on to DNP-HSA-coated cover-slips showed that SLP-76 clustering was dramatically impaired(Fig. 4B). These data suggested that SLP-76 clustering requiresits binding with Gads and, by extension, that proper subcellularlocalization of SLP-76 is likely required for FcεRI-inducedmast cell responses.
Expression of the Gads-binding fragment of SLP-76 in RBLcells inhibits Fc�RI-induced calcium flux and degranulation.To further investigate the role of the SLP-76/Gads interactionin FcεRI-induced signaling and mast cell responses, we chosea complementary approach to disrupt the SLP-76/Gads inter-action without altering the structure of the SLP-76 protein(Fig. 5A). Previous studies of Jurkat T cells demonstrated thatthe expression of 50 amino acids that correspond to the Gads-binding region of SLP-76 fused to the fluorophore DsRed2disrupted the SLP-76/Gads interaction and also inhibited T-cell-receptor-induced SLP-76 clustering, calcium flux, andCD69 upregulation (35). We hypothesized that this fragment(called the Gads-binding fragment [GBF] of SLP-76) wouldinhibit the dynamic clustering of SLP-76 and disrupt FcεRIsignaling and mast cell responses. To test whether GBF ex-pression would block clustering of SLP-76 in mast cells, GBF-DsRed2 or DsRed2 alone as a control was transiently trans-fected into GFP-SLP-76-expressing RBL cells. These cellswere then sorted for coexpression of both GFP and DsRed2and then visualized using live video confocal microscopy asthey interacted with antigen-coated coverslips (Fig. 5B). Thecollected images were analyzed and visually scored by ablinded observer for clustering of GFP-SLP-76. While SLP-76clustered in 80% of cells expressing DsRed2 alone, SLP-76clustering was observed in only 40% of cells expressing theGBF (Fig. 5C). Two representative images of cells expressing
FIG. 5. GBF inhibits SLP-76 clustering at the membrane. (A) Schematic of the GBF of SLP-76. (B) RBL cells stably expressing GFP-SLP-76were transiently transfected with either DsRed2 (vector) or GBF-DsRed2 (GBF) and then sorted by FACS for coexpression of GFP and DsRed2.These cells were rested overnight, incubated for 1 h with IgE specific for DNP-HSA, and then dropped onto a coverslip coated with either PLLor DNP-HSA. Live cell images for GFP and DsRed2 were recorded from 0 to 30 min by confocal microscopy. Two representative images of cellsexpressing DsRed2 or GBF-DsRed2 are shown. (C) SLP-76 clustering was quantitated by a blinded observer in cells coexpressing GFP-SLP-76 andGBF-DsRed2 (GBF) or GFP-SLP-76 and DsRed2 (vector). More than 75 cells were scored for both groups. Percentages of cells with GFP-SLP-76clustering were compared between GBF-DsRed2 and the DsRed2 vector by Student’s t test (P 0.01).
DsRed2 or GBF-DsRed2 are shown. Further, GFP-SLP-76clusters observed in GBF-expressing RBL cells were typicallysmaller than those observed in RBL cells expressing DsRed2only (Fig. 5C). These data indicated that the GBF disruptsappropriate subcellular localization of SLP-76 in mast cells.
We next asked whether inhibition of SLP-76 dynamic clus-tering would disrupt FcεRI signaling. To address this question,RBL cell lines stably expressing DsRed2 alone (vector) orGBF fused with DsRed2 were established. The GBF-DsRed2and vector cell lines expressed equal levels of DsRed2 fluores-cence and FcεRI receptor (Fig. 6A and B). GBF-DsRed2 andvector control RBL cells were assayed for FcεRI-induced cal-cium mobilization by flow cytometry using the calcium-sensi-tive dye Indo-1. Calcium flux was diminished in GBF-DsRed2-expressing RBL cells compared to that for vector control RBLcells, suggesting a partial disruption in FcεRI-mediated signal-ing (Fig. 7A). Importantly, ionomycin induced similar levels ofcalcium flux in GBF-DsRed2-expressing cells and vector con-trol RBL cells, indicating that cells expressing the GBF are stillcapable of mobilizing calcium (data not shown).
Since degranulation is a SLP-76-dependent FcεRI-inducedmast cell response, we investigated whether the GBF couldalso inhibit mast cell degranulation. GBF-DsRed2-expressingRBL cells exhibited significantly less degranulation than con-
trol DsRed2-expressing RBL cells (Fig. 7B). Importantly,PMA and ionomycin induced degranulation in both GBF-DsRed2 and vector control RBL cells, indicating that the de-granulation machinery is intact in both cell lines.
GBF peptide inhibits Fc�RI-induced calcium flux. Expres-sion of the 50-amino-acid GBF fused to the 248-amino-acidDsRed2 inhibited FcεRI signal transduction and mast cell re-sponses. To investigate whether the GBF could inhibit mastcell responses using a protein transduction system instead of agenetic expression system, the GBF was fused to a 17-amino-acid sequence of the antennapedia peptide, which facilitatesprotein transportation into cells (11, 23) (Fig. 7C). To test theeffect of this peptide on FcεRI signaling, RBL cells were in-cubated with the GBF-antennapedia peptide or vehicle aloneand then assessed for FcεRI-induced calcium flux. RBL cellstreated with the GBF-antennapedia peptide exhibited dimin-ished calcium flux (Fig. 7D). Further, this inhibition increasedwith the time of incubation, with inhibition of calcium flux firstobserved after 30 min of exposure to GBF peptide (data notshown). Washout experiments demonstrated that the effect ofGBF peptide disappears by 24 h (data not shown). Althoughthere is a small amount of cell death associated with peptideadministration (generally less than 10%), these cells respond
FIG. 6. Generation of GBF-DsRed2-expressing RBL cells. (A) RBL cells were transfected with DsRed2 (vector) or GBF-DsRed2 (GBF)plasmids, selected by G418, and sorted by FACS for equal levels of DsRed2 expression. Expression of DsRed2 protein was assessed by Westernblotting (WB) and FACS. (B) Histogram shows FcεRI levels. Anti-DNP IgE-sensitized RBL cells were stained with anti-IgE-biotin andstreptavidin-APC.
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completely normally to ionomycin treatment with a robustcalcium response (Fig. 7D).
GBF inhibits Fc�RI-induced calcium flux, degranulation,and cytokine production in BMMCs. GBF efficiently blocksFcεRI signaling and mast cell responses in the RBL cell line. Toextend these findings to primary mast cells, wild-type bone mar-row cells were infected with a retroviral MSCV-based vectorcontaining either the GBF-DsRed2 or DsRed2 alone and differ-entiated into BMMCs in vitro. GBF-DsRed2- or DsRed2-ex-pressing BMMCs had similar levels of DsRed2 protein and ex-pressed equal levels of FcεRI (Fig. 8A and B). Expression of theGBF inhibits FcεRI-induced calcium flux in a dose-dependentfashion, while the vector alone had no effect on calcium flux (Fig.9A). Ionomycin elicited similar responses from both GBF- andvector-expressing mast cells (data not shown).
To test the ability of the GBF to block degranulation,BMMCs expressing GBF or vector alone were sorted forDsRed2 expression. FcεRI-induced degranulation was as-
sessed by hexosaminidase release. The GBF inhibited degran-ulation relative to the vector, but PMA-ionomycin elicited sim-ilar levels of degranulation in both the GBF and vector controlcells (Fig. 9B). Cytokine secretion is also a SLP-76-dependentfunction of mast cells. We investigated whether the GBF couldinhibit FcεRI-induced IL-6 production. BMMCs expressingeither GBF or vector were stimulated for 24 h by cross-linkingthe FcεRI receptor with antigen. The GBF efficiently inhibitedIL-6 secretion relative to vector-expressing cells (Fig. 9C),demonstrating that the GBF efficiently blocks downstream sig-naling events following FcεRI engagement in primary mastcells. Importantly, PMA and ionomycin induced similar levelsof IL-6 secretion in GBF- and vector-expressing cells (Fig. 9C).
DISCUSSION
We investigated the temporal and spatial regulation ofFcεRI signaling by characterizing the subcellular localization
FIG. 7. GBF expression blocks FcεRI signaling in RBL cells. (A) Anti-DNP IgE-sensitized RBL cells sorted for equal expression of GBF-DsRed2 or the DsRed2 vector were loaded with the calcium-sensitive dye Indo-1. Calcium flux was assessed by flow cytometry with DNP-HSAadded at 30 s. (B) Anti-DNP IgE-sensitized RBL cells expressing either GBF-DsRed2 or the DsRed2 vector were stimulated with DNP-HSA for1 h, and degranulation was assessed by measuring the activity of hexosaminidase released from the cells into the supernatant divided by the totalhexosaminidase activity in the cells. Error bars represent the standard deviations. (C) Schematic of GBF peptide consisting of 17 amino acids ofthe antennapedia protein fused to the GBF. (D) Anti-DNP IgE-sensitized RBL cells were incubated with GBF peptide or dimethyl sulfoxide(DMSO) for 150 min and then loaded with the calcium-sensitive dye Indo-1. Calcium flux was assessed by flow cytometry with DNP-HSA addedat 30 s, and ionomycin was added at 225 s.
pattern of the receptor, SLP-76, LAT, Syk, and phosphoty-rosine-containing proteins. These imaging studies demon-strated that FcεRI cross-linking induces SLP-76 translocationfrom the cytosol to the cell membrane, where it forms clustersthat colocalize with the FcεRI, the tyrosine kinase Syk, themembrane adaptor LAT, and phosphotyrosine. We also ex-tended these findings to show that SLP-76 forms signalingclusters in primary mast cells. To characterize the mechanismfor SLP-76 dynamic clustering, we disrupted the SLP-76 inter-action with Gads via minimal mutation of the Gads-bindingregion of SLP-76 or overexpression of the GBF of SLP-76,which inhibits SLP-76 translocation to the membrane and sub-sequent clustering. We then demonstrated that disruption ofSLP-76 dynamic clustering inhibits FcεRI signaling in the RBLcell line and in primary mast cells.
In addition to the studies described in this paper, clusteringof signaling proteins has been observed in mast cell (17, 39–41)and T-cell lines (2, 5, 35). These findings raise a number ofquestions. For instance, what is the function of these “clusters”in FcεRI signaling? Since the clusters appear within seconds ofstimulation and are found exclusively at the sites of stimula-tion, they appear appropriately located both temporally andspatially to play a role in signal transduction. In addition,critical mediators of FcεRI signaling, such as FcεRI, Syk, LAT,and SLP-76, are found within these clusters (9, 12, 30, 32). Lossof any of these proteins severely inhibits FcεRI-induced mastcell responses. Further, these clusters are enriched in phos-phorylated proteins that are required for many critical inter-actions downstream of FcεRI engagement, such as SLP-76binding to Btk and Vav (16) and activation of PLC� (19).Disruption of the SLP-76 interaction with Gads inhibits
SLP-76 clustering, FcεRI signaling, and mast cell responses.This correlation between SLP-76 clustering and mast cell re-sponses suggests that SLP-76 clustering at the membrane maybe critical for the propagation of the FcεRI signal.
Previous studies have suggested the existence of two “sig-naling domains” downstream of the FcεRI: a primary signalingdomain that includes FcεRI, Syk, PLC�2, Gab2, and the p85regulatory subunit of PI3K and a second signaling domain thatincludes LAT, PLC�1, and p85 (39–41). SLP-76 colocalizeswith FcεRI, Syk, and LAT, indicating that SLP-76 interactswith proteins found in both signaling domains. In addition,biochemical studies have demonstrated that SLP-76 is phos-phorylated by Syk family kinases (15, 37) and associates withLAT (21), PLC� (46), and p85 (34). These results suggest thatSLP-76 may interact with members of both signaling domains,and SLP-76 may potentially regulate events mediated by bothcomplexes. Interestingly, recent studies provide biochemicaland functional evidence for two modular FcεRI signaling path-ways. One pathway, which includes Lyn and LAT, is requiredfor calcium flux and degranulation, while the other pathway,which includes Fyn, Gab2, PI3K, and AKT, is required fordegranulation but not for calcium flux (28). Perhaps the pri-mary and secondary signaling domains differentially regulatethe two signaling pathways. Biochemical and functional char-acterization of FcεRI signaling in SLP-76-deficient versus Fyn-deficient BMMCs may further shed light on the role of bothmolecules in these two pathways.
While most SLP-76 clusters colocalize with FcεRI, Syk,LAT, and phosphotyrosine, some do not. This incomplete co-localization may indicate the dynamic and heterogeneous na-ture of these signaling clusters. Since we are observing a dy-
FIG. 8. Generation of BMMCs expressing GBF-DsRed2. (A) Bone marrow was infected with an MSCV-based retroviral plasmid encodingDsRed2 vector alone or fused to the GBF, differentiated into bone marrow-derived mast cells, and then FACS sorted for expression of DsRed2.Expression of DsRed2 protein was assessed by Western blotting (WB) and FACS. (B) Following sorting, all cells were assessed for FcεRI levelsby flow cytometry.
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namic process in real time, some clusters may be in the earlystages of formation and may contain phosphorylated proximalsignaling proteins, such as FcεRI, Src kinases, Syk, and LAT,but have not yet recruited SLP-76. As these clusters mature,they may become enriched in SLP-76, which then facilitatesrecruitment and/or stabilization of its binding partners,PLC�1/2, Vav, ADAP, Btk, and Nck. There may also be sig-naling clusters that function independently of SLP-76. It will beimportant to more fully characterize the proteins present inthese signaling clusters, to define their dynamic interactions,and to investigate the mechanisms for signal termination.
Another question is what function do adaptor molecules, suchas SLP-76, play in generating these signaling clusters? Importantelements of signaling include the careful regulation of signal ini-tiation, the ability to efficiently transduce a signal once it has beeninitiated, and the appropriate signal termination. Models for sig-nal initiation include a concept of threshold for activation whichis critical for avoiding inappropriate cellular responses (31). Togenerate a signal downstream of FcεRI, perhaps SLP-76 must bephosphorylated and recruited to the membrane, must bind itspartners, and must nucleate a multimolecular signaling complex.If a signal is weak, it may not reach the threshold required forSLP-76 to form functional signaling clusters, thus avoidinginappropriate responses. If a signal is robust, it is important toefficiently transduce that signal, allowing a rapid response fromthe mast cell. In support of this model, strong FcεRI signals arerequired to phosphorylate LAT and to stimulate cytokine pro-duction, while weaker signals induce Gab2 phosphorylation
and chemokine secretion (14). One way that SLP-76 may reg-ulate the threshold for signaling is by approximating enzymesand substrates. For example, since Btk activates PLC� (38),having both proteins bound to SLP-76 may greatly enhance theefficiency of PLC� activation, thus amplifying the original sig-nal. In addition, SLP-76 localizes to membrane subdomains (4)that may be enriched for substrates of PLC�, further amplify-ing the signal.
To investigate how SLP-76 forms dynamic signaling clusters,we asked whether a SLP-76 interaction with Gads is required.Studies in T cells suggest that this interaction regulates T-cell-receptor signaling by mediating SLP-76 clustering and recruit-ment to lipid rafts (4, 35). Minimal mutation of the Gads-bindingregion of SLP-76 or overexpression of GBF, a competitive inhib-itor of this interaction, demonstrates that this interaction is re-quired for FcεRI-induced SLP-76 clustering. We then investi-gated whether disruption of the SLP-76 interaction with Gadswould inhibit FcεRI-induced mast cell responses. Overexpressionof GBF inhibited mast cell responses both in the RBL cell lineand in primary BMMCs, suggesting that the SLP-76/Gads inter-action is critical for FcεRI-induced mast cell responses. Thesedata suggest a model in which Gads recruits SLP-76 to the cellmembrane where SLP-76 mediates the formation of multimo-lecular signaling complexes that regulate FcεRI signaling.
Investigation into the mechanisms of signaling may identifyattractive therapeutic targets for pathogenic conditions whichare dependent upon mast cell function, such as asthma andallergy. In this study, we demonstrated that the GBF peptide
FIG. 9. GBF expression blocks FcεRI signaling in BMMCs. (A) Anti-DNP IgE-sensitized unsorted BMMCs expressing GBF-DsRed2 orDsRed2 vector alone were loaded with the calcium-sensitive dye Indo-1 and assayed for calcium flux by flow cytometry. Cells were gated for high,low, and negative expression levels of DsRed2 as shown in the histogram, and calcium fluxes were analyzed and compared for each population asshown in the overlay. (B) Anti-DNP IgE-sensitized BMMCs sorted for similar expression levels of GBF-DsRed2 or DsRed2 vector alone werestimulated with DNP-HSA for 1 h. Degranulation was assessed by measuring the activity of hexosaminidase released from the cells into thesupernatant divided by the hexosaminidase activity released from PMA-ionomycin-stimulated cells. (C) Anti-DNP IgE-sensitized BMMCs sortedfor similar expression levels of GBF-DsRed2 or DsRed2 alone were stimulated with DNP-HSA for 24 h, and IL-6 secretion was measured fromculture supernatant by ELISA. Error bars represent the standard deviations.
inhibits FcεRI-induced calcium flux in RBL cells. While theseresults are encouraging, the relatively high concentration ofthe peptide required for inhibition suggests that improvementsin peptide transport into the cells, peptide stability, and/orpeptide affinity will be required before attempts to utilize theGBF peptide in vivo. We are currently defining a minimal GBFsequence that will still function as a dominant negative. Asimaging technologies continue to improve, real-time simulta-neous imaging of multiple proteins in primary mast cells willenhance our understanding of FcεRI signaling and, perhaps,will lead to the discovery of novel therapeutic targets for al-lergic disorders.
ACKNOWLEDGMENTS
We thank R. Gaehlen, L. Samelson, and R. Tsien for providingreagents. Furthermore, we thank J. Rivera, F. Abtahian, M. Jordan, T.Kambayashi, J. Maltzman, and J. Stadanlick for helpful discussionsand review of the manuscript.
This work was supported by the Sandler Program for Asthma Re-search (G.A.K.). M.A.S. is a trainee under the MSTP grant at theUniversity of Pennsylvania.
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