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The Journal of Immunology, 2004, 172: 6768-6774.
Copyright © 2004 by The American Association of Immunologists

Differential Requirement for Adapter Proteins Src Homology 2 Domain-Containing Leukocyte Phosphoprotein of 76 kDa and Adhesion- and Degranulation-Promoting Adapter Protein in Fc{epsilon}RI Signaling and Mast Cell Function1

Jennifer N. Wu2,*,{dagger}, Martha S. Jordan2,*, Michael A. Silverman*,{dagger}, Erik J. Peterson{ddagger} and Gary A. Koretzky3,*

* Abramson Family Cancer Research Institute and Department of Laboratory Medicine and Pathology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; {dagger} Medical Scientist Training Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and {ddagger} Department of Medicine, Center for Immunology, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for Fc{epsilon}RI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in Fc{epsilon}RI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and Fc{epsilon}RI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in Fc{epsilon}RI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The high affinity IgE receptor (Fc{epsilon}RI) is an immunoreceptor tyrosine-based activation motif (ITAM)4-bearing multimolecular complex expressed on the surface of mast cells. Ag cross-linking of IgE-engaged Fc{epsilon}RI leads to activation of Src family kinases Lyn and Fyn and subsequent phosphorylation of the protein tyrosine kinase (PTK) Syk. Syk then associates with the ITAMs of the Fc{epsilon}RI and cooperates in the phosphorylation of multiple substrates. In striking analogy to the signaling cascades initiated by TCR engagement, Fc{epsilon}RI stimulation induces formation of a signaling complex assembled by the adapter proteins Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), Gads, Nck, adhesion- and degranulation-promoting adapter protein (ADAP), and membrane-anchored linker for activation of T cells (LAT) and containing the effector molecules phospholipase C{gamma}1 (PLC{gamma}1), PLC{gamma}2, Vav, and Btk (for reviews, see Refs. 1 and 2).

The SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is expressed in nearly all hemopoietic cells and has been shown to be critical for signaling through a variety of ITAM-bearing receptors as well as integrins expressed on T cells (3, 4), platelets (5), neutrophils (6), and mast cells (7). Structure/function analyses conducted in T cells have identified several motifs critical for SLP-76-dependent signaling (8, 9, 10). Three tyrosines near the N terminus of the protein (Y112, Y128, and Y145) are phosphorylated and mediate inducible interactions with Vav (11, 12, 13), Nck (14), and Itk (15, 16), respectively. The central, proline-rich region of SLP-76 mediates a constitutive interaction with the adapter protein Gads (17, 18) and the enzyme PLC{gamma}1 (19), whereas the C-terminal SH2 domain can bind tyrosine-phosphorylated ADAP (20, 21) and hemopoietic progenitor kinase-1 (HPK1) (22).

Despite having normal numbers of mature, granule-containing dermal mast cells, SLP-76-deficient mice have a severely blunted passive systemic anaphylactic response, as measured by heart rate elevation and serum histamine concentration. Bone marrow from these mice produces normal numbers of bone marrow-derived mast cells (BMMCs) when cultured in vitro, and these cells express normal levels of c-Kit and Fc{epsilon}RI. Consistent with their in vivo activation defect, these cells fail to degranulate or secrete IL-6 upon Fc{epsilon}RI stimulation (7).

Recently, two parallel, but intersecting, signaling pathways downstream of the Fc{epsilon}RI have been described: the canonical Lyn/Syk/LAT/SLP-76 pathway and a novel Fyn/Gab2/phosphotidylinositol 3-kinase (PI3K) pathway (23). Parravicini et al. (23) have demonstrated enhanced Fyn/Gab2/PI3K signaling and degranulation in the absence of Lyn, whereas Lyn/Syk/LAT/SLP-76 signaling is normal, but degranulation is impaired, in the absence of Fyn. Two molecules were hypothesized to mediate the apparent cross-talk/convergence of these two pathways: 1) the Tec family kinase Btk, which can be activated downstream of both PLC{gamma} and PI3K; and 2) ADAP, which can bind both SLP-76 and Fyn. Mast cells derived from mice lacking Btk have been extensively studied and demonstrate a variety of defects in Fc{epsilon}RI-induced signaling and function (24, 25). To date, however, studies of ADAP in mast cells have been limited to overexpression in the RBL-2H3 cell line and have indicated that ADAP can enhance basal mast cell adhesion to fibronectin as well as Fc{epsilon}RI-induced degranulation (26, 27).

To further elucidate the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of BMMCs expressing mutant forms of SLP-76. These experiments demonstrate that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in Fc{epsilon}RI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. To test whether the functional defects demonstrated by BMMCs expressing the SH2 domain mutant of SLP-76 are attributable to the lack of ADAP binding and to examine the postulated role of ADAP in integrating Lyn- and Fyn-mediated signals regulating adhesion and degranulation, we have also studied ADAP-deficient BMMCs. We found that ADAP-deficient mast cells demonstrate no defects in Fc{epsilon}RI-induced adhesion, granule release, cytokine production, or passive systemic anaphylaxis. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells, and ADAP is unlikely to mediate the cross-talk between Lyn- and Fyn-initiated Fc{epsilon}RI signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Bone marrow cells were cultured in RPMI 1640, 20% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.92 mg/ml glutamine, 25 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 50 µg/ml gentamicin (complete RPMI 1640) supplemented with 20 ng/ml rIL-3 (R&D Systems, Minneapolis, MN) for 2 wk. After 2 wk, medium was also supplemented with 20 ng/ml recombinant stem cell factor (SCF; PeproTech, Rocky Hill, NJ). After 4 wk of culture, >90% of cells were mast cells, as determined by flow cytometric analysis for expression of Fc{epsilon}RI. Functional assays were performed on cells that had been in culture for 4–8 wk.

cDNA constructs and production of retrovirus

SLP-76 mutants were generated from mouse SLP-76 cDNA and subcloned into the murine stem cell virus-based retroviral MiGR vector (28) as previously described (29). High titer retroviral supernatants were produced via cotransfection of 293-T cells with retroviral constructs (30) and the Helper Virus packaging construct (Imgenex, San Diego, CA).

Retroviral infection of BMMCs

Freshly isolated bone marrow from SLP-76-deficient mice was cultured overnight in complete RPMI 1640 supplemented with 10 ng/ml IL-3, 10 ng/ml IL-6 (R&D Systems), and 50 ng/ml SCF. Retroviral supernatant was then added, and cells were spin-infected at 2500 rpm for 90 min at room temperature in the presence of 8 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO). Cells were again incubated overnight at 37°C in 5% CO2, and retroviral spin infection was repeated the following day. After overnight incubation, cells were cultured as described above to generate mast cells. After 3–4 wk, green fluorescence protein-expressing cells were sorted using a FACSVantage SE flow cytometer (BD Biosciences, Mountain View, CA).

Flow cytometric analysis

Cells were stained according to standard protocols using the following labeled Abs: mouse anti-DNP IgE (Sigma-Aldrich), anti-mouse IgE-biotin (BD PharMingen, San Diego, CA), streptavidin-allophycocyanin (BD PharMingen), and anti-mouse SLP-76-PE (31). Two-color flow cytometry was performed on a FACSCalibur (BD Biosciences).

{beta}-Hexosaminidase release assay

BMMCs (1 x 106/ml) were starved of SCF overnight, then sensitized at 1 x 107/ml in complete RPMI 1640 without cytokines with 1 µg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4 h at 37°C in 5% CO2. Cells were then washed once in Tyrode’s buffer (130 mM NaCl, 10 mM HEPES, 1 mM MgCl2, 5 mM KCl, 1.4 mM CaCl2, 5.6 mM glucose, and 1 mg/ml BSA, pH 7.4) and resuspended at 2 x 106/ml in Tyrode’s buffer. Cells (200 µl) were then stimulated with varying amounts of DNP-human serum albumin (HSA) (0–1000 ng/ml) for 1 h at 37°C. Cells were spun down, and 30 µl of supernatant was transferred to a 96-well, flat-bottom plate. Thirty microliters of 1 mM p-nitrophenyl-N-acetyl-{beta}-D-glucosamide was then added to each supernatant and mixed before incubating for 1 h at 37°C. The reaction was terminated by the addition of 200 µl of 0.1 M Na2CO3-NAHCO3 buffer, and OD was read on a plate reader at a wavelength of 405 nm.

IL-6 production assay

BMMCs (1 x 106/ml) were starved of SCF overnight, then sensitized at 1 x 107/ml in complete RPMI 1640 without cytokines with 1 µg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4 h at 37°C in 5% CO2. Cells were washed once and resuspended at 1 x 106/ml in complete RPMI 1640. Cells (5 x 104) in complete RPMI 1640 were then incubated with varying concentrations of DNP-HSA overnight at 37°C in 5% CO2 in a total volume of 100 µl in a 96-well, flat-bottom plate. Each sample was assayed in triplicate. The following day, the plate was removed from the incubator and frozen at –20°C. An ELISA was performed on thawed supernatants using a murine IL-6 ELISA kit (Pierce/Endogen, Rockford, IL).

Lysate preparation and immunoblotting

BMMCs (1 x 106/ml) were starved of SCF overnight, then sensitized at 1 x 107/ml in complete RPMI 1640 without cytokines with 1 µg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4 h at 37°C in 5% CO2. Cells were washed and resuspended at 2 x 107/ml in Tyrode’s buffer. Cells were stimulated for various times with 100 ng/ml DNP-HSA. Cells were then pelleted, supernatant was aspirated, and cells were lysed in ice-cold 1% Nonidet P-40 containing proteinase (50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml pepstatin A, and 1 mM Pefablock) and phosphatase (400 µM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate) inhibitors. Western blotting was performed using the following Abs: anti-PLC{gamma}1, anti-PLC{gamma}2, 4G10, anti-phospho-PLC{gamma}2, anti-phospho-extracellular signal-regulated kinase 1/2 (anti-phospho-Erk1/2; Upstate Biotechnology, Lake Placid, NY), and anti-Erk1/2 (Zymed Laboratories, San Francisco, CA).

Calcium flux assay

BMMCs were sensitized with anti-DNP-IgE as described in the above section. Cells were then washed once in Tyrode’s buffer and resuspended at 1 x 107 in Tyrode’s buffer containing 25 mM Probenecid (Sigma-Aldrich) and 2 mg/ml Indo-1 (Molecular Probes, Eugene, OR). Cells were protected from the 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 (BD Biosciences). Baseline Ca2+ levels were measured for 30 s before addition of DNP-HSA (100 ng/ml). The sample was collected for a total of 7–8 min, collecting ~500–700 events/s. Ionomycin was added 30 s before the end of the assay. Data were analyzed using FlowJo software (TreeStar, Ashland, OR) and are represented as the mean Ca2+ flux of cells over time.

Mast cell adhesion assay

BMMCs were sensitized with anti-DNP IgE as described above. Cells were washed once in PBS and resuspended at 1 x 107 cells/ml in PBS containing 5 µg/ml calcein-AM (Molecular Probes, Eugene, OR). Cells were incubated at 37°C for 15 min for labeling, then washed twice with PBS and resuspended at 2 x 106 cells/ml in Tyrode’s buffer. Wells of a 96-well tissue culture plate were coated with varying concentrations of fibronectin (0, 0.1, 1, and 10 µg/ml) and washed once with PBS. Fifty microliters of Tyrode’s buffer containing 200 ng/ml DNP-HSA, 80 ng/ml PMA, or no stimulus was added to triplicate wells. Labeled BMMCs (1 x 105) in 50 µl of Tyrode’s buffer were then added, and the plate was incubated at 37°C for 1 h. Wells were washed five times with 200 µl of Tyrode’s buffer, and remaining cells were quantitated by calcein fluorescence on a SpectraMax 190E microplate reader (Molecular Devices, Sunnyvale, CA). The percent maximal adhesion was calculated relative to the adhesion induced by PMA stimulation. Assays were performed in triplicate for each stimulation condition and concentration of fibronectin.

Passive systemic anaphylaxis assay

Mice were anesthetized by i.p. injection of 300 µl of 2.5% 2,2,2-tribromoethanol in tert-amyl alcohol/PBS (1/40; Sigma-Aldrich). In vivo mast cells were then sensitized with 3 µg of anti-DNP IgE in 200 µl of PBS by i.v. retro-orbital injection. Twenty-four hours later mice were again anesthetized and challenged with 100 µg of DNP-HSA in 200 µl of PBS by i.v. retro-orbital injection. Ninety seconds after challenge, mice were cervically dislocated, and blood was collected by cardiac puncture. Plasma was separated from blood by centrifuging samples for 10 min at 8000 rpm at 4°C. The plasma histamine concentration was determined by competitive histamine immunoassay (Immunotech, Marseilles, France).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reconstitution of SLP-76-deficient BMMCs with SLP-76 mutants

To determine the requirements for the different domains of SLP-76 for Fc{epsilon}RI-mediated mast cell function, we expressed wild-type SLP-76 or one of three mutant forms in BMMCs by retroviral transduction. For the Y3F mutant of SLP-76, the three N-terminal tyrosines shown to mediate phosphorylation-inducible binding of Vav, Nck, and Itk have been mutated to phenylalanine (32). The {Delta}20 mutant of SLP-76 lacks aa 224–244, which are known to mediate a constitutive association with the adapter Gads (18, 33). The R448K mutant of SLP-76 has a point mutation abolishing the arginine residue known to be critical for SH2 domain binding of phosphorylated tyrosines in ADAP (20) and HPK-1 (22) (Fig. 1A). After reconstitution of SLP-76-deficient BMMCs with retrovirally expressed wild-type or mutant SLP-76, BMMCs were sorted for equivalent expression of green fluorescence protein (expressed by MIGR1 retroviral plasmid) by flow cytometry. Cells expressed comparable levels of Fc{epsilon}RI and SLP-76, as determined by cell surface and intracellular FACS, respectively (Fig. 1B).



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  		FIGURE 1. Schematic of SLP-76. A, The three functional domains of SLP-76 are shown with their binding partners. In the Y3F mutant, the three N-terminal tyrosines are mutated to phenylalanine, the {Delta}20 mutation is lacking aa 224–244, and the R448K mutation has a mutation of arginine to lysine at position 448 within the SH2 domain. B, Reconstituted BMMC lines have equivalent expression of Fc{epsilon}RI and SLP-76. After 4 h of anti-DNP-IgE sensitization, BMMC were stained with anti-IgE-biotin and strepavidin-allophycocyanin. For intracellular staining, cells were fixed, permeabilized, and stained with SLP-76-PE. Histograms show the surface expression of Fc{epsilon}RI (left histogram) and intracellular expression of SLP-76 (right histogram) in SLP-76–/– BMMC retrovirally transduced with WT (thick solid line), Y3F (thick dotted line), {Delta}20 (thick shaded line), R448K (thin dashed line), and MIGR1 (thin solid line).

 
N-terminal tyrosines and the proline-rich region are required for SLP-76 function

SLP-76 has been shown to be required for several Fc{epsilon}RI-induced mast cell functions in vitro, including granule release and cytokine production (7). We therefore investigated the ability of mast cells harboring mutant forms of SLP-76 to release the granule component hexosaminidase and to secrete IL-6. SLP-76-deficient BMMCs do not release hexosaminidase upon Fc{epsilon}RI cross-linking, whereas BMMCs reconstituted with wild-type SLP-76 respond robustly (Fig. 2A). Neither the Y3F nor the {Delta}20 mutant of SLP-76 restores significant granule release, whereas the R448K mutant rescues ~50% of wild-type function. A similar pattern of function is seen for IL-6 production, with Y3F and {Delta}20 mutants providing negligible augmentation of cytokine production over SLP-76-deficient BMMCs, and the R448K mutant restoring ~50% of wild-type function (Fig. 2B).



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  		FIGURE 2. Functional responses of SLP-76–/– reconstituted BMMC. The ability of SLP-76–/– BMMC reconstituted with WT ({blacktriangleup}), Y3F ({blacksquare}), {Delta}20 ({diamondsuit}), R448K (•), or MIGR1 (asterisks) to release {beta}-hexosaminidase (A) and secrete IL-6 (B) after Fc{epsilon}RI cross-linking was measured. A, Top panel, The percentage of hexosaminidase release was calculated relative to the hexosaminidase activity of mast cells lysed in 0.5% Triton X-100. A, Bottom panel, The percentage of wild-type degranulation was averaged for four experiments, and error bars represent the SEM. IL-6 secretion was measured in triplicate, for which the SEM was calculated and is too small to visualize.

 
In an effort to elucidate the signal transduction events underlying the functional deficits apparent in BMMCs bearing mutant forms of SLP-76, we assessed several biochemical markers of mast cell activation. SLP-76-deficient BMMCs demonstrate markedly decreased calcium flux upon Fc{epsilon}RI cross-linking, whereas restoration of wild-type SLP-76 expression restores normal flux. Here again expression of the Y3F and {Delta}20 mutants of SLP-76 restored calcium flux minimally, with the {Delta}20 mutant consistently demonstrating a marginally better calcium response than the Y3F mutant. As with hexosaminidase and cytokine production, expression of the R448K mutant resulted in an intermediate response (Fig. 3A). As mast cells express two isoforms of PLC{gamma}, PLC{gamma}1 and PLC{gamma}2, we investigated phosphorylation of these two enzymes in SLP-76 mutant BMMCs. SLP-76-deficient as well as Y3F and {Delta}20 mutant BMMCs show significantly diminished inducible phosphorylation of PLC{gamma}2. The R448K mutant again demonstrates an intermediate phenotype, partially restoring phosphorylation of PLC{gamma}2 (Fig. 3B). In contrast, SLP-76-deficient BMMCs show only slight defects in inducible phosphorylation of PLC{gamma}1 (Fig. 3B). We further investigated activation of the mitogen-activated protein kinase Erk in SLP-76-deficient mast cells, finding only mildly decreased Erk phosphorylation in these cells (Fig. 3C).



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  		FIGURE 3. Biochemical evaluation of SLP-76–/– reconstituted BMMC. A, Ca2+ flux in SLP-76–/– reconstituted BMMC was measured by flow cytometry. Anti-DNP-IgE-sensitized BMMC were stimulated with DNP at 30 s and ionomycin at 6.5 min. B, BMMC were left unstimulated or were stimulated for 2 or 10 min with 100 µg/ml DNP. Cell lysates were analyzed by Western blot using a phospho-PLC{gamma}2-specific Ab and an anti-tubulin Ab to control for protein loading (top panel) or using a phospho-PLC{gamma}1-specific Ab and a PLC{gamma}1 loading control (lower panel). Total expression of PLC{gamma}1 and PLC{gamma}2 was similar for all cell lines (data not shown). C, Erk1/2 phosphorylation was detected in whole cell lysates by Western blot using a phospho-specific Erk antibody. A Western blot for total PLC{gamma}1 was used as a loading control. Data are representative of at least three experiments.

 
ADAP-deficient BMMCs degranulate and produce cytokines normally

Given the partial defects in calcium flux, degranulation, and cytokine production seen in mast cells expressing the R448K mutant of SLP-76, and the augmentation of mast cell adhesion and degranulation reported upon overexpression of ADAP in the RBL-2H3 mast cell line (27), we investigated Fc{epsilon}RI signaling and function in ADAP-deficient BMMCs. ADAP is tyrosine-phosphorylated after Fc{epsilon}RI stimulation in wild-type cells (Fig. 4A). ADAP-deficient cells demonstrate normal surface expression of c-Kit and Fc{epsilon}RI and develop normally in vitro (data not shown). In addition, ADAP-deficient BMMCs show no defects in hexosaminidase release (Fig. 4B) or IL-6 production (Fig. 4C) after Fc{epsilon}RI stimulation.



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  		FIGURE 4. Function of ADAP+/+ and ADAP–/– BMMC. A, ADAP is phosphorylated after stimulation of BMMC through the Fc{epsilon}RI. Cell lysates from anti-DNP-IgE-sensitized ADAP+/+ BMMC were left unstimulated or were stimulated with 30 ng/ml DNP for various time periods and analyzed by Western blot using 4G10. Arrows indicate the 120- and 130-kDa forms of ADAP. The ability of ADAP+/+ and ADAP–/– BMMC to release {beta}-hexosaminidase (B) and secrete IL-6 (C) after Fc{epsilon}RI cross-linking was measured. Data are representative of four experiments, and IL-6 secretion was measured in triplicate for which the SEM was calculated and is too small to visualize. Differences in degranulation and IL-6 production between ADAP+/+ and ADAP–/– were not statistically significant. D, BMMC were stimulated through the Fc{epsilon}RI and analyzed for their ability to adhere to various concentrations of fibronectin. Adhesion of unstimulated cells, which was comparable for all samples, was subtracted. The percentage of maximal adhesion was calculated relative to PMA-induced adhesion, which was equivalent among all samples. For ADAP+/+ and ADAP–/– BMMC, the average of five experiments is shown. For SLP-76–/– reconstituted BMMC, the average of three experiments is shown. Error bars represents the SEM.

 
ADAP-deficient BMMCs have normal Fc{epsilon}RI-induced adhesion to fibronectin, but SLP-76-deficient BMMCs do not

In T cells, ADAP deficiency has been shown to impair TCR-induced clustering of the integrin LFA-1 ({alpha}L{beta}2) as well as adhesion to integrin substrates ICAM-1 and VCAM-1 (34, 35). Recently, others reported that overexpression of ADAP in RBL-2H3 cells results in increased VLA-4 ({alpha}4{beta}1) clustering and enhanced basal adhesion to fibronectin (26, 27). To test whether ADAP deficiency impairs the ability of BMMCs to inducibly adhere, we performed static adhesion assays. Mast cells were stimulated through the Fc{epsilon}RI, via phorbol ester, or were left unstimulated, and adhesion to fibronectin-coated plates was assessed. Surprisingly, ADAP-deficient BMMCs demonstrate a robust Fc{epsilon}RI-induced adhesion to fibronectin, similar to wild-type BMMCs (Fig. 4D). We then questioned whether SLP-76 was required for mast cell adhesion. BMMCs from SLP-76-deficient mice show decreased adhesion to fibronectin upon Fc{epsilon}RI stimulation, although some inducible adhesion can be detected at the highest dose of fibronectin (10 µg/ml). However, BMMCs expressing the R448K mutant of SLP-76 adhere normally to fibronectin (Fig. 4D). This result indicates that inside-out activation of {beta}1 integrins on BMMCs requires SLP-76, but is independent of both ADAP and the SH2 domain of SLP-76, and may therefore be mechanistically distinct from TCR-induced integrin activation.

Passive systemic anaphylactic response is intact in ADAP-deficient mice

Finally, to extend these results in vivo, we examined the passive systemic anaphylactic response in ADAP-deficient and wild-type mice. Mast cells were sensitized in vivo by i.v. injection of monoclonal {alpha}-DNP-IgE and later challenged by i.v. injection of DNP. Mice were sacrificed 90 s after Ag challenge and blood was collected for analysis of histamine levels. As shown in Fig. 5, ADAP-deficient and wild-type mice developed comparable levels of histamine in the blood after Ag administration, whereas SLP-76-deficient mice demonstrated very low histamine response, as previously reported (7). Thus, we have detected no defects in Fc{epsilon}RI-mediated mast cell function in vitro or in vivo in the absence of ADAP.



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  		FIGURE 5. ADAP–/– mice retain the ability to exhibit a passive systemic anaphylactic response. Anti-DNP-IgE-sensitized ADAP+/+ (n = 3), ADAP–/– (n = 5), and SLP–/– (n = 3) mice were challenged with DNP for 90 s. Plasma histamine levels were determined by ELISA. The SEM is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated in this study that the N-terminal tyrosines and the central proline-rich region of SLP-76 are required for its function downstream of the Fc{epsilon}RI in mast cells. BMMCs expressing SLP-76 that lacks either of these domains flux calcium, degranulate, and produce IL-6 only marginally better than cells lacking expression of SLP-76 entirely (7). The SH2 domain of SLP-76 is also required for optimal mast cell function, and BMMCs expressing this mutant have ~50% the functional response of wild-type cells. We have further shown that the phenotype of this SH2 domain mutant of SLP-76 is probably not due to an inability to associate with the adapter protein ADAP. ADAP-deficient BMMCs show no functional defects in Fc{epsilon}RI-induced adhesion, degranulation, or cytokine production, and ADAP-deficient mice have an intact passive systemic anaphylactic response. Taken together, these data suggest that the formation of a multimolecular signaling complex anchored by SLP-76, Gads, and LAT and recruiting Vav, Nck, and Btk is an important element of signaling downstream of the Fc{epsilon}RI in mast cells. In contrast, ADAP is dispensable for these Fc{epsilon}RI-induced mast cell functions and is unlikely to mediate its postulated role as an integrator of Lyn- and Fyn-mediated signaling.

A structure/function analysis of SLP-76 in mast cells has recently been reported (36). Although the results reported in this study are largely in accord with those findings, there are some notable differences. Most strikingly, Kettner et al. (36) have reported that BMMCs expressing a Y3F mutant of SLP-76 retain the ability to produce normal amounts of IL-6 and to flux calcium and degranulate significantly better than SLP-76-deficient mast cells. In contrast, the expression of a mutant of SLP-76 lacking the entire N-terminal acidic region, including all three phosphorylated tyrosines, fails to restore significant Fc{epsilon}RI function in mast cells. Although we have not tested an N-terminal truncation mutant, we observed minimal cytokine production, degranulation, and calcium flux for the Y3F mutant of SLP-76. We do not believe that these discrepancies are due to differences in the degree of Fc{epsilon}RI cross-linking, because the Y3F mutant of SLP-76 functions poorly across a wide range of Ag doses in our experiments (Fig. 2 and data not shown). Notably, our studies were performed with the mouse monoclonal anti-DNP IgE SPE7, whereas Kettner et al. (36) used polyclonal rat IgE. SPE7 has been reported to induce mast cell signaling, survival, and cytokine production in the absence of Ag cross-linking (37, 38). This reagent and other methodologic differences may account for the divergent results. Thus, our results indicate that if there exists an additional domain in the N-terminal region of SLP-76 contributing to mast cell function, it is unable to compensate for the absence of the tyrosine phosphorylation motifs in our experimental approach.

The R448K point mutant of SLP-76 is able to only partially restore each of the aberrant functional responses we measured in SLP-76-deficient BMMCs, suggesting that recruitment of a binding partner by the SH2 domain of SLP-76 is necessary for optimal Fc{epsilon}RI signaling. ADAP is known to bind this region of SLP-76 (20), is phosphorylated after Fc{epsilon}RI stimulation in BMMCs (Fig. 4A), and has been reported to modulate adhesion and Fc{epsilon}RI-induced degranulation in cell lines (26, 27). However, ADAP-deficient BMMCs have no demonstrable defects in adhesion, degranulation, or cytokine production, and ADAP-deficient mice produce a robust passive systemic anaphylactic response. Thus, we postulate that a protein other than ADAP is responsible for SLP-76 SH2 domain-dependent Fc{epsilon}RI function. One possible explanation for the intact Fc{epsilon}RI-induced functions of ADAP-deficient BMMCs is the presence of a homologous protein that may compensate for the absence of ADAP. We were unable to detect mRNA or protein expression of the only known homologue of ADAP, PRAM-1 (39), in mast cells (data not shown). A more likely candidate is HPK-1 (22), a known binder of the SLP-76 SH2 domain in T cells. Furthermore, we submit that ADAP is unlikely to mediate the cross-talk between the Lyn and Fyn pathways described by Parravicini et al. (23). Nonetheless, confirmation of this will require studying degranulation and calcium flux in mast cells doubly deficient for Lyn and ADAP or for Fyn and ADAP.

Two main pathways for activation of Erk have been described in mast cells and T cells: a PLC{gamma}/1,2-diacylglycerol/Ras-guanine nucleotide-releasing protein (40) cascade and a LAT/Grb2/Sos (41) pathway. Consistent with the previous report (36), we found that Erk is significantly inducibly phosphorylated in SLP-76-deficient mast cells. Although assessment of Erk activation in SLP-76-deficient T cells is not possible, varying levels of Erk phosphorylation can be observed in the SLP-76-deficient Jurkat T cell line, J14 (our unpublished observation). Thus, in both Jurkat and mast cells, partial Erk activation is possible in the absence of SLP-76. In contrast, Erk phosphorylation is absent in LAT-deficient BMMCs and in two models of LAT-deficient Jurkat cells, J.CaM2 and ANJ3 (42, 43). Given that SLP-76-deficient mast cells, much like LAT-deficient mast cells, retain some residual ability to inducibly phosphorylate PLC{gamma}1, these data suggest the possibility that Erk activation in primary mast cells is largely dependent on the LAT/Grb2/Sos pathway.

We also report that SLP-76-deficient BMMCs have a more pronounced defect in phosphorylation of PLC{gamma}2 than of PLC{gamma}1. Studies have suggested that these two isoforms of PLC{gamma} are differentially localized and activated in RBL-2H3 cells upon Fc{epsilon}RI stimulation (44, 45). Barker et al. (45) reported that inhibition of PI3K activity by wortmannin suppresses phosphorylation and lipase activity of PLC{gamma}1, but not PLC{gamma}2. Furthermore, Wilson et al. (44) describe the formation of distinct signaling domains in Fc{epsilon}RI-stimulated RBL-2H3 cells: primary signaling domains containing Fc{epsilon}RI, Syk, PLC{gamma}2, Vav, and a variety of other proteins; and secondary domains characterized by the presence of LAT and PLC{gamma}1. Preferential localization of SLP-76 to either of these domains has not yet been described, but our data suggest that SLP-76 may be more important for recruitment or stabilization of PLC{gamma}2 in close proximity to its activating kinase within primary signaling domains.

SLP-76-deficient BMMCs have been reported to retain the ability to inducibly phosphorylate a number of substrates, including LAT, PLC{gamma}, Vav, Btk, and Erk (7, 36), suggesting that, as in LAT-deficient BMMCs, several components of Fc{epsilon}RI signaling, perhaps along the Fyn/Gab2/PI3K pathway, are intact. Future studies will continue to address the integrity of these pathways and their relevance to in vitro and in vivo mast cell functions.


   Footnotes
 
1 This work was supported by the National Institutes of Health (to G.A.K., E.J.P., J.N.W., and M.A.S.), the Sandler Foundation for Asthma Research (to G.A.K.), the Arthritis Foundation (to E.J.P.), the Cancer Research Institute (to M.S.J.), and the Abramson Family Cancer Research Institute. Back

2 J.N.W. and M.S.J. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Gary A. Koretzky, 415 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: koretzky{at}mail.med.upenn.edu Back

4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ADAP, adhesion- and degranulation-promoting adapter protein; BMMC, bone marrow-derived mast cell; Erk, extracellular signal-regulated kinase; HSA, human serum albumin; LAT, linker for activation of T cell; PI3K, phosphotidylinositol 3-kinase; PLC{gamma}1, phospholipase C{gamma}1; PTK, protein tyrosine kinase; SCF, stem cell factor; SH2, Src homology 2; SLP-76, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa. Back

Received for publication January 8, 2004. Accepted for publication March 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kinet, J. P.. 1999. The high-affinity IgE receptor (Fc{epsilon}RI): from physiology to pathology. Annu. Rev. Immunol. 17:931.[Medline]
  2. Rivera, J., R. Arudchandran, C. Gonzalez-Espinosa, T. S. Manetz, S. Xirasagar. 2001. A perspective: regulation of IgE receptor-mediated mast cell responses by a LAT-organized plasma membrane-localized signaling complex. Int Arch. Allergy Immunol. 124:137.[Medline]
  3. Clements, J. L., J. R. Lee, B. Gross, B. Yang, J. D. Olson, A. Sandra, S. P. Watson, S. R. Lentz, G. A. Koretzky. 1999. Fetal hemorrhage and platelet dysfunction in SLP-76-deficient mice. J. Clin. Invest. 103:19.[Medline]
  4. Pivniouk, V., E. Tsitsikov, P. Swinton, G. Rathbun, F. W. Alt, R. S. Geha. 1998. Impaired viability and profound block in thymocyte development in mice lacking the adaptor protein SLP-76. Cell 94:229.[Medline]
  5. Judd, B. A., G. A. Koretzky. 2001. The role of the adapter molecule SLP-76 in platelet function. Oncogene 20:6291.[Medline]
  6. Newbrough, S. A., A. Mocsai, R. A. Clemens, J. N. Wu, M. A. Silverman, A. L. Singer, C. A. Lowell, G. A. Koretzky. 2003. SLP-76 regulates Fc{gamma} receptor and integrin signaling in neutrophils. Immunity 19:761.[Medline]
  7. Pivniouk, V. I., T. R. Martin, J. M. Lu-Kuo, H. R. Katz, H. C. Oettgen, R. S. Geha. 1999. SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells. J. Clin. Invest. 103:1737.[Medline]
  8. Musci, M. A., D. G. Motto, S. E. Ross, N. Fang, G. A. Koretzky. 1997. Three domains of SLP-76 are required for its optimal function in a T cell line. J. Immunol. 159:1639.[Abstract]
  9. Kumar, L., V. Pivniouk, M. A. de la Fuente, D. Laouini, R. S. Geha. 2002. Differential role of SLP-76 domains in T cell development and function. Proc. Natl. Acad. Sci. USA 99:884.[Abstract/Free Full Text]
  10. Myung, P. S., G. S. Derimanov, M. S. Jordan, J. A. Punt, Q. H. Liu, B. A. Judd, E. E. Meyers, C. D. Sigmund, B. D. Freedman, G. A. Koretzky. 2001. Differential requirement for SLP-76 domains in T cell development and function. Immunity 15:1011.[Medline]
  11. Wu, J., D. G. Motto, G. A. Koretzky, A. Weiss. 1996. Vav and SLP-76 interact and functionally cooperate in IL-2 gene activation. Immunity 4:593.[Medline]
  12. Tuosto, L., F. Michel, O. Acuto. 1996. p95vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells. J. Exp. Med. 184:1161.[Abstract/Free Full Text]
  13. Onodera, H., D. G. Motto, G. A. Koretzky, D. M. Rothstein. 1996. Differential regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76 to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase. J. Biol. Chem. 271:22225.[Abstract/Free Full Text]
  14. Wunderlich, L., A. Farago, J. Downward, L. Buday. 1999. Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes. Eur. J. Immunol. 29:1068.[Medline]
  15. Su, Y. W., Y. Zhang, J. Schweikert, G. A. Koretzky, M. Reth, J. Wienands. 1999. Interaction of SLP adaptors with the SH2 domain of Tec family kinases. Eur. J. Immunol. 29:3702.[Medline]
  16. Bunnell, S. C., M. Diehn, M. B. Yaffe, P. R. Findell, L. C. Cantley, L. J. Berg. 2000. Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade. J. Biol. Chem. 275:2219.[Abstract/Free Full Text]
  17. Asada, H., N. Ishii, Y. Sasaki, K. Endo, H. Kasai, N. Tanaka, T. Takeshita, S. Tsuchiya, T. Konno, K. Sugamura. 1999. Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT. J. Exp. Med. 189:1383.[Abstract/Free Full Text]
  18. Liu, S. K., N. Fang, G. A. Koretzky, C. J. McGlade. 1999. The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors. Curr. Biol. 9:67.[Medline]
  19. Yablonski, D., T. Kadlecek, A. Weiss. 2001. Identification of a phospholipase C-{gamma}1 (PLC-{gamma}1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-{gamma}1 and NFAT. Mol. Cell. Biol. 21:4208.[Abstract/Free Full Text]
  20. Musci, M. A., L. R. Hendricks-Taylor, D. G. Motto, M. Paskind, J. Kamens, C. W. Turck, G. A. Koretzky. 1997. Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases. J. Biol. Chem. 272:11674.[Abstract/Free Full Text]
  21. da Silva, A. J., Z. Li, C. de Vera, E. Canto, P. Findell, C. E. Rudd. 1997. Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production. Proc. Natl. Acad. Sci. USA 94:7493.[Abstract/Free Full Text]
  22. Sauer, K., J. Liou, S. B. Singh, D. Yablonski, A. Weiss, R. M. Perlmutter. 2001. Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes. J. Biol. Chem. 276:45207.[Abstract/Free Full Text]
  23. Parravicini, V., M. Gadina, M. Kovarova, S. Odom, C. Gonzalez-Espinosa, Y. Furumoto, S. Saitoh, L. E. Samelson, J. J. O’Shea, J. Rivera. 2002. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat. Immunol. 3:741.[Medline]
  24. Setoguchi, R., T. Kinashi, H. Sagara, K. Hirosawa, K. Takatsu. 1998. Defective degranulation and calcium mobilization of bone-marrow derived mast cells from Xid and Btk-deficient mice. Immunol. Lett. 64:109.[Medline]
  25. Kawakami, Y., J. Kitaura, A. B. Satterthwaite, R. M. Kato, K. Asai, S. E. Hartman, M. Maeda-Yamamoto, C. A. Lowell, D. J. Rawlings, O. N. Witte, et al 2000. Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation. J. Immunol. 165:1210.[Abstract/Free Full Text]
  26. Geng, L., C. Rudd. 2001. Adaptor ADAP (adhesion- and degranulation-promoting adaptor protein) regulates {beta}1 integrin clustering on mast cells. Biochem. Biophys. Res. Commun. 289:1135.[Medline]
  27. Geng, L., S. Pfister, S. K. Kraeft, C. E. Rudd. 2001. Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain. Proc. Natl. Acad. Sci. USA 98:11527.[Abstract/Free Full Text]
  28. Pear, W. S., J. P. Miller, L. Xu, J. C. Pui, B. Soffer, R. C. Quackenbush, A. M. Pendergast, R. Bronson, J. C. Aster, M. L. Scott, et al 1998. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92:3780.[Abstract/Free Full Text]
  29. Judd, B. A., P. S. Myung, A. Obergfell, E. E. Myers, A. M. Cheng, S. P. Watson, W. S. Pear, D. Allman, S. J. Shattil, G. A. Koretzky. 2002. Differential requirement for LAT and SLP-76 in GPVI versus T cell receptor signaling. J. Exp. Med. 195:705.[Abstract/Free Full Text]
  30. Pear, W. S., G. P. Nolan, M. L. Scott, D. Baltimore. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90:8392.[Abstract/Free Full Text]
  31. Clements, J. L., S. E. Ross-Barta, L. T. Tygrett, T. J. Waldschmidt, G. A. Koretzky. 1998. SLP-76 expression is restricted to hemopoietic cells of monocyte, granulocyte, and T lymphocyte lineage and is regulated during T cell maturation and activation. J. Immunol. 161:3880.[Abstract/Free Full Text]
  32. Fang, N., D. G. Motto, S. E. Ross, G. A. Koretzky. 1996. Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. J. Immunol. 157:3769.[Abstract]
  33. Motto, D. G., S. E. Ross, J. Wu, L. R. Hendricks-Taylor, G. A. Koretzky. 1996. Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production. J. Exp. Med. 183:1937.[Abstract/Free Full Text]
  34. Griffiths, E. K., C. Krawczyk, Y. Y. Kong, M. Raab, S. J. Hyduk, D. Bouchard, V. S. Chan, I. Kozieradzki, A. J. Oliveira-Dos-Santos, A. Wakeham, et al 2001. Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/Slap. Science 293:2260.[Abstract/Free Full Text]
  35. Peterson, E. J., M. L. Woods, S. A. Dmowski, G. Derimanov, M. S. Jordan, J. N. Wu, P. S. Myung, Q. H. Liu, J. T. Pribila, B. D. Freedman, et al 2001. Coupling of the TCR to integrin activation by Slap-130/Fyb. Science 293:2263.[Abstract/Free Full Text]
  36. Kettner, A., V. Pivniouk, L. Kumar, H. Falet, J. S. Lee, R. Mulligan, R. S. Geha. 2003. Structural requirements of SLP-76 in signaling via the high-affinity immunoglobulin E receptor (Fc{epsilon}RI) in mast cells. Mol. Cell. Biol. 23:2395.[Abstract/Free Full Text]
  37. Kitaura, J., J. Song, M. Tsai, K. Asai, M. Maeda-Yamamoto, A. Mocsai, Y. Kawakami, F. T. Liu, C. A. Lowell, B. G. Barisas, et al 2003. Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the Fc{epsilon}RI. Proc. Natl. Acad. Sci. USA 100:12911.[Abstract/Free Full Text]
  38. Kalesnikoff, J., M. Huber, V. Lam, J. E. Damen, J. Zhang, R. P. Siraganian, G. Krystal. 2001. Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 14:801.[Medline]
  39. Moog-Lutz, C., E. J. Peterson, P. G. Lutz, S. Eliason, F. Cave-Riant, A. Singer, Y. Di Gioia, S. Dmowski, J. Kamens, Y. E. Cayre, et al 2001. PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor {alpha} in acute promyelocytic leukemia cells. J. Biol. Chem. 276:22375.[Abstract/Free Full Text]
  40. Ebinu, J. O., S. L. Stang, C. Teixeira, D. A. Bottorff, J. Hooton, P. M. Blumberg, M. Barry, R. C. Bleakley, H. L. Ostergaard, J. C. Stone. 2000. RasGRP links T-cell receptor signaling to Ras. Blood 95:3199.[Abstract/Free Full Text]
  41. Jabril-Cuenod, B., C. Zhang, A. M. Scharenberg, R. Paolini, R. Numerof, M. A. Beaven, J. P. Kinet. 1996. Syk-dependent phosphorylation of Shc. A potential link between Fc{epsilon}RI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2. J. Biol. Chem. 271:16268.[Abstract/Free Full Text]
  42. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, A. Weiss. 1998. LAT is required for TCR-mediated activation of PLC{gamma}1 and the Ras pathway. Immunity 9:617.[Medline]
  43. Zhang, W., B. J. Irvin, R. P. Trible, R. T. Abraham, L. E. Samelson. 1999. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11:943.[Abstract/Free Full Text]
  44. Wilson, B. S., J. R. Pfeiffer, Z. Surviladze, E. A. Gaudet, J. M. Oliver. 2001. High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc{epsilon}RI and LAT. J. Cell Biol. 154:645.[Abstract/Free Full Text]
  45. Barker, S. A., K. K. Caldwell, J. R. Pfeiffer, B. S. Wilson. 1998. Wortmannin-sensitive phosphorylation, translocation, and activation of PLC{gamma}1, but not PLC{gamma}2, in antigen-stimulated RBL-2H3 mast cells. Mol. Biol. Cell 9:483.[Abstract/Free Full Text]



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