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Overcoming the Signaling Defect of Lyn-Sequestering, Signal-Curtailing FcRI Dimers: Aggregated Dimers Can Dissociate from Lyn and Form Signaling Complexes with Syk¹

Martha Lara^*, Enrique Ortega^2,*, Israel Pecht, Janet R. Pfeiffer, A. Marina Martinez, Rebecca J. Lee, Zurab Surviladze, Bridget S. Wilson and Janet M. Oliver

^* Departamento de Inmunologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autónoma de Mexico, Mexico DF, Mexico; Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel; and Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM 87131

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clustering the tetrameric (₂) IgE receptor, FcRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcRI and subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcRI dimers formed by a particular anti-FcRI mAb (H10) initiate signaling through Lyn activation and FcRI subunit phosphorylation, but cause only modest activation of Syk and little Ca²⁺ mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcRI multimers, induced by adding F(ab')₂ of goat anti-mouse IgG to H10-treated cells, support strong Ca²⁺ mobilization and secretion. Accompanying the recovery of signaling, H10-FcRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase C2. Immunogold electron microscopy showed that H10-FcRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic "signaling domains" that accumulate FcRI and Syk in Ag-treated cells. In contrast, H10-FcRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcRI from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In basophils and mast cells, clustering the type I receptor for IgE, FcRI, activates a signaling sequence that leads within minutes to degranulation and membrane/cytoskeletal responses, including actin polymerization, ruffling, spreading, integrin activation, and actin plaque assembly, and leads within hours to increased cytokine synthesis (reviewed in Ref. 1).

Previous studies with the rat mucosal-type mast cell line RBL-2H3 have established the probable sequence of early events by which cross-linking this tetrameric (₂) immunoreceptor leads to cell activation. RBL-2H3 cells contain two FcRI-associated protein-tyrosine kinases, the Src-related enzyme, Lyn (2), whose principal substrates are the receptor’s and subunits (3, 4), and the 72-kDa protein-tyrosine kinase, Syk (5), which phosphorylates a wide range of downstream signaling molecules including phospholipase C (PLC)³ isoforms, the p85 subunit of phosphatidylinositol 3 (PI 3)-kinase, p95^vav, Grb2, Cbl, linker for activation of T cells, Src homology 2 domain-containing leukocyte protein of 76 kDa, and others (reviewed in Refs. 1 and 6). Biochemical and morphological studies showed that a portion of the Lyn in resting RBL-2H3 cells associates with the FcRI subunit (7, 8). FcRI cross-linking permits Lyn to phosphorylate tyrosines located within immunoreceptor tyrosine-based activation motifs (ITAMs) in the and subunits of adjacent receptors (9). The doubly phosphorylated FcRI ITAMs serve as binding sites for the tandem Src homology 2 domains of Syk, resulting in its autophosphorylation and activation (10). A similar sequential activation of FcRI-associated kinases and downstream signaling molecules has been observed in mouse bone marrow-derived mast cells (11) and in human blood basophils (12). A very similar biochemical cascade of successive Src and Syk kinase activation leading to downstream responses is also initiated by ligating other immunoreceptors, including the TCR, the B cell receptor, and several members of the FcR family (reviewed in Ref. 13).

Recent studies have emphasized the importance of membrane topography in FcRI signaling. From sucrose gradient centrifugation studies, Field et al. (14, 15, 16) suggested that clustered FcRI may encounter Lyn in detergent-resistant microdomains that are also enriched for the glycerophosphatidylinositol-linked protein Thy-1, glycosphingolipids, gangliosides, and cholesterol. Stauffer and Meyer (17) used fluorescence microscopy to suggest that Syk also associates with aggregated FcRI in ganglioside-enriched membrane patches. Wilson et al. (8) used immunogold electron microscopy on native membrane sheets obtained from RBL-2H3 cells to show that FcRI interacts with Lyn and Syk in topographically distinct microdomains. In unstimulated cells, FcRI and Lyn are loosely colocalized in small, dispersed membrane clusters that are rarely adjacent to coated pits. FcRI cross-linking with multivalent Ag induces a separation of receptor from Lyn, apparently by Lyn segregation to the periphery of larger receptor clusters. These Lyn-excluding clusters characteristically form on membrane patches that stain intensely with osmium and are very often found adjacent to coated pits. Syk shows no association with FcRI in resting cells but is dramatically recruited to the FcRI aggregates that form on osmiophilic membrane patches in Ag-stimulated cells. The presence of Syk and other signaling molecules (PLC2, PI 3-kinase, Gab2, and others; Refs. 8 and 18) identifies the osmiophilic membrane patches as likely sites of active signaling to downstream responses.

Previously, we have explored the signaling properties of a series of anti-FcRI mAbs that compete with each other and with IgE for binding sites on the subunit of the FcRI expressed on RBL-2H3 cells. Comparison of the secretory dose-response curves with the extent of FcRI dimerization demonstrated that anti-FcRI mAb H10-receptor dimers elicit substantially less secretion than dimers induced by several other anti-FcRI mAbs (19). mAb H10-receptor dimers also induce very little inositol 1,4,5-trisphosphate synthesis, Ca²⁺ mobilization, spreading, ruffling, and actin plaque assembly in comparison with dimers generated with the other anti-FcRI mAbs and with multivalent Ag (20).

Studies of the FcRI-associated kinases showed that although H10-receptor dimers activate Lyn and support FcRI and subunit phosphorylation, they are poor Syk activators in comparison with Ag and the other anti-FcRI mAbs (20). The apparent curtailment of signaling downstream of and subunit phosphorylation in mAb H10-treated cells was linked to the formation of unusual detergent-resistant complexes between activated Lyn and receptor subunits. We hypothesized from these studies that the signal curtailing properties of H10-receptor dimers may result from the failure of Lyn dissociation from receptor subunits, a previously unrecognized regulatory step in the FcRI signaling cascade needed for Syk activation and signal progression.

Here we show that H10-FcRI multimers generated by adding F(ab')₂ of goat anti-mouse IgG (GaM) to cross-link the H10-FcRI dimers are able to elicit near-normal signaling responses. The multimers more effectively support the dissociation of Lyn from phosphorylated FcRI subunits, the phosphorylation and activation of Syk, and the progression of the signaling cascade to PLC2 phosphorylation and Ca²⁺ mobilization and secretion. Immunogold electron microscopy of membrane sheets prepared from RBL-2H3 cells treated with mAb H10 dimers and multimers suggests that H10-receptor dimers become trapped in signal-initiating Lyn microdomains, and that added GaM restores signaling by enabling the redistribution of H10-multimers to separate membrane microdomains that accumulate the signal-propagating kinase, Syk.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

The mAb H10 has been previously described (19). The purification of monoclonal mouse anti-DNP IgE from ascites and the preparation of rabbit anti-mouse IgE was also done as previously described (21, 22, 23). Mouse mAb JRK against the FcRI subunit was a gift from Dr. J. Rivera (National Institutes of Health, Bethesda, MD), sheep polyclonal Ab against the FcRI subunit was a gift from Dr. J.-P. Kinet (Harvard Medical School, Boston, MA), and polyclonal rabbit anti-Syk Ab was a gift from Dr. P. Draber (Institute of Cell Biology, Prague, the Czech Republic). Mouse anti-phosphotyrosine mAb PY20 (anti-PY) and PY20-HRP were obtained from Transduction Laboratories (Lexington, KY); rabbit polyclonal anti-Lyn, anti-Syk, and anti-PLC2 Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); and HRP-conjugated secondary Ab and GaM were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). DNP₂₄-BSA (DNP-BSA) was obtained from Molecular Probes (Eugene, OR), and colloidal gold particles (3–10 nm in diameter) conjugated to anti-rabbit IgG and anti-mouse IgG were obtained from Nanoprobes (Stony Brook, NY) and Amersham Pharmacia Biotech (Piscataway, NJ).

Cell activation

Conditions for RBL-2H3 cell culture were previously described (23). For stimulation, cell suspensions or monolayers were usually washed three times with modified Hanks’ buffer (24) containing 0.1% BSA and activated for 5 min at 37°C by the addition of 0.1 or 1.0 µg/ml of DNP-BSA (Ag, XL), 7 nM H10 (H10, H10-D), or 7 nM H10 plus 10 nM GaM (H-10M), all in Hanks’-BSA buffer. The Ag-treated cells, but not the H10-treated cells, were previously incubated overnight with 1 µg/ml of anti-DNP-IgE.

Secretion

Release of the granule enzyme -hexosaminidase was measured as previously described (19). All measurements were done in triplicate. To calculate the percentage of total enzyme released under every experimental condition, total cell -hexosaminidase content was measured by Triton X-100 lysis of an equivalent number of unstimulated cells.

Ca²⁺ mobilization

Untreated or IgE-primed RBL-2H3 cells were placed on coverslips in a stage microincubator (TC202A, Harvard Apparatus, Holliston, MA) and loaded for 30 min with 2 µM fura 2-AM (Molecular Probes) at room temperature under 5% CO₂. After loading, extracellular dye was removed by solution exchange with Hanks’-BSA, and the temperature was increased to 37°C. Experiments were done on a Zeiss IM35 inverted microscope equipped with a 200 W Hg/xenon combination lamp and computer-controlled filter wheels and shutters that allow excitation light to pass alternately through 10 nm bandpass filters centered at 350 and 385 nm (Zeiss, Oberkochen, Germany). Emitted fluorescence was collected at >510 nm using an intensified Sony CCD camera (Sony, Tokyo, Japan) interfaced to a Compix image analysis system (Compix, Cranberry Township, PA). After acquisition of baseline fluorescence for 2 min, stimuli were added by pipette to give final concentrations of (approximately) 7 nM H10, 7 nM H10 plus 10 nM GaM, and 0.1 µg/ml DNP-BSA, and fluorescence emissions were measured for an additional 6–8 min. Data were corrected for background, and average ratio values for each cell in a field were calculated for user-defined areas within each cell as previously described (25, 26). Average ratio values were converted to intracellular Ca²⁺ concentration on the basis of calibration solutions containing maximal and minimal Ca²⁺ levels. Each experiment provided time-resolved analysis of Ca²⁺ levels for between 10 and 40 individual cells. The extent of the increase in intracellular Ca²⁺ concentration was determined using the Prism "area under the curve" analysis. A 240-s integral was calculated for each cell beginning at its initial response. Appropriate baselines for each cell were determined and subtracted from the calculated areas. Results reported are averages of the stimulated Ca²⁺ responses for the indicated number of individual cells.

Immune complex kinase assays

Cell culture dishes (6 x 10⁶ cells/100-mm dish) were activated as described above and then lysed in 1 ml ice-cold 50 nM Tris-HCl (pH 7.4), 150 nM NaCl, 1% Brij-96, and 1 µg/ml each of leupeptin, antipain, PMSF, and NaVO₄. Protein concentration in the lysis supernatants was determined by the DC Protein Assay (Bio-Rad, Hercules, CA) following the manufacturer’s instructions. Lysates were cleared of any protein A/G-reactive proteins by incubation for 1 h at 4°C twice with protein A/G-Sepharose beads. Precleared cell lysates were incubated for 1 h at 4°C with specific Ab prebound to protein A/G-Sepharose beads. After washing six times, kinase activity was determined from the incorporation of ³²P into specific proteins during a 10-min incubation at 37°C with 5 µCi of [-³²P]ATP as described in Ref. 23 .

Immunoblotting

Cells were activated, lysed, and precleared, and target proteins were immunoprecipitated with specific Abs as described above for immune complex kinase assays. Ab-protein complexes were released from the washed beads by boiling in Laemmli sample buffer, separated by 10% SDS-PAGE, and transferred to nitrocellulose. The membranes were blocked overnight by incubation with 1% BSA plus 3% milk for anti-PY blots, or with 5% milk for blotting with other Abs. After blocking, the membranes were incubated for 1 h at room temperature with the blotting Ab, washed, incubated with HRP-conjugated secondary Ab for 1 h at room temperature, and washed again. The membranes were developed by 5-min incubation with SuperSignal substrate (Pierce, Rockford, IL) and exposed to Biomax film (Eastman Kodak, Rochester, NY).

Densitometry

Digitized images of the autoradiograms (from in vitro kinase assays) or from the photographic films used to capture chemiluminescent signals from the immunoblots were obtained with the Gel-Doc 2000 System (Bio-Rad) and analyzed with the Bio-Rad Quantity One software. In most cases, signal intensities were normalized to the intensity measured in the H10-D treatment samples.

Immunoelectron microscopy

RBL-2H3 cells were allowed to settle overnight onto 15-mm round, clean glass coverslips. FcRI were cross-linked by incubation for 5 min with mAb H10 with and without goat anti-mouse IgG as described above. Alternatively, cells were primed with anti-DNP IgE and activated for 2 min with DNP-BSA (0.1–1 µg/ml). Plasma membrane sheets were prepared by a modification of the method of Sanan and Anderson (27) as previously described (8). Briefly, cell monolayers on coverslips were rapidly chilled by immersion in ice-cold HEPES buffer (25 mM HEPES (pH 7), 25 mM KCl, and 2.5 mM magnesium acetate) and were then inverted onto formvar and carbon-coated nickel electron microscopy grids that, on the day of the experiment, had been glow-discharged and floated on (poly)L-lysine (0.8 mg/ml for 30 min), followed by 10 s rinsing in distilled water and air-drying. Excess liquid was removed by blotting, and pressure was applied for 20 s by bearing down with a cork. The coverslips were lifted, leaving sections of the upper cell surface adherent to the (poly)L-lysine-coated grid. Membranes were rinsed in 4°C HEPES buffer, fixed in 2% paraformaldehyde for 7 min, and receptor subunits and kinases were labeled by sequential incubation with primary Abs and gold-conjugated secondary reagents, with intermediate washes, by inverting the grids onto droplets. Primary Abs were diluted in PBS and 0.1% BSA at the following concentrations: FcRI , 28 µg/ml; Lyn, 2 µg/ml; and Syk, 10 µg/ml. Gold-conjugated secondary reagents were diluted 1/20 from commercial stocks in PBS-BSA. The samples were postfixed in 2% glutaraldehyde in PBS and processed for TEM analysis using an Hitachi 600 transmission electron microscope (Hitachi, Tokyo, Japan).

Detergent extraction, sucrose gradient centrifugation, and analysis of membrane fractions

IgE-primed RBL-2H3 cells (40 x 10⁶ cells/treatment condition) were harvested from culture dishes with 1.5 mM EDTA in Hanks’-buffered saline without divalent cations. Washed cells were resuspended in Hanks’-buffered saline and incubated for 5 min at 37°C with no addition or with 1 µg/ml DNP-BSA (XL), 7 nM H10 (H10-D), or 7 nM H10 plus 10 nM GaM (H10-M). Cells were collected by centrifugation at 4°C, cell pellets were resuspended in 750 µl ice-cold lysis buffer containing low concentrations of detergent (10 mM Tris-HCl (pH 8.0), 0.05% Triton X-100, 50 mM NaCl, 10 mM EDTA, 10 mM glycerophosphate, 1 mM NaV0₄, and 1x protease inhibitor mixture from Roche Molecular Chemicals, Indianapolis, IN). Lysates were mixed with 750 µl 80% sucrose (prepared in 10 mM Tris-HCl (pH 8.5), 50 mM NaCl, and 2 mM EDTA) and overlaid onto 0.5 ml 80% sucrose in polyallomer tubes (13 x 51 mm), followed by 0.5-ml layers of 35, 25, and 20% and 0.6-ml aliquots of 15 and 10%. The gradient was centrifuged in a SW 55 rotor (Beckman Coulter, Fullerton, CA) at 200,000 x g for 16 h at 4°C. Fractions (0.5 ml) were harvested sequentially from the top of the gradient. For analyses of protein composition, aliquots (35 µl) were mixed with equal volume of 2x SDS sample buffer, boiled for 5 min, and separated by 8 or 10% SDS-PAGE. Proteins were transferred to nitrocellulose using a semidry blotting system (Labconco, Kansas City, MO). Blots were probed with anti-Lyn and anti-FcRI Abs followed by HRP-conjugated secondary Abs. Detection by chemiluminescence was performed as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Different secretory activities of H10-FcRI dimers and multimers

Due to its bivalency and its stoichiometry of binding to the FcRI, intact mAb H10 can aggregate FcRI only into dimers (H10-D). mAb H10 differs from some other FcRI-specific IgG mAbs by inducing only low levels of mediator secretion (19, 20). To confirm previous evidence (19) that larger aggregates of H10-FcRI complexes can induce more robust FcRI-mediated secretion, we incubated RBL-2H3 cells with intact mAb H10 plus GaM to generate H10-FcRI multimers (H10-M). The secretion of -hexosaminidase from RBL-2H3 cells stimulated with Ag, H10-D, and H10-M is shown in Fig. 1A. Approximately 1% of total -hexosaminidase was released spontaneously. H10-FcRI dimers stimulated the release of 32% of total enzyme. mAb H10 alone did not induce more secretion at any of a range of concentrations tested (0.7–100 nM, data not shown; see also Ref. 19). In contrast, H10-FcRI multimers induced the release of 60% of total -hexosaminidase, twice the release induced by dimers and similar to the 70% release induced by IgE plus Ag. Similar results were observed in multiple independent experiments during the course of the studies reported here.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Signaling competence of H10-FcRI dimers and multimers. A, RBL-2H3 cell monolayers on culture plates (3 x 107 cells in 10 ml Hanks’-BSA) were incubated for 30 min at 37°C with 1 µg/ml of DNP-BSA (XL) or with 7 nM H10 (H10-D), or 7 nM H10 plus 10 nM GaM (H10-M). Reactions were stopped with 10 ml ice-cold PBS, and -hexosaminidase activity was measured in three replicate portions of the resulting supernatants as well as in replicate portions of lysates prepared from the corresponding cell monolayers. Results are given as percentage of total enzyme content (± SEM). The experiment is representative of multiple degranulation assays performed routinely in conjunction with the biochemical analyses. B–D, RBL-2H3 cell monolayers on glass coverslips were loaded with fura 2-AM and mounted in an environmental chamber on the stage of a microscope configured for ratio imaging microscopy (25 ). Baseline Ca2+ levels were measured for 2–3 min. Cells were then activated by the addition of 7 nM mAb H10 (B), 7 nM H10 plus 10 nM GaM (C), or 0.1 µg/ml of DNP-BSA (D) directly to the incubation chamber, and measurement was continued for 6–8 min. Traces are the averaged Ca2+ mobilization responses for all cells in the experiment. The area under the curve (± SEM) is averaged from individual determinations for each cell as described in Materials and Methods. Numbers of cells per condition are indicated. Results are typical of three separate experiments.

Different Ca²⁺ mobilization induced by H10-FcRI dimers and multimers

FcRI cross-linking by multivalent Ag and by signaling-competent anti-FcRI mAbs induces the release of cytoplasmic stored Ca²⁺ and the influx of extracellular Ca²⁺ (20). Because the mobilization of Ca²⁺ is essential for secretion, we predicted that H10-FcRI multimers might be more effective than H10-FcRI dimers at inducing Ca²⁺ responses. This is confirmed in the averaged Ca²⁺ responses from groups of 30 cells, illustrated in Fig. 1, B–D. H10-FcRI dimers induce a small sustained increase in cytoplasmic Ca²⁺ levels (Fig. 1B). Additionally, the traces from individual cells published in Ref. 20 revealed that H10-FcRI dimers induce repetitive Ca²⁺ spikes that are obscured by averaging the responses for a field of cells. H10-FcRI multimers (Fig. 1C) induced a much larger and more sustained Ca²⁺ response in RBL-2H3 cells than the H10-FcRI dimers. Multivalent Ag consistently induced the largest Ca²⁺ responses (Fig. 1D).

Effects of H10-FcRI dimers and H10-FcRI multimers on the phosphorylation of Lyn, Syk, and PLC2

Previous in vitro kinase assays indicated that H10-FcRI dimers activate Lyn but are poor activators of Syk (20). In Fig. 2, anti-PY immune complexes prepared from resting and activated cells were incubated with [-³²P]ATP, and the incorporation of radiolabel into Lyn and Syk was determined by SDS-PAGE and autoradiography. Predictably, IgE plus Ag increased the intensity of in vitro-phosphorylated Lyn and Syk in these assays. Fig. 2A (and the accompanying densitometry in the legend to Fig. 2A) shows that Lyn phosphorylation measured by the in vitro kinase assay is similarly increased over resting levels whether cells are stimulated with H10-FcRI dimers or with H10-FcRI multimers. In contrast, Fig. 2B (and its accompanying densitometry) shows that Syk phosphorylation measured in the same in vitro kinase assay is increased substantially more in cells stimulated with H10-FcRI multimers than with H10-FcRI dimers.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Effect of H10-FcRI dimers and multimers on Lyn and Syk in vitro phosphorylation. RBL-2H3 cells were incubated with no addition (0), and with 1 µg/ml of DNP-BSA (XL), 7 nM H10 (H10-D), and 7 nM H10 plus 10 nM GaM (H10-M) for 5 min at 37°C. Cells were lysed, the lysates precleared, and PY-20-reactive proteins were immunoprecipitated from equivalent protein amounts of precleared lysates by incubation with mAb PY-20 bound to protein A/G-Sepharose beads. Immune complexes were incubated for 10 min at 37°C with [-32P]ATP, and the resulting phosphoproteins were resolved by SDS-PAGE and detected by autoradiography. Results show the Lyn (A) and Syk (B) bands, respectively, from one of four similar in vitro kinase assays. Relative signal intensities obtained by densitometric analysis of these autoradiograms were, for Lyn: XL, 2.5; H10-D, 1.0; and H10-M, 0.98; and for Syk: XL, 2.9; H10-D, 1.0; and H10-M, 1.98. Signal intensities in the control lanes (0) were below the limits of detection.

View larger version (47K): [in this window] [in a new window]   FIGURE 3. Effect of H10-FcRI dimers and multimers on the in vivo phosphorylation of Lyn, Syk, and PLC2. RBL-2H3 cells were incubated with no addition, and with XL, H10-D, and H10-M as in Fig. 2. Proteins were immunoprecipitated from equivalent amounts of protein from each of the precleared lysates by incubation with mAb PY-20 (A and B) or anti-PLC2 (C) conjugated to protein A/G-Sepharose beads. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected on the blots using anti-Lyn (A), anti-Syk (B), and anti-PY (C) Abs followed by HRP-anti-rabbit IgG. Blots were developed with an ECL reagent. Relative signal intensities obtained by densitometric analysis of this autoradiogram were, for Lyn: 0, 0.54; XL, 2.10; H10-D, 2.04; and H10-M, 2.17; and for Syk: 0, 0; XL, 4.33; H10-D, 1.0; and H10-M, 2.24. For PLC2, relative intensities were, for 0, 0; XL, 3.17; H10-D, 1.0; and H10-M, 1.78. Similar results were obtained twice.

H10-FcRI dimers, but not H10-FcRI multimers, form stable complexes with Lyn

Previously, we suggested that the poor signaling activity of H10-FcRI dimers may be related to the formation of stable complexes between Lyn and phosphorylated FcRI and subunits that impair signal propagation (20). The results in Figs. 4 and 5 show the effect of converting dimers to multimers on these unusual detergent-resistant FcRI-Lyn complexes.

View larger version (66K): [in this window] [in a new window]   FIGURE 4. Stronger association of FcRI -chains with Lyn in H10-FcRI dimers, but not multimers, revealed by immune complex kinase activities. RBL-2H3 cells were activated as in Fig. 2. The supernatants were precleared by incubation with protein A/G-Sepharose beads. Proteins were then immunoprecipitated from equivalent amounts of total protein from each lysate using anti-Lyn Abs coupled to protein A/G-Sepharose beads, and the immunoprecipitates were incubated with [-32P]ATP before SDS-PAGE and autoradiography. The :Lyn ratios, calculated from the densitometric signals of the corresponding bands in the H10-D and H10-M lanes, were, respectively, >2:1 and 0.5:1. Results are from one of three independent experiments with similar results.

View larger version (56K): [in this window] [in a new window]   FIGURE 5. Association of FcRI with Lyn in H10-FcRI dimers but not multimers revealed by immunoblotting. RBL-2H3 cells were activated as in Fig. 2 and then lysed, and Lyn and its associated proteins were immunoprecipitated from equivalent amounts of total protein from precleared lysates by incubation with anti-Lyn mAb. Proteins were separated on 12% SDS-PAGE gels and transferred to nitrocellulose for Western blotting using Abs to Lyn followed by anti-rabbit IgG-HRP (A) or to FcRI followed by anti-mouse-HRP (B). Blots were developed with an ECL reagent. Lyn is detected in all lanes (overloaded in the control sample, in similar amounts (XL, 0.9; H10-D, 1.0; and H10-M, 0.8) by densitometry of the three treatment samples). In this experiment, FcRI and subunits were detectable only in lysates prepared from H10-treated cells. In a separate experiment, the FcRI :Lyn ratios between treatments were, for 0, 0.08; XL, 0.29; H10-D, 1.0; and H10-M, 0.03.

To obtain an independent assessment of the association of the FcRI and subunits with Lyn, we used Western blotting to probe for the presence of and in anti-Lyn immunoprecipitates from variously activated cells (Fig. 5). The results in Fig. 5A show that anti-Lyn immunoprecipitates from differently activated cells all contain Lyn. Fig. 5B shows that FcRI and subunits are present in anti-Lyn immunoprecipitates from cells stimulated with mAb H10 alone, but are essentially undetectable in anti-Lyn immunoprecipitates from resting cells or from cells stimulated with H10 plus GaM or with Ag.

Membrane topography of H10-FcRI dimers and multimers and their associated kinases

The topography of H10-FcRI dimers and multimers and of the FcRI-associated tyrosine kinases Lyn and Syk was determined by immunogold labeling and transmission electron microscopic analysis of native membrane sheets prepared from the dorsal surface of RBL-2H3 cells.

The micrographs in Fig. 6 show the typical distributions of gold particles marking FcRI and Lyn in variously treated cells. Replicating previous work (8), gold particles marking FcRI and Lyn are frequently colocalized in unstimulated cells as singlets and small dispersed clusters on apparently unspecialized membrane regions (Fig. 6A). Treatment with IgE plus Ag causes a rapid redistribution of gold particles marking FcRI to osmiophilic membrane patches (Fig. 6B). Coated pits occur at the periphery of a high proportion of these osmiophilic patches. Lyn segregates from FcRI during this clustering and becomes concentrated in topographically separate membrane patches.

View larger version (105K): [in this window] [in a new window]   FIGURE 6. Membrane topography of H10-induced FcRI dimers and multimers and of the FcRI-activated kinase Lyn. Cell monolayers on glass coverslips were incubated at 37°C with no addition (A) or for 2 min with DNP-BSA (B), or for 5 min with H10-D (C) or H10-M (D). Membrane sheets were prepared from these cells and labeled from the inside with 10 nm protein A colloidal gold particles conjugated to anti-FcRI mAb and with 5 nm protein A gold particles conjugated to rabbit Abs to Lyn. Control samples (not shown) were incubated with unconjugated protein A gold particles. There was essentially no nonspecific labeling in these experiments. Representative clusters of receptor and Lyn, some mixed and some separate, on bulk membrane are circled. Arrows point to Lyn that appears to be specifically excluded from FcRI -containing osmiophilic membrane patches. Bar = 0.1 µm.

The micrographs in Fig. 7 replicate these labeling conditions, except that the smaller gold particles now reveal the distribution of Syk. In resting cells, receptor is again distributed as dispersed clusters. There are relatively few gold particles marking Syk on resting membranes and no Syk-FcRI colocalization (Fig. 7A). As previously described (8), gold particles marking FcRI and Syk are strongly colocalized in osmiophilic patches after treatment with IgE plus Ag (Fig. 7B).

View larger version (106K): [in this window] [in a new window]   FIGURE 7. Membrane topography of H10-induced FcRI dimers and multimers and of the FcRI-activated kinase, Syk. Cells were incubated at 37°C with no addition (A) or for 2 min with DNA-BSA (B), or for 5 min with H10-D (C) or H10-M (D). Membrane sheets prepared from these cells were labeled from the inside with 10 nm protein A-colloidal gold particles conjugated to anti-FcRI mAb and with 5 nm protein A gold particles conjugated to rabbit Abs to Syk. Representative clusters of receptor and Syk, some mixed and some separate, are circled. Arrows point to Syk that appears to be specifically included with FcRI in osmiophilic membrane patches. Bar = 0.1 µm.

The association of coated pits with a high proportion of osmiophilic patches provides a marker for locating these putative signaling domains. The impression that H10-FcRI multimers redistribute to membrane signaling domains more often than H10-FcRI dimers was confirmed by identifying coated pits on all the micrographs from three separate experiments and counting the numbers of gold particles marking FcRI in the immediately adjacent membrane. Fig. 8, A and B, shows that >80% of coated pits from both unstimulated and H10-stimulated cells had no adjacent FcRI -gold particles. In contrast, <40% of coated pits from H10 plus GaM-stimulated cells and <30% of gold particles from Ag-stimulated cells had no adjacent FcRI -gold particles (Fig. 8, C and D). The size of FcRI -gold aggregates associated with pits was also different between resting and H10-activated cells and cells stimulated with H10 plus GaM or Ag. Thus, 2 gold particles was the largest gold cluster seen adjacent to coated pits in resting cells, and 3–5 FcRI -gold particles adjacent to coated pits was the largest cluster seen in cells stimulated with H10-FcRI dimers. In contrast, clusters of >6 FcRI -gold particles adjacent to coated pits were frequently seen in cells stimulated with H10-FcRI multimers (Fig. 8C), and multivalent Ag commonly induced clusters of 11–25 gold particles adjacent to coated pits (Fig. 8D).

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Association of H10-FcRI dimers and multimers with membrane adjacent to coated pits. Cell monolayers on glass coverslips were incubated for 5 min at 37°C with no addition (A), with H10-D (B), or with H10-M (C) or for 2 min at 37°C with XL (D). Membrane sheets were prepared from these cells and labeled with 10 nm protein A-colloidal gold particles conjugated to anti-FcRI mAb. Coated pits that form at the periphery of osmiophilic signaling patches and serve as markers for these membrane domains were identified on randomly selected micrographs, all photographed at x20,000, from three separate experiments. Each pit was scored for the number of gold particles marking FcRI on osmiophilic membrane continuous with the pit or on unspecialized membrane that fell within one radius of the pit. The bar graphs show the fraction of total coated pits with zero, 1–2, 3–5, 6–10, or 11–25 adjacent FcRI -gold particles. Results are based on analysis of 41 micrographs (43 pits) from resting cells, 54 micrographs (103 pits) from cells treated for 5 min with 7 nM mAb H10, 61 micrographs (148 pits) from cells treated for 5 min with 7 nM H10 plus 10 nM GaM, and 38 micrographs (104 pits) from Ag-stimulated cells.

The evidence that H10-FcRI dimers may induce a more stable association of FcRI with Lyn than either IgE plus multivalent Ag or H10-FcRI multimers was further investigated by analysis of the biochemical composition of membrane rafts isolated by detergent extraction and sucrose density gradient centrifugation analysis of variously activated cells. The results in Fig. 9A reproduce published evidence (14, 15, 16) that a portion of FcRI is colocalized with Lyn in the light fractions containing detergent-resistant membranes (DRMs or rafts) of resting cells and that additional FcRI is distributed in the heavy (soluble) fractions of the gradient. Stimulation with mAb H10 alone causes a marked redistribution of FcRI into the light (Lyn-containing) fractions of the gradient (Fig. 9B). FcRI cross-linking with either H10 plus GaM (Fig. 9C) or IgE plus multivalent Ag (Fig. 9D) also causes an increase in the portion of FcRI recovered in the Lyn-enriched raft fractions in comparison with unstimulated cells. However, the extent of the redistribution is less than the redistribution induced by H10 alone. Densitometric analyses of the distribution of FcRI in the individual gradient fractions confirms that, by far, the greatest shift of FcRI into the light (DRM) fractions is induced by H10-FcRI dimers. Close to 90% of is found in the Lyn-containing fractions 3, 4, and 5 after stimulation with H10 alone, vs 40% in resting cells and 60% in cells stimulated by IgE plus Ag or H10 plus GaM. This result is consistent with both biochemical and microscopic evidence (as described above) that H10-FcRI dimers associate stably with Lyn, and that formation of higher order aggregates reduces the proportion of FcRI that associates with Lyn.

View larger version (17K): [in this window] [in a new window]   FIGURE 9. Analysis of proteins distributed across sucrose density fractions. RBL-2H3 cells were incubated for 5 min at 37°C with no addition (A), with H10-D (B), or with H10-M (C) or for 2 min at 37°C with DNP-BSA (D). Cells were solubilized in 0.05% Triton-X-100, and lysates were loaded onto 80% sucrose cushions, followed by layers of 35, 25, 20, 15, and 10% sucrose. Following ultracentrifugation, fractions were collected from the top to the bottom of the gradient (from the lightest to heaviest fractions). Proteins in equal aliquots of the fractions were separated by SDS-PAGE, followed by immunoblot analysis using Abs to Lyn and FcRI . The percentage of FcRI in each fraction was calculated from densitometric quantitation of the corresponding bands. In resting cells (A), 40% of FcRI is found in the Lyn-containing fractions 3, 4, and 5. The percentage of total FcRI found in the Lyn-containing fractions increases to almost 90% after activation with H10 (B) and to 60% after activation with H10 plus GaM (C) and with IgE plus Ag (D). Similar results were obtained in two separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

If the orientation of receptors is indeed crucial for proper signaling, it was predicted that creating higher order aggregates of H10-FcRI complexes might increase the frequency of two receptors contacting each other with the appropriate relative orientation to promote signaling. Here, we confirm the prediction by showing that cross-linking the H10-FcRI dimers into multimers stimulates not only secretion (reported previously in Ref. 19) but also the mobilization of Ca²⁺ and the phosphorylation of PLC2. Accompanying the recovery of cell signaling, the and subunits of the H10-FcRI multimers no longer form stable complexes with Lyn. Furthermore, the weak phosphorylation and activation of Syk characteristic of cells activated with H10-FcRI dimers is replaced by strong Syk phosphorylation and activation when cells are stimulated with H10-FcRI multimers.

The results of immunoelectron microscopic studies provided a separate perspective on the properties of H10-FcRI dimers and multimers. Previous studies in IgE plus Ag-treated cells showed that non-cross-linked FcRI occur in mast cell membranes in loose association with Lyn but not Syk, and that the addition of cross-linking agent induces a striking redistribution of receptor aggregates to osmiophilic patches. These membrane patches exclude Lyn and accumulate Syk (8). It was proposed that the osmiophilic patches represent domains of FcRI signaling to downstream responses.

Here we show that H10-FcRI dimers are impaired in their ability to separate from Lyn and redistribute to osmiophilic membrane patches. In contrast, H10-FcRI multimers are frequently observed without Lyn in osmiophilic membrane patches. Thus, the hypothesized changes in the configuration of the H10-FcRI dimers that result in the formation of stable Lyn-receptor complexes also arrest the sequence of topographical events that initiate FcRI signaling. This sequence of events is restored by aggregating the dimers with GaM to form H10-FcRI multimers.

As expected, gold particles marking the H10-FcRI multimers in osmiophilic membrane patches are consistently colocalized with markers for Syk. Surprisingly, H10-FcRI dimers also seem to interact with Syk even though they rarely enter the putative signaling domains. It is possible that this recruited Syk has reduced catalytic activity due to its incorrect topography. There is precedent for this in work showing that H-Ras becomes biologically inert by treatments that modify its topography (31). At least in Ag-stimulated cells, the osmiophilic patches also accumulate signaling molecules such as PLC2, PI 3-kinase, and Gab2 (18). Thus, H10-FcRI dimers that recruit and activate Syk, but cannot enter the putative signaling domains, would have reduced access to a number of Src homology 2 domain-containing proteins that are phosphorylated directly or indirectly by Syk and mediate downstream signaling.

Other investigators have used detergent extraction and gradient centrifugation rather than microscopy on native membranes to explore the microdomain organization of FcRI signaling (15, 16). These experiments have identified specialized fractions called DRMs or lipid rafts as sites that are inherently enriched for Lyn and are foci for the redistribution and phosphorylation of cross-linked FcRI during signaling. Our experiments show that H10-FcRI dimers cause an extensive redistribution of FcRI from heavy (soluble) to light (raft, DRM, Lyn microdomain) fractions, and that H10-FcRI multimers cause a less complete recruitment of FcRI to DRMs. The more extensive redistribution of FcRI to Lyn-containing fractions in H10-treated cells provides independent support for the hypothesis that H10-FcRI dimers are impaired in their ability to separate from Lyn. Because signaling is poorest when the association of FcRI with Lyn microdomains is greatest, these results also support the hypothesis that Lyn microdomains are intermediates in the formation of the osmiophilic patches that accumulate FcRI and Syk and appear to represent sites of signal propagation in mast cells.

Previously, we proposed that the Lyn-sequestering, signal-curtailing properties of mAb H10-FcRI dimers may result from an unfavorable dimer configuration (19, 29, 30) whose consequences include the inability of Lyn to dissociate from H10-FcRI complexes (20). Extending the orientational hypothesis to other tyrosine kinase-coupled receptors, the efficiency of signaling through cytokine receptors is now known also to depend critically on the separation, orientation, and relative disposition of receptor dimers (32, 33). Here we show that these orientational or configurational constraints in the H10-FcRI dimers are expressed as changes in the sequence of topographical as well as in the biochemical events that initiate FcRI signaling. Specifically, H10-FcRI dimers have very little access to the osmiophilic membrane patches that normally accumulate Syk and downstream signaling molecules. Importantly, these restraints are reversible. Further cross-linking to form H10-FcRI multimers results in a renewed ability of cross-linked receptors both to dissociate from Lyn and to redistribute to membrane domains specialized for Syk-mediated signal propagation.

Although mAb H10 allows some signaling, and so is not itself an ideal signal-blocking drug, the Lyn-sequestering, signal-curtailing properties of mAb H10-FcRI dimers do suggest new approaches for the treatment of allergic inflammation. Work in the Metzger group (34, 35, 36) has established that Lyn is rate limiting for FcRI signaling in RBL-2H3 cells. Assuming the same is true in human basophils and mast cells, stringently Lyn-sequestering cross-linkers of the human FcRI should not only block the subset of ligated receptors but, by sequestering the initiating kinase, Lyn, might also inhibit allergen-induced FcRI signaling through independently cross-linked IgE- receptor complexes. The inhibition might be relatively long-lived because, in contrast to FcRI multimers, FcRI dimers are not internalized by receptor-mediated endocytosis (20).

	Acknowledgments

 
We thank Claudia Garay for excellent technical assistance, the University of New Mexico School of Medicine for resources for electron microscopy, and the University of New Mexico Cancer Research and Treatment Center for resources for ratio imaging microscopy.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grants RO3 TW00440, RO1 GM49814, and P50 HL56384, by American Cancer Society Grant RPG992330CIM, by Consejo Nacional de Ciencia y Tecnología Grant 31783N, and by Universidad Nacional Autónoma de México Grant IN208399.

² Address correspondence and reprint requests to Dr. Enrique Ortega, Departamento de Immunología, Universidad Nacional Autónoma de México, Ap Postal 70228, Cd. Universitaria, CP 04510, Mexico D.F., Mexico. E-mail address: ortsoto{at}servidor.unam.mx

³ Abbreviations used in this paper: PLC, phospholipase C; PI 3, phosphatidylinositol 3; ITAM, immunoreceptor tyrosine-based activation motif; GaM, F(ab')₂ of goat anti-mouse IgG; anti-PY, anti-phosphotyrosine Ab; DRM, detergent-resistant membrane.

Received for publication January 17, 2001. Accepted for publication August 21, 2001.

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