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Spatial Raft Coalescence Represents an Initial Step in FcR Signaling¹

Hajime Kono^*, Takeshi Suzuki^*, Kazuhiko Yamamoto^*, Masato Okada, Tadashi Yamamoto and Zen-ichiro Honda^2,*

^* Department of Allergy and Rheumatology, Graduate School of Medicine and Faculty of Medicine, and Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Department of Oncogene Research, Research Institute for Microbial Disease, Osaka University, Osaka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of lipid rafts as separated membrane microdomains consist of heterogeneous proteins suggesting that lateral assembly of rafts after Ag receptor cross-linking represents the earliest signal generating process. In line with the concept, cross-linked Ag receptors have been shown to associate with detergent-insoluble raft fraction without the aid of Src family kinases. However, it has not been established whether spatial raft coalescence could also precede Src family kinase activation. In this study, we showed that spatial raft coalescence after low-affinity FcR cross-linking in RAW264.7 macrophages is independent of Src family kinase activity. The lateral raft assembly was found to be ascribed to the action of ligand-binding subunits, rather than to immunoreceptor tyrosine-based activation motif-bearing signal subunits, because monomeric murine FcRIIb expressed in rat basophilic leukemia cells successfully induced spatial raft reorganization after cross-linking. We also showed that extracellular and transmembrane region of FcRIIb is sufficient for raft stabilization. Moreover, this receptor fragment triggers rapid calcium mobilization and linker for activation of T cells phosphorylation, in a manner sensitive to Src family kinase inhibition and to cholesterol depletion. Presence of immunoreceptor tyrosine-based inhibitory motif and addition of immunoreceptor tyrosine-based activation motif to the receptor fragment abolished and enhanced the responses, respectively, but did not affect raft stabilization. These findings support the concept that ligand-binding subunit is responsible for raft coalescence, and that this event triggers initial biochemical signaling.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Receptors for Ig Fc region (FcR) belong to a family of multichain immune recognition receptors (MIRRs)³ composed of TCR and B cell Ag receptors (BCRs) and the FcRs (1, 2). Upon the recognition of multivalent ligands or multiple MHC-peptide complexes, MIRRs trigger Src family kinase-dependent phosphorylation of tyrosine in immune receptor tyrosine-based activation motifs (ITAMs) (1, 3). Syk/Zap-70 tyrosine kinases are recruited to the ITAMs and then phosphorylate central adapter proteins such as linker for activation of T cells (LAT) or B cell linker protein (4, 5). These scaffolding proteins subsequently recruit phospholipase C, Grab family proteins, and phosphatidylinositol 3-kinases, thus forming signal transduction machinery (6).

In addition to these molecular assemblies via protein-protein interactions, recent studies have revealed the significance of protein compartmentalization that relies on spatially segregated plasma membrane domain, referred to as detergent-insoluble membranes (DIM) or lipid rafts (7). Several of Src family kinases and LAT constitutively associate with lipid rafts, and TCR and FcRI signaling is transduced solely by the raft-associating Src family members (8, 9). Clustering-dependent association of MIRRs and downstream signaling molecules with lipid rafts has been shown in a variety of systems (10, 11, 12, 13). Upon the contact with APCs, T cells polarize to form spatially organized molecular assembly, called immunological synapse, at the contact site (14, 15). This reorganization process also uses lipid rafts as vehicles (16). Besides the roles in such late signaling events as immunological synapse formation, rafts have been claimed to function in the earliest stage of MIRR signaling. For instance, partition of FcRI or BCR into lipid raft fractions was not prevented by the inhibition of Src family kinase activity (17, 18), and the FcRI association with rafts was not affected by the deletion of ITAMs in and subunits (19). These observations indicate that receptor-detergent insoluble membrane (DIM) association represents the earliest event after the receptor multimerization, although the mechanisms to how this process links to signal generation are still elusive.

Recent biophysical analysis of rafts showed that raft size is small enough to expect that each raft possesses different protein constituents (20). Through the pioneering studies in FcRI system (17, 19, 21), Baird et al. (22) hypothesized that FcRI is brought into association with Lyn after raft coalescence, and that FcRI is then phosphorylated by Lyn. In a previous work, we showed that Lyn molecules in quiescent rat basophilic leukemia (RBL) 2H3 cells are mixture of active and inactive forms (23). Therefore, it is also possible that raft coalescence provides a field for Lyn transactivation. If the hypothesis holds, spatial raft coalescence should precede intracellular signaling. However, this problem has not been fully examined. In this study, we first showed that FcR-mediated spatial raft coalescence is independent of Src family kinase activity. One of the common features in receptor-DIM association is that ligand-binding subunits frequently devoid of signal-generating ITAMs actively translocate into DIM, and that signaling subunits such as FcRI and subunits, and TCR- subunits are constitutively recovered from DIM (9, 12, 17). We presumed that it reflects active participation of ligand binding subunits in raft coalescence. By using monomeric FcRIIb as a model system, we provided evidence supporting that ligand-binding subunit-mediated spatial raft coalescence represents initial and productive signaling process.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

HRP-conjugated cholera toxin B (CTxB), FITC-conjugated CTxB, and methyl--cyclodextrin (MCD) were from Sigma-Aldrich (St. Louis, MO). PP2 was from Calbiochem (Darmstadt, Germany). Rhodamine-conjugated streptavidin was from Molecular Probes (Eugene, OR). Protein G-Sepharose, ECL Protein Biotinylation system, and streptavidin-HRP conjugate were from Amersham Pharmacia Biotech (Buckinghamshire, U.K.). All the culture media and Geneticin were purchased from Life Tech Oriental (Osaka, Japan). FCS was from Equitec Bio (Ingram, TX). The FcRI subunit knockout C57BL/6 mice (24) were purchased from Taconic Farms (Germanstown, NY).

A rat anti-mouse FcRIIb/IIIa mAb, 2.4G2, was purified from culture supernatant with protein G-Sepharose chromatography. Biotinylation of 2.4G2 and preparation of Fab were performed using ECL protein biotinylation system (Amersham Pharmacia Biotech) and immobilized papain (Pierce, Rockford, IL), respectively. FITC-conjugated 2.4G2 and FITC-conjugated rat mAb against mouse CD45 were from BD PharMingen (San Diego, CA). A mouse monoclonal anti-DNP IgE, SPE-7, was from Sigma-Aldrich. A polyclonal Ab against CTxB was purchased from Calbiochem. Anti-phosphotyrosine mAb, 4G10, was from ICN Biochemicals (Costa Mesa, CA). Polyclonal Abs against Lyn, c-Src, and Syk were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal Ab against LAT was from Upstate Biotechnology (Lake Placid, NY). Polyclonal Abs against rat IgG and mouse IgE were from ICN (Aurora, OH). Polyclonal Abs against FcRIIb and common subunit of FcR/FcRI were kindly donated by Dr. T. Takai (Tohoku University, Sendai, Japan), and by Dr. R. P. Siraganian (National Institutes of Health, Bethesda, MD), respectively.

Cell culture

RAW264.7 and RBL2H3 cells were cultured as a monolayer in DMEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FCS. RAW264.7 cells stably expressing a membrane-anchored Csk (mCsk) and RBL2H3 cells stably expressing platelet activating factor (PAF) receptor (25) were described previously (9, 26).

Bone marrow cells were prepared from FcRI ^+/+ or ^-/- C57BL6 mouse, and bone marrow-derived macrophages (BMMCs) were elicited using 10% L929 cell-conditioned medium in DMEM with 10% FCS as described (27).

Preparation of RBL2H3 cells expressing FcRIIb mutants

Truncation of murine FcRIIb cytoplasmic domain (FcRIIb-truncated) and replacement of the cytoplasmic domain with murine FcRI subunit bearing ITAM (FcRIIb-ITAM) were conducted as described (28). To create FcRIIb- ITAM chimera, cDNAs of murine FcRIIb2 and subunit were subcloned into pBlueScript II in sequence, and FcRIIb2 extracellular and transmembrane domains were connected with subunit cytoplasmic domain by PCR-based techniques (28). Primers used for truncated FcRIIb were 5'-TGGAACCTGCTTTTTCTTGA-3' and 5'-TAGTCTCCCTTGGCGAATTC-3'. Those for FcRIIb- ITAM chimera were 5'-CGAAAGGCAGCTATAGCCAG-3' and 5'-TGGAACCTGCTTTTTCTTGA-3'. cDNAs were sequenced, subcloned into pCXN2 (29), and multiple RBL2H3 cell clones stably expressing wild-type (WT) and the mutated FcRIIb were established as described (9, 30). Surface expression of WT and mutated FcRIIbs was analyzed with flow cytometry after staining cells with FITC-conjugated 2.4G2 as described (26).

Cell stimulation and cell lysis

To stimulate cells via FcRs, adherent RAW cells, BMMCs, or RBL cells expressing FcRIIb or its mutants were sensitized with 2.4G2 mAb (10 µg/ml) or with 2.4G2 Fab (10 µg/ml) in ice-cold assay medium (DMEM containing 10 mM HEPES-NaOH (pH 7.4) and 1 mg/ml BSA) for 30 min. In the case of FcRI stimulation, RBL transfectants were incubated with anti-DNP IgE (1 µg/ml) in the ice-cold assay medium for 30 min. Cells were washed twice with ice-cold assay medium, and clustering of FcRs and FcRI was initiated by replacing the medium with prewarmed (37°C) medium containing goat anti-rat IgG (30 µg/ml) and with DNP-BSA (100 ng/ml), respectively. After indicated periods, medium was aspirated, and cells were solubilized with 500 µl of ice cold Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 20 mM -glycerophosphate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 0.2 mM PMSF). Insoluble materials were removed by centrifugation at 12,000 rpm for 10 min at 4°C, and the supernatant was used as total cell lysate.

Immunoprecipitation and immunoblotting

Total cell lysates were first incubated with 15 µl of protein G Sepharose beads (50% slurry) to separate 2.4G2-bound materials. After continuous rotation for 1 h at 4°C, samples were centrifuged at 2500 rpm at 4°C for 2 min, and the supernatants were saved. Beads were washed three times with 500 µl of 0.1% Nonidet P-40 lysis buffer, and the 2.4G2-bound materials were eluted by boiling in 2% SDS sample buffer. The saved supernatants were incubated with various first Abs for 1 h and then with 15 µl suspension of protein G-Sepharose beads for 30 min at 4°C under continuous rotation. The beads were washed and bound materials were eluted as described above. Samples were analyzed by Western blotting using ECL detection system (Amersham Pharmacia Biotech) as described previously (9).

To analyze subunit composition of intrinsic FcRI and transfected FcRs in RBL cells, 1.5 x 10⁷ cells in the assay medium were sensitized with biotinylated IgE (2 µg) or with biotinylated 2.4G2 (10 µg) on ice for 60 min. Cells were lysed in 2% digitonin lysis buffer (20 mM Tris-HCl (pH 7.4), 2% digitonin, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 20 mM -glycerophosphate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.2 mM PMSF), and the receptors were immunoprecipitated with anti-mouse IgE or with anti-rat IgG and with protein G-Sepharose beads as described above.

Raft fractionation by sucrose density gradient centrifugation

Triton X-100 solubilization of cells and raft fractionation were conducted essentially following the method reported by Field et al. (17). Cell suspension at 1 x 10⁷/ml was sensitized with 2.4G2 (10 µg/ml) or with 2.4G2 Fab (10 µg/ml) in ice-cold assay medium for 30 min, washed twice and resuspended at 1 x 10⁷/ml in stimulation buffer (20 mM HEPES-NaOH (pH 7.4), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 1 mg/ml BSA). Cells were prewarmed at 37°C for 5 min, challenged with goat anti-rat IgG (30 µg/ml) for indicated periods, and then solubilized by mixing with an equal volume of ice-cold 0.1% Triton X-100 lysis buffer (0.1% Triton X-100, 80 mM HEPES-NaOH (pH 7.4), 20 mM EDTA, 0.1%NaN₃, 2 mM NA₃VO₄, 20 mM -glycerophosphate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM PMSF). The cell lysate was mixed with an equal volume of ice-cold 80% sucrose buffer (80% sucrose, 25 mM HEPES-NaOH (pH 7.4), 125 mM NaCl, 2 mM EDTA), and sucrose density gradients were made in 13 PA tube (1.5 x 9.6 cm; Hitachi Koki, Hitachi, Japan) by sequential layering of 0.625 ml of 80%, 1.25 ml of 60%, 3.75 ml of the cell lysate adjusted to 40% sucrose, 1.875 ml of 30%, 1.25 ml of 20%, and 1.5 ml of 10% sucrose buffers. The gradients were centrifuged at 38,000 rpm at 4°C for 18 h in RPS40T rotor (Hitachi Koki). A total of 1 ml of fractions were collected from the bottom; proteins were extracted following the methods by Wessel and Flugge (31) and analyzed by Western blotting.

Confocal microscopic analysis

Adherent RAW cells or RBL2H3 cells on Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY) were first sensitized with ice-cold assay medium containing biotinylated 2.4G2 (5 µg/ml) for 30 min. In the studies to compare the distributions of FcRs to those of CD45, FITC-conjugated anti-CD45 mAb (5 µg/ml) was also included in the medium. Cells were washed twice with ice-cold assay medium, and then treated with prewarmed (37°C) assay medium containing rhodamine-conjugated streptavidin (10 µg/ml) to initiate FcR cross-linking. The reaction was continued at 37°C for 3 min, and terminated by the fixation of cells with of 3.7% formaldehyde in PBS. Cells were solubilized with 0.01% Triton X-100 for RAW cells or with 1% Triton X-100 for RBL2H3 cells for 3 min at room temperature, washed twice with PBS, and ganglioside GM1 and unligated FcRs were stained with FITC-conjugated CTxB (10 ng/ml) and with rhodamine-conjugated streptavidin (10 µg/ml), respectively. Confocal microscopic observation was performed using Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany) with x63 objective lens. For excitation, a 488-nm Ar laser was used for FITC and a 543-nm HeNe laser for rhodamine. Emission band path was set at 505 nm for FITC, and at 560 nm for rhodamine.

Codistribution of FcR with GM1 or with CD45 was quantified as described (21). The two line profiles of plasma membrane fluorescence for FITC and rhodamine were obtained from confocal images of cross sections using NIH Image (version 1.62; http://rsb.info.nih.gov/nih-image/; Ref. 32). The correlation coefficient () of the two profiles was calculated from equitation below using StatView 4.5 software (Abacus Concepts, Berkeley, CA).

In this equation, x_i and y_i are the intensities of FITC and rhodamine at the pixel, respectively, and <x> and <y> indicate the corresponding mean fluorescence intensities.

Copatching of CTxB with FcRs was conducted as described by Janes et al. (33). Cells were first sensitized with biotinylated 2.4G2 Fab (5 µg/ml) and CTxB-FITC (10 µg/ml) in ice-cold assay medium for 30 min. Next, patching of CTxB was induced by the addition of anti-CTxB Ab (diluted by 1/250) in ice-cold assay medium for 30 min. Last, FcRs were cross-linked and visualized with streptavidin-rhodamine (10 µg/ml) at 37°C for 3 min before (FcR cross-linking (+)) or after (FcR cross-linking (-)) cell fixation with 3.7% formaldehyde in PBS. Cell permeabilization with Triton X-100 was not applied to the copatching experiment.

Fluorometric imaging of intracellular Ca²⁺ concentration ([Ca²⁺]_i)

[Ca²⁺]_i was measured as described (9). Adherent cells on glass coverslips were sensitized with biotinylated 2.4G2 Fab (5 µg/ml) for 15 min or with anti-DNP IgE (1 µg/ml) for 1 h, loaded with fura-2 AM (5 µM) for 1 h at 37°C, and stimulated with streptavidin (100 nM) or DNP-BSA (100 ng/ml) in HEPES-Tyrode buffer (25 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 12 mMNaHCO₃, 5.6 mM D-glucose, 0.49 mM MgCl₂, 0.37 mM NaH₂PO_4, 1 mg/ml BSA). Fluorometric images of cells (340/380 nm) were sequentially recorded by Argus-50 system (Hamamatsu Photonics, Hamamatsu, Japan). For presentation, 25 cells in a field were randomly assigned, and the calculated average of [Ca²⁺]_i was expressed as a line graph.

Cholesterol depletion

To remove cholesterol, cells were incubated for 30 min at 37°C in the presence or absence of indicated concentrations of MCD in HEPES-tyrode solution containing 1 mg/ml fatty acid-free BSA as described (34). For the recovery of cholesterol, cells were incubated with 2.5 mM of MCD/cholesterol (8:1, mole/mole) complexes for 2 h at 37°C (34), or with 10% FCS in HEPES-tyrode buffer (12) for 6 h at 37°C. Total cellular cholesterol was measured using cholesterol oxidase-based assay kit (Cholesterol C-II test; WAKO, Richmond VA) after chloroform/methanol extraction of cellular lipids.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
FcRIIIa and IIb association with DIM is independent of Src family kinase activity

Previous studies in FcRI and BCR systems using Triton X-100 cell lysis followed by density gradient centrifugation technique revealed that association of oligomerized receptors with DIM is independent of intracellular signaling (17, 18). Thus, we first evaluated whether FcRIIIa and IIb subunits associate with DIM after the receptor cross-linking in RAW264.7 cells, and whether the receptor redistribution is independent of Src family kinase activity. FcRIIIa and IIb subunits were probed with biotinylated 2.4G2 mAb, cross-linked with or without second Ab, and solubilized with 0.05% Triton X-100. The total cell lysate was directly subjected to ultracentrifugation on sucrose density gradients. Fig. 1, A and B show distributions of cell surface-bound biotinylated 2.4G2, which correspond to those of FcR- subunits. Upon cross-linking, FcR- subunits became partly associated with low-density DIM fractions and codistributed with ganglioside GM1 (CTxB blot). Lyn was in part associated with DIM, and c-Src was excluded from DIM, as observed previously (Fig. 1A; Ref. 9). Fig. 1B shows the time course of FcR- redistribution. In this figure and in the following ones, bottom fraction (B: bottom, fraction 1 in Fig. 1A), combined soluble fractions (M: middle, factions 3–5) and combined DIM fractions (T: top, fractions 8–10) were analyzed. FcR subunits rapidly redistributed to DIM fraction (T) and also to bottom fraction (B) within 30 s after clustering. The receptor redistribution to the B fraction is ascribed to FcR- association with residual F-actin, because it was prevented by latrunculin A that depolymerize residual F-actin in vivo, but not by cytochalasin D that inhibits de novo actin polymerization (data not shown). This fraction was not further characterized in the current study. FcR- subunit distribution to DIM was transient; it peaked at 3 min, and declined thereafter.

View larger version (72K): [in this window] [in a new window]   FIGURE 1. Redistribution of cross-linked FcR to DIM. A, FcRIIIa and IIb subunits in RAW264.7 cells were probed with biotinylated 2.4G2 mAb, and cells were treated with (cross link +) or without (-) second Ab. Cells were solubilized with 0.05% Triton X-100, and subjected to sucrose density gradient centrifugation. Fractions were analyzed by Western blotting. 2.4G2-probed FcR subunits and GM1 were detected by HRP-streptavidin and HRP-CTxB, respectively. Blottings for Lyn, c-Src, and GM1 (CTxB blot) were those obtained from unstimulated cells. FcR cross-linking did not alter their distributions (data not shown). B, Kinetics of FcR subunit redistribution to DIM and to the highest density fraction. In this presentation, the highest density fraction (B: bottom), combined soluble fractions (M: middle) and combined DIM fractions (T: top) are shown. C, Src protein tyrosine kinase (PTK)-independent FcR subunit redistribution to DIM. WT cells pretreated with (+) or without (-) 50 µM PP2, and mCsk-overexpressing cells were stimulated via FcRs for 3 min, and FcR- redistribution was analyzed as above. PP2 and mCsk overexpression did not affect FcR subunit association with DIM after cross-linking. D, Suppression of protein tyrosine phosphorylation by PP2 treatment or by mCsk overexpression. RAW cells pretreated with (+) or without (-) 50 µM PP2, or mCsk-overexpressing cells were stimulated via FcRs for 3 min, and tyrosine phosphorylated Syk (pY-Syk), -subunit (pY-), and FcRIIb (pY- FcRIIb) were analyzed by immunoprecipitation followed by Western blotting. The same membranes were reprobed with cognate first Abs after stripping. PP2 treatment and mCsk overexpression strictly suppressed these Src kinase-derived signaling.

Spatial raft coalescence is independent of Src kinase activity

We next examined the roles of Src family kinase activity in spatial coalescence of lipid rafts by using confocal microscopic techniques. To this end, PP2 could not be applied, because acute PP2 treatment considerably affects cell adherence to substratum. Therefore, we down-regulated Src family kinase activity by mCsk overexpression. Control RAW cells carrying vector alone or cells overexpressing mCsk adhered to Lab-Tek chamber slide were sensitized with biotinylated 2.4G2, and treated with or without rhodamine-streptavidin to cross-link FcRs or not. Cells were fixed with formaldehyde, treated with Triton X-100 to remove detergent-sensitive materials following the methods of Janes et al. (33), and unligated FcRs were then stained with rhodamine-streptavidin. The fixation procedures did not appreciably affect biotinylated 2.4G2 binding to streptavidin (data not shown). GM1 detected with FITC-CTxB was used as a raft marker. CD45 was regarded as a marker that is not concentrated at rafts (33).

Distributions of FcR subunits GM1 and CD45 before and after FcR cross-linking were shown in Fig. 2A. As shown in the left panel, FcR subunits were sensitive to the detergent treatment before cross-linking and readily solubilized. After cross-linking with rhodamine-streptavidin, they accumulated as discrete Triton X-100-resistant patches along cell membrane in both control cells (WT-RAW) and in mCsk-overexpressing cells (Fig. 2A, left panel, upper row). Concurrently, coaccumulation of GM1 patches with FcR patches became distinguishable in control cells. Of note, formation of GM1 patches and their colocalization with ligated FcR subunits were well-preserved in mCsk cells, thereby indicating that suppression of Src family kinase activity did not affect lateral clustering of GM1. Almost identical results were obtained when biotinylated 2.4G2 was cross-linked with 2nd Ab (data not shown). As control experiments, distributions of FcR subunits were compared with those of CD45 (Fig. 2A, right panel). After cross-linking, FcR subunits again accumulated as detergent-resistant patches along cell membranes in control cells and in mCsk overexpressing cells (Fig. 2A, right panel, upper row). In contrast to GM1, CD45 staining along plasma membrane did not codistribute with FcR patches (Fig. 2A, right panel, lower row).

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Spatial raft coalescence is independent of Src PTK activity. A, Analyses of spatial raft reorganization in control (WT-RAW) and mCsk-expressing RAW cells by fluorescent confocal microscopy. FcR subunits were probed with biotinylated 2.4G2, cross-linked (+) or not (-) with streptavidin-rhodamine for 3 min, and fixed with 3.7% formaldehyde. Cells were then solubilized with 0.01% Triton X-100. Unligated FcR subunits were stained with streptavidin-rhodamine after the cell fixation. GM1 and CD45 were visualized with CTxB-FITC and with FITC-conjugated anti-CD45 mAb, respectively. Left panel, FcR subunits and GM1 accumulated as discrete patches after FcR cross-linking in WT and mCsk-overexpressing RAW cells. Right panel, CD45 was almost homogenously distributed before and after FcR cross-linking, and did not colocalize with FcR patches. The confocal images were taken by the identical setting in each series for comparison. The excitation/emission wavelengths were at 488/505 nm for FITC and 543/560 nm for rhodamine. Bars, 5 µm. B, Representative line profiles of fluorescence intensity of FcR- and GM1 (left panels), and those of FcR- and CD45 (right panels). These profiles are obtained from the confocal images in A. FcR- and GM1 signals were augmented after receptor cross-linking at discrete segments of cell membrane and these segments were colocalized in both control and mCsk-expressing RAW cells. In contrast, CD45 signal did not give detectable peaks before and after FcR cross-linking. Codistribution of cross-linked FcR- with GM1 or with CD45 were quantitatively analyzed by calculating correlation coefficient () from line profiles. = 1 corresponds to complete colocalization. The mean ± SD of value was calculated from 10 individual cells.

FcRIIb associates with DIM in subunit -/- macrophages

We next tested whether ligand-binding subunits play roles in raft reorganization. To this end, monomeric FcRIIb was used as a model system, since this receptor does not require associating subunits for cell surface expression. In addition, FcRIIb possesses immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic region, and FcRIIb clustering alone does not induce tyrosine phosphorylation signaling (24, 36). These characteristics are desirable to test the hypothesis of tyrosine phosphorylation signal-independent raft coalescence.

We first examined whether cross-linking of FcRIIb alone is sufficient for its association with DIM. BMMCs elicited from ^-/- mice were used in the experiments, because surface expression of FcRIIIa is absent in the cells (24). FcRIIIa and IIb on WT BMMCs or FcRIIb on ^-/- BMMCs were probed with 2.4G2, cross-linked with or without second Ab, and changes in their distributions were examined by the raft-floating assay. As shown in Fig. 3A, 2.4G2 signal was increased at DIM fraction (T) after receptor clustering in WT cells, and this process was well-preserved in ^-/- cells. As shown in Fig. 3B, clustering of FcRIIb alone in ^-/- cells did not induce detectable Syk tyrosine phosphorylation. These findings revealed that clustering of FcRIIb subunit is sufficient for its association with DIM, and the receptor redistribution is independent of tyrosine phosphorylation signaling.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. FcRIIb redistribute to DIM after cross-linking in -/- macrophages. A, BMMCs obtained from WT or -/- C56BL/6 mice were stimulated (+) or not (-) via FcRs, solubilized with 0.05% Triton X-100, and subjected to density gradient centrifugation assay as described in the legend for Fig. 1B. Cross-linking of FcRIIb alone induced its association with DIM. B, Defective Syk tyrosine phosphorylation in -/- macrophages. WT and -/- macrophages were sensitized with 2.4G2, stimulated with (cross link (+)) or without (-) second Ab, and tyrosine phosphorylation of Syk (pY-Syk) and -subunit (pY-) were analyzed. Syk tyrosine phosphorylation was undetectable in -/- macrophages.

FcRIIb extracellular and transmembrane domain is sufficient for raft reorganization

We next studied whether monomeric FcRIIb could induce spatial raft reorganization, by using heterologous expression system. To test whether the presence of cytoplasmic positive (ITAM) or negative (ITIM) signal generating modules affects spatial raft reorganization, we also prepared FcRIIb lacking almost the entire cytoplasmic region except for juxtamembrane basic cluster (aa 247–252, KKKQVP; FcRIIb-truncated), and FcRIIb- ITAM chimera, whose cytoplasmic region is replaced with that of common subunit possessing ITAM. Schematic representation of these structures was shown in Fig. 4A. These constructs were overexpressed in RBL2H3 mast cells, and multiple clones with similar expression levels of the receptors were obtained. Flow cytometric analysis of FITC-2.4G2 binding in representative cell lines and in RAW macrophages was shown in Fig. 4B. As compared with RAW cells, these RBL2H3 clones expressed the FcRIIbs at around seven times higher levels.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. FcRIIb and its mutated forms expressed in RBL2H3 cells. A, Schematic representation of murine WT FcRIIb2, truncated FcRIIb, and FcRIIb- ITAM chimera. Structure of subunit is also shown. The extracellular (EC), transmembrane (TM), and intracellular (IC) domains are shown. Numbering of amino acids for FcRIIb2 is according to Lewis et al. (49 ) and that of subunit is according to Ra et al. (50 ). Truncated FcRIIb lacks almost the entire cytoplasmic region except for juxtamembrane basic cluster (247-252, KKKQVP). FcRIIb- ITAM chimera was created by replacing intracellular region (253-293) of FcRIIb with corresponding FcR- subunit bearing ITAM (50-86). B, Analysis of surface expression of WT and the mutated FcRIIbs expressed in RBL cells. RBL transfectants and RAW cells intrinsically expressing FcRIIb/IIIa were stained with FITC-conjugated 2.4G2 mAb or with isotype-matched Ab. Surface fluorescence was analyzed by EPICS-XL flow cytometer (Coulter, Hialeah, FL). The expression of WT and mutated FcRIIbs in RBL clones were almost comparable and around seven times higher than that of intrinsic FcRIIb/IIIa in RAW cells. C, Lack of association of FcRI and subunits with the FcRIIbs. RBL transfectants were sensitized with biotinylated IgE or biotinylated 2.4G2 mAb, lysed in digitonin buffer, and the receptors were immunoprecipitated with corresponding second Abs. FcRI- and FcRI- were probed with specific Abs and stained with HRP-conjugated second Ab. Immunoprecipitation efficiency was verified by streptavidin staining.

The WT and mutated FcRIIbs were tested for their ability to associate with DIM. To this end, we used 2.4G2 Fab to probe the receptors instead of 2.4G2 whole molecule, because divalent ligation by 2.4G2 was found to induce significant translocation of the FcRIIbs to DIM without second Ab addition (data not shown). We tentatively speculated that the premature association of dimerized receptors with DIM is due to their high expression levels. Cells sensitized with 2.4G2 Fab were treated with or without second Ab for 3 min, solubilized with 0.05% Triton X-100, and subjected to density gradient fractionation assay. As shown in Fig. 5A, small amounts of FcRIIb, FcRIIb- ITAM chimera, and truncated FcRIIb were distributed at DIM fraction (T) before clustering, and the receptor cross-linking significantly augment their association with DIM.

View larger version (52K): [in this window] [in a new window]   FIGURE 5. Raft reorganization by FcRIIb and its mutated forms expressed in RBL2H3 cells. A, Association of the FcRIIbs with DIM after cross-linking. The FcRIIbs were probed with biotinylated 2.4G2 Fab, treated with (cross-link (+)) or without (-) second Ab, and association of the receptors with DIM was analyzed, as described in the legend for Fig. 1B. Small amounts of the FcRIIbs were associated with DIM before clustering, but receptor cross-linking significantly enhanced the association. B, Raft stabilization by cross-linked FcRIIbs. RBL2H3 cells expressing WT and mutated FcRIIbs were treated as above, fixed with 3.7% formaldehyde, and solubilized with 1% Triton X-100 for 3 min. FcRIIbs and GM1 were stained with streptavidin-rhodamine and CTxB-FITC, respectively, and observed by confocal microscopy. After cross-linking, all the FcRIIbs became resistant to the detergent extraction, and accumulated along cell membrane. Rafts were also stabilized, as evidenced by clear GM1 staining at along cell membrane (see the x6 magnified views below) after receptor cross-linking. Bars, 5 µm. C, Colocalization of the FcRIIbs with patched GM1 after receptor cross-linking. GM1 was patched by incubation with CTxB-FITC and anti-CTxB Ab. The FcRIIbs were probed with biotinylated 2.4G2 Fab, and treated with streptavidin-rhodamine before fixation (receptor cross-link (+)) or after fixation (receptor cross-link (-)) with 3.7% formaldehyde. Solubilization with Triton X-100 was not applied. Top and middle rows, The FcRIIbs (red) and GM1 (green), respectively. In the overlay images (bottom row), colocalized segments of the FcRIIbs with GM1 are yellow colored. See text for details.

The homogeneous distributions of the FcRIIb-derived receptors and GM1 prevented us to assess their spatial codistributions. To confirm their colocalizations, we used raft patching study (33). GM1 was patched by FITC-conjugated CTxB and anti-CTxB Ab. The FcRIIbs were concurrently sensitized with biotinylated 2.4G2 Fab. To cross-link the receptors, cells were treated with streptavidin-rhodamine before fixation. To visualize uncross-linked ones, streptavidin-rhodamine was added after fixation (Fig. 5C). As shown in the middle row of Fig. 5C, CTxB-FITC staining showed discontinuous, patchy distribution of GM1 along plasma membrane. In the absence of FcRIIb-clustering, FcRIIbs stained with streptavidin-rhodamine almost evenly distributed. After cross-linking, patchy distributions of the FcRIIb, FcRIIb- ITAM chimera, and truncated FcRIIb became apparent (Fig. 5C, upper row). As shown in the overlay images (Fig. 5C, bottom row), the FcRIIbs partly codistributed with GM1 (yellow-colored segments) before the receptor cross-linking, due to their even distributions, but red-colored segments representing FcRIIbs outside lipid rafts were clearly visible. After cross-linking, red-colored segments were almost disappeared, and discontinuous yellow patches became prominent in all the RBL transfectants. These results indicate that FcRIIb, FcRIIb- ITAM chimera, and truncated FcRIIb almost completely colocalized with patched rafts after cross-linking.

FcRIIb extracellular and transmembrane domain represents cell activation domain

The above findings showed that extracellular and transmembrane segment of FcRIIb subunit is sufficient to induce spatial raft reorganization. We subsequently tested whether this process is responsible for the generation of intracellular signaling. Fig. 6A shows elevation of [Ca²⁺]_i after receptor cross-linking. Control RBL cells and the cells expressing the FcRIIbs were sensitized with biotinylated 2.4G2 Fab, challenged with streptavidin, and changes in [Ca²⁺]_i in 25 randomly assigned cells were recorded. In vector control cells and the cells expressing WT FcRIIb, this treatment did not elicit detectable calcium mobilization. Clustering of FcRIIb- ITAM chimera induced rapid and sustained [Ca²⁺]_i elevation. Of note, clustering of truncated FcRIIb also elicited small but rapid calcium transient. As shown in the control experiments, streptavidin alone did not elicit detectable [Ca²⁺]_i elevation in the RBL2H3 clones, and clustering of intrinsic FcRI induced almost comparable [Ca²⁺]_i elevation in the clones. These results were reproducible in two sets of RBL clones expressing the FcRIIb-derived molecules. The peak [Ca²⁺]_i increase in two sets of clones was presented in Fig. 6B. The peak [Ca²⁺]_i increase was 1677 ± 44 nM and 652 ± 104 nM in cells expressing FcRIIb- ITAM and truncated FcRIIb, respectively.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Biochemical signaling elicited by FcRIIb and by mutated FcRIIbs in RBL2H3 cells. A, Calcium mobilization in RBL2H3 cells expressing control vector, WT FcRIIb, and mutated FcRIIbs. RBL2H3 clones were sensitized with biotinylated 2.4G2 Fab and stimulated with streptavidin (left panel). The line graphs represent the average of [Ca2+]i in 25 cells randomly selected. Right panels, The same clones were sensitized with anti-DNP IgE, and stimulated with streptavidin, subsequently with DNP-BSA Ag. B, [Ca2+]i elevation via the FcRIIbs in multiple clones (n = 3). Mean ± SD of peak [Ca2+]i increase is shown. Calcium response was not detectable in control and FcRIIb2 expressing RBL cells. FcRIIb- ITAM and truncated FcRIIb elicited [Ca2+]i elevation whose peak increases were 1677 ± 44 nM and 652 ± 104 nM, respectively. C, Phosphorylation of LAT. The RBL2H3 clones were sensitized with biotinylated IgE or Fab of biotinylated 2.4G2, stimulated with (+) or without (-) streptavidin, and tyrosine phosphorylation of LAT (pY-LAT) were analyzed. The same membrane was reprobed with anti-LAT Ab after stripping. IgE and FcR- ITAM chimera stimulation gave strong signals. Truncated FcRIIb also induced small but discrete LAT tyrosine phosphorylation. D, Effects of Src PTK inhibition by PP2 on the truncated FcRIIb-mediated calcium mobilization. RBL2H3 cells expressing truncated FcRIIb were pretreated with 50 µM of PP2 for 30 min (+) or not (-) and stimulated via FcR as described above (n = 3). RBL2H3 cells expressing PAF receptor were used as a control. PP2 suppressed peak [Ca2+]i increase by 71%, whereas it marginally affected PAF-induced response. *, p < 0.01 compared with PP2-untreated cells by t test. E, Effects of Src PTK inhibition by PP2 on the truncated FcRIIb-mediated LAT phosphorylation. RBL2H3 cells expressing truncated FcRIIb were pretreated with PP2 and stimulated via FcR as described above, and tyrosine phosphorylation of LAT (pY-LAT) was analyzed. PP2 decreased LAT tyrosine phosphorylation. F, Effects of cholesterol depletion on the truncated FcRIIb-mediated calcium mobilization. RBL cells expressing truncated FcRIIb were treated with MCD at 0, 10, or 15 mM for 30 min, and then with (recovery +) or without (-) DMEM containing 10% FCS. Cells were stimulated as above. Upper panel, Peak [Ca2+]i increase (mean ± SD) is presented. A total of 15 mM MCD treatment significantly suppressed peak [Ca2+]i increase by 61%, and this inhibition was reversible. Lower panel, The amount of cell cholesterol was determined. Ten and 15 mM MCD treatment significantly decreased the cellular cholesterol to 50 and 37% of control, respectively, and DMEM containing 10% FCS completely restored cholesterol content. *, p < 0.01 compared with MCD-untreated cells by t test.

We next examined by pharmacological manipulations whether the biochemical signaling elicited by truncated FcRIIb is dependent on Src family kinase activity or on the integrity of lipid rafts. As shown in Fig. 6D, treatment of the cells with a Src family kinase-selective inhibitor, PP2 at 50 µM, suppressed truncated FcRIIb-mediated peak [Ca²⁺]_i elevation by 71%, whereas this reagent did not significantly affect the response elicited by heterotrimeric G protein-coupling PAF receptor expressed in RBL cells (25, 39). PP2 treatment also decreased LAT tyrosine phosphorylation mediated by truncated FcRIIb (Fig. 6E). To study the involvement of rafts in the calcium signaling, we examined the effects of cholesterol depletion by MCD (40). As shown in Fig. 6F, 15 mM MCD suppressed peak [Ca²⁺]_i elevation by 61%. Subsequent treatment of the cells with 10% FCS-containing DMEM almost completely recovered the extent of calcium response. For the recovery study, MCD/cholesterol complex could not be applied, because addition of 2.5 mM MCD/cholesterol complex (8/1, by mole) to MCD-treated cells induced significant elevation of basal [Ca²⁺]_i, presumably due to extensive membrane perturbation by the acute cholesterol addition (data not shown). As shown in Fig. 6F, MCD at 10 and 15 mM decreased net cholesterol content to 50 and 37% of that in untreated cells, respectively, and cholesterol repletion with 10% FCS almost completely recovered the cholesterol content. Therefore, cross-linking of extracellular and transmembrane region of FcRIIb elicits LAT tyrosine phosphorylation and calcium mobilization in a manner dependent on Src family kinase activity and on the integrity of lipid rafts. In addition, tandem ligation of ITAM and ITIM to the receptor fragment augments and suppresses the signal amplitude, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Initiation of intracellular signaling from FcR involves conversion of physical clustering of the receptors to Src family kinase-mediated ITAM phosphorylation. Recent characterization of lipid rafts has provided a clue to consider the initial mechanisms. Findings that TCR and FcRI signaling is catalyzed solely by raft-associating Src family kinases indicate roles of rafts in providing fields for signal generation (8, 9). It has also been shown that cross-linked FcRI and BCR associate with DIM under the condition that intracellular signaling is suppressed (17, 18). These observations raised the possibility that receptor-DIM association represents the upstream signaling. This early process is presumed to be responsible for bringing receptors into the association with the signal initiating Src family kinases (41). This concept is well applicable to BCR system, because both surface IgM and Ig/Ig are incorporated into DIM after clustering (13). What is apparently contradictory to the notion is that, in TCR and FcRI systems, ITAM-bearing TCR- and FcRI and subunits seems to constitutively associate with DIM, and that ligand-binding subunits lacking ITAM actively translocate into DIM after receptor clustering (9, 10, 12, 17). We presumed that this behavior of ligand-binding subunits reflects their active roles in raft coalescence. Therefore, we addressed three problems in this study. First, is spatial raft coalescence independent of Src family kinase activity? Second, are ligand-binding subunits responsible for raft reorganization? Third, does the initial raft reorganization represent productive signaling?

Although raft redistribution to the site of TCR engagements has been shown to require tyrosine kinase signaling and actin polymerization (42), it is still possible that raft reorganization in its early phase is signal-independent. We tested the first hypothesis by cytological observation of rafts after detergent treatment, following the methods by Janes et al. (33). The current findings in mCsk-overexpressing RAW cells quantitatively showed that spatial raft coalescence after FcR clustering is independent of Src family kinase activity. These findings indicate that raft coalescence as well as FcR association with DIM could be positioned at upstream of intracellular signaling. Signaling-independent spatial raft coalescence is further supported by efficient raft stabilization by the clustering of WT FcRIIb ectopically expressed in RBL2H3 cells.

We next tested whether ligand-binding subunits are responsible for raft reorganization. To avoid complexity derived from associating subunits, monomeric FcRIIb was used as a model system, because signaling subunits are frequently required for surface expression of MIRRs including FcRIIIa, TCR, and FcRI (2). The current findings using FcRIIb chimeras provided an example supporting the roles of ligand-binding subunits in raft reorganization. We also showed that the extracellular and transmembrane region of FcRIIb is sufficient for stabilizing rafts at cell membrane. Field et al. (19) showed that FcRI and/or transmembrane segment is responsible for its association with DIM fraction. The current data are consistent with their findings, and further emphasized the roles of ligand-binding subunits in spatial raft reorganization. Obviously, these findings in FcRIIb are not sufficient to deduce general mechanisms of MIRR-mediated raft coalescence, but it is intriguing to speculate that extracellular and transmembranous segments of ligand-binding subunits possess roles in raft reorganization besides their roles in ligand sensing.

We finally examined whether raft coalescence induced by the truncated FcRIIb is responsible for the generation of intracellular signaling. A previous study showed that clustering of FcRIIb alone does not lead to tyrosine phosphorylation signaling (24). We confirmed that clustering of ectopically overexpressed FcRIIb did not induce detectable calcium mobilization or LAT tyrosine phosphorylation. We presumed that FcRIIb-mediated raft reorganization potentially includes positive signaling, but that simultaneous ITIM condensation hindered it. Consistent with the notion, extracellular and transmembrane segment of FcRIIb elicited discrete LAT tyrosine phosphorylation and calcium response after clustering, in a manner dependent on Src family kinase activity and on raft integrity. Tandem ligation of ITAM to the receptor segment augmented the response. Under our experimental conditions, coimmunoprecipitation of the extracellular and transmembrane segment of FcRIIb with subunit was nearly undetectable (see Fig. 4C). Although the lack of coimmunoprecipitation did not completely exclude the potential protein-protein interaction between the truncated FcRIIb with subunit, the present findings support the notion that raft coalescence induced by the receptor segment triggers productive signaling. Pearse et al. (36) showed that FcRIIb cross-linking induces B cell apoptosis, and assumed that transmembrane segment catalyzes the functions through membrane perturbation. These findings together with ours suggest significant roles of the transmembrane segment in biological functions. It could be presumed that raft reorganization is also involved in the B cell apoptosis. Obviously, the observed productive signaling by the "ITAM-less" FcRIIb fragment does not preclude the involvement of ITAMs. It is likely that indirect condensation of ITAM-bearing subunits after raft coalescence plays roles in signal generation.

Chimera strategy as used in this study has been used to examine the roles of ITAM in MIRR signal transduction (43, 44, 45). In those studies, IL-2R subunit (Tac) was frequently used as a source of extracellular and transmembrane domain. Tac- ITAM chimeras reproducibly induced prompt signaling such as calcium mobilization or granule release, consistent with FcRIIb- ITAM chimera in this study. What is apparently contradictory to our findings is that Tac constructs lacking cytoplasmic region were unable to induce early biochemical signaling (43, 44, 45). We tentatively presumed that this difference is simply due to different expression levels, but not to receptor sources, because IL-2R was shown to associate with DIM after clustering (19).

It has long been recognized that mechanical clustering of rafts by cross-linking of GPI-anchored proteins induces Src family kinase-mediated signaling (46). The current study indicates that inducible raft coalescence triggered by FcR cross-linking could also be positioned at the upstream of Src family kinase activation, and that ligand-binding subunits are responsible for the initial process. It is also suggested that raft coalescence itself generates initial productive signaling. However, how raft coalescence leads to biochemical signaling is still undetermined. One of the possible mechanisms is that raft coalescence provides fields for one Lyn molecule to transactivate another (20). Given that that unperturbed raft is so small as estimated by biophysical analysis (20), Lyn molecules might be separated from each other. Of note, we have previously shown that Lyn molecules in RBL2H3 cells are a mixture of C-terminal tyrosine phosphorylated and dephosphorylated forms (23). Therefore, it might be possible that rafts are chimeric in terms of Lyn activity, and that the Lyn activity is dependent on the presence of Cbp/PAG-Csk complex (47, 48). As mentioned above, it is also possible that lipid rafts function as vehicles of the other signaling molecules including subunit, and that raft coalescence indirectly causes localized ITAM condensation. These possibilities should be evaluated in various model systems including ^-/- cells.

	Acknowledgments

 
We thank Dr. R. P. Siraganian and Dr. T. Takai for FcRI Ab and FcRIIb Ab, respectively. We also thank H. Ota-Ichijo for excellent technical assistance.

	Footnotes

 
¹ This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government, by AstraZeneca Asthma Research Award, and by Manabe Research Foundation.

² Address correspondence and reprint requests to Dr. Zen-ichiro Honda, Department of Allergy and Rheumatology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: honda-phy{at}h.u-tokyo.ac.jp

³ Abbreviations used in this paper: MIRR, multichain immune recognition receptor; DIM, detergent insoluble membrane; RBL, rat basophilic leukemia; CTxB, cholera toxin B; MCD, methyl--cyclodextrin; BCR, B cell Ag receptor; ITAM, immune receptor tyrosine-based activation motif; mCsk, membrane-anchored Csk; PAF, platelet-activating factor; [Ca²⁺]_i, intracellular Ca²⁺ concentration; WT, wild type; BMMC, bone marrow-derived macrophage; ITIM, immunoreceptor tyrosine-based inhibitory motif; PTK, protein tyrosine kinase.

Received for publication October 9, 2001. Accepted for publication April 30, 2002.

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