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The Journal of Immunology, 2007, 178: 3048-3058.
Copyright © 2007 by The American Association of Immunologists, Inc.

Transmembrane Mutations to Fc{gamma}RIIA Alter Its Association with Lipid Rafts: Implications for Receptor Signaling1

Erick García-García*, Eric J. Brown{dagger} and Carlos Rosales2,*

* Immunology Department, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico; and {dagger} Program in Microbial Pathogenesis and Host Defense, University of California, San Francisco, CA 94158


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor Fc{gamma}RIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) Fc{gamma}RIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT Fc{gamma}RIIA and mutants that associated with lipid rafts efficiently activated NF-{kappa}B, in an ERK-dependent manner. In contrast, WT Fc{gamma}RIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of Fc{gamma}RIIA as responsible for regulating its inducible association with lipid rafts and suggest that Fc{gamma}RIIA-mediated responses, like NF-{kappa}B activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lipid rafts were initially described in the field of epithelial cell biology as lipidic structures responsible for the transport of proteins from Golgi to the apical membrane. Hence, the name "lipid rafts" was coined (1, 2, 3). Though the precise structure and composition of lipid rafts remains a matter of intense debate (2, 4, 5, 6), evidence indicating that these membrane domains are important in cell function is growing at an accelerated rate (1, 2, 7, 8). Besides their role in the transport of proteins in epithelial cells, lipid rafts have been shown to have an important role in the regulation of intracellular signaling processes (7, 8, 9). Although methods for identification of lipid rafts are still evolving, one property generally agreed upon is that they contain low-density membranes that, because of high cholesterol content, are resistant to solubilization by nonionic detergents such as Triton X-100 (3, 10). Although these detergent-resistant membrane domains (DRM)3 may not be identical with lipid rafts, they are derived from them. A wide array of membrane receptors associates with DRM and signaling events regulating many cell responses appear to depend on DRM integrity (1, 7, 10). Within the immune system it has been shown that many immunoreceptors, including the AgRs of B and T lymphocytes, as well as various receptors for the Fc portion of Igs (FcRs), associate with DRM in response to ligand-induced cross-linking (8, 9, 11, 12). Though the list of DRM-associated membrane receptors of importance to immune functions is large, the way in which this association is regulated is still obscure.

Human Fc{gamma}RIIA is a member of the FcR family that binds IgG molecules (13). Fc{gamma}RIIA is composed of a single polypeptide chain bearing an ITAM on its cytoplasmic domain (Cyt) (13). This ITAM confers on Fc{gamma}RIIA the ability to initiate signaling events that regulate cell responses, including phagocytosis, respiratory burst, cytokine production, and Ab-dependent cell-mediated cytotoxicity (13, 14). The different signaling pathways that regulate Fc{gamma}RIIA-mediated cell responses have been relatively well-described (15, 16, 17). It was recently found that Fc{gamma}RIIA, like other immunoreceptors, associates with DRM upon ligand-induced cross-linking (18, 19, 20). The way in which Fc{gamma}RIIA association with DRM is regulated is not known.

Experiments performed in other systems have shown that various membrane proteins use different domains, and/or posttranslational modifications, such as acylation of amino acid residues located close to the inner leaflet of the plasma membrane, to regulate their association with DRM. For example, the hemagglutinin A (HA) of the influenza virus is a protein constitutively associated to DRM. HA relies on the sequence of its transmembrane domain (TM) for DRM association (21). In addition, palmitoylation of residues located on its Cyt was also required for DRM association (22). Other molecules, such as CD4 (23, 24) and CD20 (25), required their Cyt for DRM association. In the case of CD4, it has also been reported that palmitoylation of cysteines located downstream of its TM was required for DRM association (23). Other molecules, such as the epidermal growth factor receptor (26) and the prion protein (27), require the EC of the protein to mediate DRM association.

In the present report, we evaluated the possible roles for the cytoplasmic and TMs of Fc{gamma}RIIA in facilitating receptor association with DRM (lipid rafts). We transfected human wild-type (WT) Fc{gamma}RIIA, and a series of cytoplasmic and transmembrane mutants of the receptor, into a cell line of rat basophilic cells (RBL-2H3). Detergent extraction and standard sucrose density gradient centrifugation methods (6, 19, 28, 29) were used to analyze the association of the transfected receptors with DRM or lipid rafts, before and after receptor ligation and cross-linking. This analysis demonstrated that the cytoplasmic tail of the receptor, including its palmitoylation site (30), was dispensable for DRM association. We identified one transmembrane mutant (A224S) that lacks the ability to associate with DRM upon receptor cross-linking, and two transmembrane mutants (VA231-2MM and VVAL234-7GISF), that were capable of associating with DRM in the absence of receptor cross-linking. Either inducible or constitutive Fc{gamma}RIIA-DRM association was required for the activation of the NF-{kappa}B. Fc{gamma}RIIA-mediated phagocytosis could be triggered by Fc{gamma}RIIA transmembrane mutants, irrespective of their ability to associate with DRM.

Our data identify the TM of Fc{gamma}RIIA as responsible for regulating the cross-linking-dependent association of the receptor with DRM and suggest that some Fc{gamma}RIIA-mediated responses, such as NF-{kappa}B activation, require DRM association, while others, such as phagocytosis, can proceed independently of, but be modulated by, DRM association.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

The following Abs were used: specific anti-human Fc{gamma}RII (CD32) mAb IV.3 (catalog no. 025-1) obtained from Medarex; goat polyclonal Ab N-20 anti-CD32 (catalog no. sc-12808), rabbit polyclonal anti-ICAM-1 (catalog no. sc-7891), rabbit polyclonal anti-Syk (catalog no. sc-573), rabbit polyclonal anti-NF-{kappa}B p50 (catalog no. sc-114), and rabbit polyclonal anti-ERK (catalog no. sc-94), obtained from Santa Cruz Biotechnology. The rabbit polyclonal anti-phospho-ERK (catalog no. 9101) was obtained from Cell Signaling Technology. The anti-HA mAb 12CA5 was obtained from Roche Molecular Biochemals. HRP-conjugated F(ab')2 goat anti-mouse IgG (catalog no. 55468), FITC-conjugated F(ab')2 goat anti-mouse IgG (catalog no. 55522), and FITC-conjugated F(ab')2 goat anti-rabbit IgG (catalog no. 55665) were obtained from Cappel. Anti-transferrin receptor mAb L5.1 was obtained from American Type Culture Collection. The anti-phosphotyrosine mAb AFT8 has been previously described (31, 32). Protein A, FITC-, and HRP-conjugated cholera toxin B subunit were obtained from Sigma-Aldrich. The Syk inhibitor piceatannol was obtained from Calbiochem, and the MEK/ERK inhibitor PD98059 was obtained from New England Biolabs. All other chemicals were obtained from Sigma-Aldrich.

Generation of Fc{gamma}RIIA cytoplasmic and transmembrane mutants

The full cDNA codifying the human Fc{gamma}RIIA was a gift from Dr. J. C. Unkeless (Mount Sinai School of Medicine, New York, NY). Fc{gamma}RIIA cDNA was cloned into the EcoRI site of pcDNA3 (Invitrogen Life Technologies) using standard molecular biology techniques. The Fc{gamma}RIIA-pcDNA3 construct was used for the subsequent generation of the receptor mutants. Deletion of the Fc{gamma}RIIA Cyt was performed by selective PCR amplification of the cDNA coding for amino acids 1–245 of Fc{gamma}RIIA. The 5' oligo was 5'-GCGTGGATAGCGGTTTGACTCACG-3' (bases 644–667 of the pcDNA3 promoter), and the 3' oligo was 5'-CTGAAATCCGCTTTTTCCTGCA-3'. To generate all other Fc{gamma}RIIA mutants, the GeneEditor Site-Directed Mutagenesis System (Stratagene) was used according to the manufacturer’s instructions.

Cell culture

Human monocytic cell line THP-1 and rat basophilic cell line RBL-2H3 were maintained in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies), 20 mM glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin at 37°C in a 5% CO2 atmosphere.

Transfection and generation of stable transfectants

RBL-2H3 cells were transfected using Lipofectamine Plus Reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. For the generation of stable transfectants, RBL-2H3 cells were cultured in supplemented RPMI 1640 medium containing 500 µg/ml G418 (Invitrogen Life Technologies) for ~2 wk. Selection of cells expressing higher levels of the transfected receptors was performed by FACS.

FACS

Cells were labeled for 30 min at 4°C with 10 µg/ml the specific anti-human Fc{gamma}RIIA mAb IV.3 in labeling buffer (PBS plus 0.5% BSA plus 2 mM EDTA). Cells were washed three times with labeling buffer to remove unbound Ab and then labeled for 15 min at 4°C with FITC-conjugated F(ab')2 goat anti-mouse IgG. Cells were washed again three times with labeling buffer and analyzed and separated in a FACSCalibur apparatus (BD Biosciences).

Cell stimulation by Fc{gamma}RIIA cross-linking

Fc{gamma}RIIA-transfected RBL-2H3 cells, or THP-1 monocytes (2 x 107 cells/ml), were labeled for 30 min on ice with 10 µg/ml IV.3 Ab in labeling buffer (PBS plus 0.5% BSA plus 2 mM EDTA). Unbound Ab was washed three times with cold labeling buffer. Cells were stimulated by resuspending them at 1 x 107 cells/ml in warm labeling buffer containing 30 µg/ml F(ab')2 goat anti-mouse IgG and incubating them at 37°C for the indicated times. Unbound Ab was then washed twice and samples were processed for subsequent isolation of DRM or analysis of intracellular signaling events. In selected experiments, cells were treated with 50 µM piceatannol (Syk inhibitor) or 50 µM PD98059 (ERK inhibitor) for 30 min, before the labeling and stimulation steps. When inhibitors were used control cells were treated only with the solvent DMSO.

DRM isolation

Detergent-resistant membrane (lipid raft) isolation was performed essentially as described (28, 33). Cells were lysed for 20 min with 300 µl of cold TNE buffer (20 mM Tris, 140 mM NaCl, 2 mM EDTA) containing 0.05% Triton X-100, and protease inhibitors (complete EDTA-free protease inhibitor mixture; Roche Applied Science). Lysates were then mixed with 375 µl of 80% sucrose in TNE-Triton X-100 buffer and transferred to ultracentrifuge tubes (catalog no. 347356; Beckman Coulter). Cell lysates, now in 45% sucrose, were overlaid with 1 ml of 35% sucrose in TNE-Triton X-100 buffer and this latter fraction was overlaid with 400 µl of 5% sucrose in TNE-Triton X-100 buffer. Samples were then centrifuged at 4°C for 16 h at 170,000 x g in an Optima TLX ultracentrifuge using the TLS 55 rotor (Beckman Coulter). After centrifugation, seven 300-µl fractions were collected (bottom to top).

Detection of Fc{gamma}RIIA and ganglioside GM1

Sucrose density gradient fractions were diluted 2-fold with PBS and the content of each fraction was coupled to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore) using a dot-blot apparatus (Bio-Rad). A total of 400 µl of the diluted samples were applied to the PVDF membrane for Fc{gamma}RIIA detection and 50 µl for the detection of ganglioside GM1. PVDF membranes were blocked overnight at 4°C with blocking buffer (PBS plus 0.05% Tween 20, 1% BSA, and 5% nonfat dry milk; Carnation/Nestle Food). For Fc{gamma}RIIA detection, a modification of a described protocol (19) was used as follows: PVDF membranes, prepared with samples of unstimulated cells (labeled only with the IV.3 Ab), were incubated with a 1/10,000 dilution of HRP-conjugated F(ab')2 goat anti-mouse IgG, in blocking buffer, for 40 min. Membranes were then washed and developed by chemiluminescence. PVDF membranes, prepared with samples of Fc{gamma}RIIA-stimulated cells (labeled with the IV.3 Ab and cross-linked with HRP-conjugated F(ab')2 goat anti-mouse IgG), were just developed by chemiluminescence immediately after blocking. For GM1 detection, PVDF membranes were incubated for 1 h at room temperature in blocking buffer, with a 1/10,000 dilution of HRP-conjugated cholera toxin B subunit. The membrane was then washed and developed by chemiluminescence.

Fc{gamma}R phosphorylation analysis

Fc{gamma}RIIA phosphorylation status was determined as follows: after Fc{gamma}RIIA stimulation, RBL-2H3 cells (5 x 106) were lysed with 100 µl of ice-cold lysis buffer (1% Nonidet P-40, 150 mM NaCl, and 50 mM Tris (pH 7.4)), containing protease inhibitors (complete EDTA-free protease inhibitor mixture; Roche Applied Science), 1 mM sodium orthovanadate, and 1 mM p-nitrophenyl phosphate. Seventy-five microliters of lysates were mixed with 30 µl of protein G-Sepharose, prebound to 1 µg of anti-phosphotyrosine Ab AFT8. Lysis buffer was then added to a final volume of 500 µl. Lysates were incubated overnight at 4°C on a rotating wheel. Sepharose beads were then washed five times with cold lysis buffer. Immunoprecipitated proteins were resolved by SDS-PAGE using a 10% gel. Proteins were then transferred to PVDF membranes and Western blotted against Fc{gamma}RIIA with 200 ng/ml anti-human CD32 Ab N-20.

Syk in vitro kinase assay

Analysis of Syk kinase activity was performed by in vitro kinase assays as described for RBL-2H3 cells (34). Briefly, after Fc{gamma}RIIA stimulation, RBL-2H3 cells (1 x 107) were lysed with 400 µl of ice-cold lysis buffer (1% Nonidet P-40, 150 mM NaCl, and 50 mM Tris (pH 7.4)), containing protease inhibitors (complete EDTA-free protease inhibitor mixture; Roche Applied Science) and 1 mM sodium orthovanadate. Lysates were mixed with 1.5 µg of rabbit anti-Syk Ab prebound to 30 µl of protein G-Sepharose and incubated for 1 h at 4°C on a rotating wheel. Sepharose beads were washed once with cold lysis buffer, twice with kinase buffer (25 mM HEPES and 10 mM MnCl2 (pH 7.5)), and resuspended in 40 µl of kinase buffer containing 10 µCi [{gamma}-32P]ATP (0.11 TBq/mM; 2 mCi/ml) (Amersham Biosciences). After a 5-min incubation at 30°C beads were washed three times with cold kinase buffer. Proteins were then eluted by the addition of 45 µl of Laemmli buffer containing 5% 2-ME and boiling for 5 min. Proteins were finally resolved by SDS-PAGE using a 7.5% gel. Syk autophosphorylation was analyzed by autoradiography of the gels using a Phosphoimager model STORM 480 (Molecular Probes). Gels were stained with Coomassie or Western blotted against Fc{gamma}RIIA with Ab N-20 to detect immunoprecipitated proteins.

Nuclear staining of NF-{kappa}B

Nuclei were isolated from 2 x 106 RBL-2H3 cells essentially as described for EMSAs (35). Briefly, after centrifugation and complete removal of supernatant, cells were fast-frozen for 10 min in a dry ice-ethanol bath, and then disrupted by resuspending them in 100 µl of a hypotonic buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, and 1 mM fresh DTT (pH 7.9)). Nuclei were recovered by centrifugation for 10 min at 3000 rpm in a cooled Eppendorf microcentrifuge and resuspended in 100 µl of 4% paraformaldehyde in PBS. After a 20-min incubation on ice, nuclei were centrifuged at 6000 rpm for 1 min in an Eppendorf microcentrifuge and permeabilized for 10 min on ice by resuspending them in 100 µl of 0.1% Triton X-100 and 4% paraformaldehyde in PBS. Nuclei were then blocked with 100 µl of 1% BSA in PBS for 40 min on ice, centrifuged, and resuspended in 100 µl of 1% BSA and 2.5 µg/ml anti-NF-{kappa}B (p50) Ab in PBS. Nuclei were incubated on ice for 45 min. Unbound Ab was washed with 1 ml of PBS and nuclei were resuspended in 100 µl of 1% BSA plus 3 µl of FITC-conjugated F(ab')2 goat anti-mouse (1 µg/ml stock) in PBS. After 45 min on ice, unbound Ab was washed and nuclei were finally resuspended in 500 µl of 1% paraformaldehyde in PBS. To analyze NF-{kappa}B by flow cytometry, 10,000 nuclei were acquired per sample. NF-{kappa}B activation was calculated by multiplying the proportion of NF-{kappa}B-positive nuclei (nuclei whose fluorescence value was higher than that of nuclei stained with the unspecific Ab anti-ICAM-1) times the mean fluorescence intensity value of the positive nuclei population.

ERK phosphorylation

Detection of ERK and phospho-ERK was done by Western blotting exactly as described (31, 32) and also by FACS. Briefly, stimulated or unstimulated RBL-2H3 cells (1 x 106 cells) were fixed for 20 min on ice with 100 µl of 2% paraformaldehyde in PBS. After an additional 10 min at 37°C, cells were permeabilized by immediately adding 900 µl of ice-cold methanol. Cells were gently vortexed and incubated on ice for 30 min. Cells were centrifuged at 6000 rpm in an Eppendorf microcentrifuge for 1 min and resuspended in 1.5 ml of PBS containing 4% FCS (PBS-FCS). Cells were gently vortexed, centrifuged, and resuspended in 100 µl of PBS-FCS with a 1/100 dilution of anti-ERK or anti-phospho-ERK Abs. After a 20-min incubation on ice, unbound Ab was washed by adding 1.5 ml of PBS-FCS and vortexing gently for 15 s. Cells were centrifuged and resuspended in 100 µl of PBS-FCS containing 3 µl of FITC-conjugated F(ab')2 goat anti-mouse (1 mg/ml stock). Next, cells were incubated on ice for 20 min and unbound Ab washed with 1.5 ml of PBS-FCS. Cells were finally centrifuged, resuspended in 700 µl of 1% paraformaldehyde in PBS, and analyzed by flow cytometry. Numeric analysis was performed by subtracting the mean fluorescence intensity (MFI) value of cells stained with a control Ab (anti-HA) from the MFI value of cells stained for the molecule of interest. This corrected MFI value for cells in the resting state was considered 100%; the change induced by Fc{gamma}RIIA cross-linking was plotted as a percentage of the resting state value.

Phagocytosis assays

Opsonization of 4.8-µm fluorescent latex particles (catalog no. 16592; Polysciences) with protein A and the IV.3 Ab was performed by following the manufacturer’s recommendations. Phagocytosis assays were performed with a target:cell ratio of 3:1. For phagocytosis assays, 1.8 x 106 RBL-2H3 cells in 2.5 ml of supplemented RPMI 1640 medium were seeded onto a well of a 6-well plaque, on the day before the assay. The next day, cells (80–90% confluent) were harvested and resuspended at 1 x 107 cells/ml in cold phagocytosis buffer (2 mM calcium chloride, 1.5 mM magnesium chloride, 1% human serum albumin in PBS). In a prechilled Eppendorf tube, 90 µl of cold phagocytosis buffer were mixed with 10 µl of the cell suspension (1 x 104 cells), and 3.3 µl of a suspension (1 x 108 particles/ml) of IV.3-opsonized or control-opsonized (only protein A) fluorescent latex particles. Cells were vortexed gently and incubated on ice or at 37°C for the indicated times. Cells were centrifuged at 6000 rpm for 1 min in an Eppendorf microcentrifuge and resuspended in 100 µl of ice-cold trypsin-EDTA solution (0.05% trypsin, 1 mM EDTA in PBS) to detach uninternalized particles from the cells. After a 15-min incubation on ice, cells were washed with 1 ml of cold PBS plus 0.5% BSA plus 2 mM EDTA and finally resuspended in 500 µl of cold 1% paraformaldehyde in PBS. To analyze phagocytosis by flow cytometry, latex particles were gated out during sample acquisition and 10,000 cells were acquired per sample. Phagocytic score was calculated by multiplying the proportion of positive cells (cells whose fluorescence value was equal or higher than that of a single fluorescent latex particle) times the mean fluorescence value of the positive cell population. For microscopy analysis, cells were labeled on ice for 30 min with 1 µg/ml FITC-conjugated cholera toxin B subunit in phagocytosis buffer and then mixed with IV.3-opsonized nonfluorescent latex particles as indicated for the standard phagocytosis assay. Cells were incubated for 20 min at 37°C and then fixed with ice-cold 1% paraformaldehyde in PBS. Images were captured with a fluorescence microscope (OptipHot-2) and a Nikon Cool Pix 4300 charge-coupled device camera (Nikon Instruments).

Statistical analysis

Data were compared by an unpaired Student’s t test or a one-way ANOVA test using the computer program KaleidaGraph, version 3.6.2 for Macintosh (Synergy Software). Differences were considered to be statistically significant when p values were 0.03 or less.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation and expression Fc{gamma}RIIA mutants

Human WT Fc{gamma}RIIA and its mutants (Fig. 1) were expressed in RBL-2H3 cells (Fig. 2). The WT Fc{gamma}RIIA is composed of an extracellular domain (EC), a TM, and a Cyt. Because either the Cyt or the TM could be responsible for Fc{gamma}RIIA association with DRM upon receptor cross-linking, we decided first to eliminate features within its Cyt. Fc{gamma}RIIA contains a palmitoylated cysteine close to the inner leaflet of the plasma membrane (30) and two phosphorylatable tyrosines within its ITAM (13). In the Pal– mutant, cysteine 240 was replaced for serine, in the ITAM– mutant, tyrosines 287 and 303 were replaced for phenylalanines, and in the Cyt– mutant amino acids 246–316 were deleted (Fig. 1A). Then, we compared the TM of Fc{gamma}RIIA with the TM of HA of the influenza virus, a protein constitutively associated with DRM (21), and found that they showed some degree of conservation (Fig. 1B). Comparison of HA and several FcRs showed about the same level of conservation between HA and each FcR (Fig. 1C). The HA transmembrane showed less similarity to various proteins reported not to associate with DRM (Fig. 1D). In contrast, HA showed marked similarity with other proteins known to associate constitutively with DRM, especially in the transmembrane amino acids closer to the cytosol (Fig. 1E). Based on these comparisons, various mutations were introduced in the TM of Fc{gamma}RIIA (Fig. 1F). Some mutations changed residues that were similar between HA and Fc{gamma}RIIA or between Fc{gamma}RIIA and other FcRs, for example A220G. Other mutations made Fc{gamma}RIIA almost identical with HA in the transmembrane end closer to the cytosol (Fig. 1G). The RBL-2H3 cell line was selected as a system that allowed analysis of the function of WT Fc{gamma}RIIA and its mutants, in the absence of endogenously expressed Fc{gamma}RIIA. After transfection and antibiotic selection, stably transfected cells expressing high levels of Fc{gamma}RIIA and mutants were selected by FACS and used for all subsequent experiments. Cells expressed WT Fc{gamma}RIIA and all mutants at equivalent levels (Fig. 2B).


Figure 1
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  		FIGURE 1. Fc{gamma}RIIA cytoplasmic and transmembrane mutants. A, WT Fc{gamma}RIIA (WT) is composed of an EC, a TM, and a Cyt. Fc{gamma}RIIA Cyt contains two phosphorylatable tyrosines (Y) within its ITAM, and a palmitoylated cysteine close to the inner leaflet of the plasma membrane. In the Pal– mutant, cysteine 240 was replaced for serine. In the ITAM– mutant, tyrosines 287 and 303 were replaced for phenylalanines. In the Cyt– mutant, amino acids 246–316 were deleted. B–E, Protein alignment of the TMs of various proteins. Black boxes indicate positions where amino acids are identical and white boxes indicate positions where the functional group is conserved. B, Comparison of HA of influenza virus and Fc{gamma}RIIA. C, Comparison of HA with several FcRs. D, Comparison of HA with several proteins nonassociated with DRM. HA (21 ), dipeptidyl peptidase IV (DPP IV) (50 ), CD45 (51 ), H+/K+-ATPase (fourth transmembrane segment) (52 ), ectonucleotide pyrophosphatase/phosphodiesterase (NPP3) (53 ), enteropeptidase (54 ), Na+/K+-ATPase (52 ), transferrin receptor (transferrin R) (55 ). E, Alignment of HA with other DRM-associated proteins. Neuraminidase (NA) (38 ), megalin (56 ), connexin 43 (57 ), aminopeptidase N (APN) (58 ). F, Mutations to the TM of Fc{gamma}RIIA reported in this study. Changes are indicated by gray boxes. G, Mutations of Fc{gamma}RIIA that make it similar to HA at the carboxyl end of the TM.

 

Figure 2
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  		FIGURE 2. Expression of Fc{gamma}RIIA in RBL-2H3 cells. The cDNA coding for Fc{gamma}RIIA or its mutants, cloned in the pcDNA3 plasmid, was transfected into RBL-2H3 cells. A, Stable Fc{gamma}RIIA transfectants, or untransfected cells, were labeled with anti-human Fc{gamma}RIIA mAb IV.3 and FITC-conjugated goat anti-mouse IgG. Transfected cells within the M1 marker were selected for higher expression levels of Fc{gamma}RIIA by cell sorting. B, MFI values of sorted cells expressing the WT Fc{gamma}RIIA or its mutants at equivalent levels.

 
WT Fc{gamma}RIIA expressed in RBL-2H3 cells associates with DRM

To analyze the association of the transfected Fc{gamma}RIIA with DRM, cold detergent extraction and sucrose gradient centrifugation methods were used as described in Materials and Methods. In the resting state, Fc{gamma}RIIA distributed to high-density fractions 1–4 of the sucrose gradient (Fig. 3A). These fractions correspond to the nonlipid rafts part of the membrane, as indicated by the presence of the transferrin receptor, a protein that does not associate with DRM, and by the lack of the classic DRM marker ganglioside GM1 (Fig. 3A). In contrast, after Fc{gamma}RIIA cross-linking, the receptor almost entirely redistributed to the low-density fractions 5–7 of the gradient (Fig. 3A). These fractions correspond to DRM, as indicated by the presence of ganglioside GM1. As a control, sucrose density gradient analysis was performed to evaluate the distribution of the monocyte endogenously expressed Fc{gamma}RIIA into the gradient fractions. As observed with the transfected WT Fc{gamma}RIIA in RBL-2H3 cells, monocyte Fc{gamma}RIIA was found in the non-DRM fractions of the gradient in the resting state, but after cross-linking it distributed to the DRM fractions of the gradient (Fig. 3B). These results were in agreement with reports showing that monocyte and neutrophil endogenously expressed Fc{gamma}RIIA translocates to DRM after cross-linking (18, 19), and indicated that the Fc{gamma}RIIA was functional when transfected into RBL-2H3 cells.


Figure 3
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  		FIGURE 3. WT Fc{gamma}RIIA associates with DRM. A, Sucrose density gradient fractions from Fc{gamma}RIIA-expressing RBL-2H3 cells. In absence of cross-linking, Fc{gamma}RIIA is found in high-density (non-detergent resistant; NR) fractions 1–4. Transferrin receptor (TfR) a protein known to be excluded from DRM is also found in these fractions. After cross-linking, Fc{gamma}RIIA is found in low-density (detergent-resistant; R) fractions 5–7. The DRM marker ganglioside GM1 is also found in these fractions. IF, Interface between 35 and 5% sucrose. B, DRM were isolated from THP-1 monocytes or RBL-2H3 cells. The distribution of Fc{gamma}RIIA was analyzed as in A, except that nonrafts (NR) and DRM (rafts; R) fractions were pooled. Data are representative of three separate experiments.

 
The Cyt of Fc{gamma}RIIA is not necessary to regulate receptor association with DRM

It has been reported that the Cyt of some membrane proteins is required to mediate their association with DRM or lipid rafts (23, 24, 25, 36). In some cases, posttranslational modifications on the cytoplasmic portion of these molecules, such as acylation of residues proximal to the inner leaflet of the plasma membrane, have also been found to be important for association of the molecule with lipid rafts (23, 33). Because the cytoplasmic portion of Fc{gamma}RIIA contains a palmitoylation site close to the inner leaflet of the plasma membrane (30), we tested whether this site was necessary for DRM association. A palmitoylation-deficient mutant, Pal–, was made by changing the palmitate-acceptor cysteine 240 for serine (Fig. 1A). The Pal– mutant distributed to the DRM fractions upon receptor cross-linking (Fig. 4). This result indicated that palmitoylation of the Cyt of Fc{gamma}RIIA was not required for lipid raft association. Inhibition of tyrosine kinases has suggested that translocation of Fc{gamma}RIIA to DRM is independent of signaling events triggered by receptor stimulation (37). In agreement with this hypothesis, an Fc{gamma}RIIA mutant lacking the phosphorylatable tyrosines located within the receptor ITAM, also distributed to DRM fractions after receptor cross-linking (Fig. 4). Because the Fc{gamma}RIIA ITAM is essential to trigger signaling events upon receptor aggregation (13), these results support the idea that receptor translocation to DRM occurs before the initiation of signaling events. To determine whether other motifs present in the Cyt of Fc{gamma}RIIA were responsible for mediating receptor association with DRM, a mutant lacking the whole Cyt (Fig. 1A), was tested. This mutant, termed Cyt–, was also found in DRM fractions after receptor cross-linking (Fig. 4). This indicated that the Cyt of human Fc{gamma}RIIA is not required for receptor association with lipid rafts.


Figure 4
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  		FIGURE 4. The Cyt of Fc{gamma}RIIA is not required for receptor association with DRM. Distribution in non-detergent-resistant (NR) and detergent-resistant (R) gradient fractions of WT Fc{gamma}RIIA, palmitoylation-deficient Fc{gamma}RIIA (Pal–), ITAM-defective Fc{gamma}RIIA (ITAM–), and Fc{gamma}RIIA lacking the Cyt (Cyt–) in RBL-2H3 cells. Data are representative of three separate experiments.

 
The TM of Fc{gamma}RIIA regulates receptor association with DRM

It has been reported that some membrane proteins require information within the TM to associate with DRM (21, 38, 39). To investigate whether the TM of Fc{gamma}RIIA was also relevant for the receptor association with DRM, we tested the localization in sucrose gradient fractions of the transmembrane mutants described above. Like the WT Fc{gamma}RIIA, most of the transmembrane mutants also distributed to DRM fractions upon receptor cross-linking (Fig. 5A). In contrast, the mutant A224S failed completely to redistribute to DRM fractions (Fig. 5A). These data indicated that the alanine 224 is critical for the capacity of the receptor to associate with lipid rafts.


Figure 5
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  		FIGURE 5. The TM of Fc{gamma}RIIA regulates its association with DRM. Distribution in non-detergent-resistant (NR) and detergent-resistant (R) gradient fractions of WT Fc{gamma}RIIA, and five transmembrane mutants (A220G, A224S, T225A, VA231-2MM, and VVAL234-7GISF) of Fc{gamma}RIIA in RBL-2H3 cells, after (A) or before (B) receptor cross-linking. Data are representative of three separate experiments.

 
In the absence of receptor cross-linking, WT Fc{gamma}RIIA was found in the nonlipid rafts fraction (Figs. 3 and 5B). In contrast, the transmembrane mutant VA231-2MM was present in both nonlipid rafts and lipid raft (DRM) fractions in about the same proportion (Fig. 5B), while the transmembrane mutant VVAL234-7GISF was distributed almost entirely to DRM fractions (Fig. 5B). These data indicated that the TM of Fc{gamma}RIIA regulates the capacity of the receptor to associate with lipid rafts both in the unligated state and also after receptor cross-linking.

Activation of Syk by Fc{gamma}RIIA cross-linking is independent of DRM association

It has been suggested that receptor redistribution to lipid rafts is important for immunoreceptor signaling (8, 9, 11, 12). Some of our Fc{gamma}RIIA mutants presented abnormal DRM localization, so it was possible that their signaling capacities were defective. To explore the signaling capabilities of the Fc{gamma}RIIA mutants, we first examined Syk activation, because this is thought to be an early step in signal transduction, in response to the stimulation of various immunoreceptors, including Fc{gamma}Rs (13, 40, 41). Cross-linking of the WT Fc{gamma}RIIA transfected into RBL-2H3 cells induced Syk activation (Fig. 6). Similarly, the transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF all induced Syk activation at equivalent levels (Fig. 6). Syk activation depends in part on the phosphorylation of the receptor. Thus, the phosphorylation status of these Fc{gamma}RIIA mutants was analyzed. WT Fc{gamma}RIIA transfected into RBL-2H3 cells was not phosphorylated in the resting state. Upon cross-linking, the receptor was tyrosine phosphorylated. Phosphorylation of the receptor could be detected by one minute after stimulation and reached a maximum sustained level ~3–5 min (Fig. 7). The transmembrane mutant A224S, which fails to associate with lipid rafts, was also phosphorylated upon receptor cross-linking with similar kinetics as the WT receptor (Fig. 7). In contrast, the transmembrane mutants VA231-2MM and VVAL234-7GISF were phosphorylated with different kinetics from the WT receptor. Receptor phosphorylation reached a maximum level ~1 min after cross-linking and then tended to decay (Fig. 7). In fact, the mutant VVAL234-7GISF, which is constitutively associated with DRM, was already tyrosine phosphorylated before receptor cross-linking (Fig. 7). Because A224S does not associate with DRM upon cross-linking, these data suggested that at least one of the earliest steps in the Fc{gamma}RIIA-signaling pathway does not require lipid raft localization. However, receptor association with lipid rafts seems to increase the tyrosine phosphorylation status of the receptor.


Figure 6
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  		FIGURE 6. Fc{gamma}RIIA association with lipid rafts is not required for Syk activation. A, WT Fc{gamma}RIIA or its transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF, in RBL-2H3 cells, were cross-linked (+), or not (–), for 5 min. Cell lysates were then prepared and Syk activity detected by an in vitro kinase assay. Upper panels, Autoradiography of phosphorylated Syk (pSyk). Lower panels, Syk immunoprecipitated at equivalent amounts in all samples. Graph represents the densitometric analysis of Syk activity of four experiments. Data are mean ± SEM. *, Differences from control were statistically significant at p ≤ 0.02. B, Cell lysates immunoprecipitated with an anti-HA irrelevant Ab did not have any protein (lower panels) or kinase activity (upper panels) in the region of the gel where Syk (72 kDa) should appear.

 

Figure 7
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  		FIGURE 7. Fc{gamma}RIIA tyrosine phosphorylation upon receptor cross-linking. WT Fc{gamma}RIIA or its transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF, in RBL-2H3 cells, were cross-linked for the indicated times. Cell lysates were then prepared and tyrosine-phosphorylated proteins immunoprecipitated (IP) with anti-phosphotyrosine (PY) mAb AFT8. Fc{gamma}RIIA was then detected by Western blotting (WB) with Ab N-20. Lower panels, Fc{gamma}RIIA present at equivalent amounts in all whole cell lysates (WCL). Data are representative of two experiments.

 
Activation of NF NF-{kappa}B by Fc{gamma}RIIA requires lipid raft association

To determine whether signaling events downstream of Syk activation required DRM association, we examined activation of a NF important for transcription of cytokine genes, the NF-{kappa}B. Cross-linking of the WT Fc{gamma}RIIA induced a small NF-{kappa}B activation (Fig. 8A). Significantly, the A224S transmembrane mutant, which could not associate with DRM, did not activate NF-{kappa}B (Fig. 8A). In contrast, the constitutively DRM-associated mutants, VA231-2MM and VVAL234-7GISF, induced higher activation of NF-{kappa}B than WT Fc{gamma}RIIA after cross-linking (Fig. 8A). Activation of NF-{kappa}B in response to cross-linking of DRM-associated Fc{gamma}RIIA variants was blocked by piceatannol (Fig. 8B), thus indicating that, as expected, the activation of this NF required Syk activity. These data indicated that only lipid raft-associated Fc{gamma}RIIA variants were capable of inducing NF-{kappa}B activation in a Syk-dependent manner.


Figure 8
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  		FIGURE 8. Fc{gamma}RIIA-induced activation of NF-{kappa}B requires lipid raft association. A, WT Fc{gamma}RIIA, or its transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF, in RBL-2H3 cells, were cross-linked or not (control) for 20 min, in the absence (A) or presence of 50 µM piceatannol (B). Nuclei were then isolated, fixed, and stained for NF-{kappa}B. Relative NF-{kappa}B activation (fluorescence intensity) was then quantified by flow cytometry. In B, the fluorescence value of cells in the resting state (control) was considered 100%, and the change induced by Fc{gamma}RIIA cross-linking was plotted as a percentage of the resting state value. Data are mean ± SEM of 8–10 determinations. *, Differences from control were statistically significant at p ≤ 0.02.

 
To further investigate the mechanistic basis for the requirement of lipid raft association of Fc{gamma}RIIA for NF-{kappa}B activation, we assessed the phosphorylation state of the upstream molecule ERK. Previously, we had reported that Fc{gamma}R-mediated NF-{kappa}B activation in monocytes required phosphorylation of ERK (32). In RBL-2H3 cells expressing the WT Fc{gamma}RIIA, ERK showed a constitutive high level of phosphorylation. This high level of ERK phosphorylation was also found in untransfected cells (data not shown). Upon WT Fc{gamma}RIIA cross-linking, the level of ERK phosphorylation was reduced (Fig. 9A). In cells expressing the A224S transmembrane mutant, which does not associate with lipid rafts, ERK had also a higher level of activity (Fig. 9A). However, in contrast to WT Fc{gamma}RIIA, cross-linking the A224S mutant did not affect ERK phosphorylation (Fig. 9A). We confirmed these results by determining the levels of active ERK by flow cytometry, before and after receptor stimulation, in transfected RBL-2H3 cells. Consistent with the Western blot data, cells expressing the WT Fc{gamma}RIIA showed a reduction in active ERK upon receptor cross-linking (Fig. 9B). The level of total ERK expressed was similar in all cases (Fig. 9B, lower panel). In contrast to either the WT Fc{gamma}RIIA or the A224S transmembrane mutant, cross-linking the constitutively DRM-associated mutants VA231-2MM and VVAL234-7GISF in RBL-2H3 cells induced a marked increase in the level of active ERK (Fig. 9C). The MEK/ERK inhibitor PD98059 did not have any effect on the small NF-{kappa}B activation induced by the WT Fc{gamma}RIIA (Fig. 9D). In contrast, activation of NF-{kappa}B in response to cross-linking of constitutively DRM-associated Fc{gamma}RIIA variants was completely blocked by PD98059 (Fig. 9D). Thus, efficient activation of NF-{kappa}B also required ERK activity. These data indicated that only lipid raft-associated Fc{gamma}RIIA variants were capable of inducing strong NF-{kappa}B activation through activation of ERK.


Figure 9
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  		FIGURE 9. Fc{gamma}RIIA-induced activation of ERK requires lipid raft association. WT Fc{gamma}RIIA, or its transmembrane mutant A224S, was cross-linked (+) or not (–) for 20 min. Cell lysates were then prepared and phosphorylated ERK (p-ERK) or total ERK were detected by Western blotting (A) or by flow cytometry (B). Lower panels show total ERK in equal amounts in all cell lysates. C and D, WT Fc{gamma}RIIA, or its transmembrane mutants A224S,VA231-2MM, and VVAL234-7GISF, were cross-linked or not (control) for 20 min, in the absence (C) or presence of 50 µM PD98059 (PD) (D). Whole cells (in C) or isolated nuclei (in D) were then fixed, permeabilized, and stained for phosphorylated ERK (p-ERK) or for NF-{kappa}B, respectively. Relative activation (fluorescence intensity) was then quantified by flow cytometry. The fluorescence value of cells in the resting state (control) was considered 100%, and the change induced by Fc{gamma}RIIA cross-linking was plotted as a percentage of the resting state value. Data are mean ± SEM of six determinations. *, Differences from control were statistically significant at p ≤ 0.03 (for C) and at p ≤ 0.02 (for D).

 
Fc{gamma}RIIA-mediated phagocytosis can occur in the absence of receptor association with lipid rafts

Because Fc{gamma}RIIA-mediated activation of the NF NF-{kappa}B depended on the ability of the receptor to associate with DRM, we assessed whether Fc{gamma}RIIA-mediated phagocytosis, another Syk-dependent function, was also DRM dependent. Phagocytosis of IV.3-coated particles occurred at very low levels in untransfected RBL-2H3 cells (Fig. 10A). Cells expressing WT Fc{gamma}RIIA phagocytosed very efficiently (Fig. 10A). The A224S transmembrane mutant promoted phagocytosis to a similar extent as the WT Fc{gamma}RIIA (Fig. 10A). Kinetics of phagocytosis was also very similar for the WT Fc{gamma}RIIA and the A224S transmembrane mutant (Fig. 10A). In contrast, phagocytosis through the constitutively DRM-associated mutants VA231-2MM and VVAL234-7GISF followed similar kinetics to the WT Fc{gamma}RIIA and A224S transmembrane mutant, but occurred at lower levels (Fig. 10A). Phagocytosis of latex particles coated only with protein A occurred at a very low level, which was similar in all transfectants (data not shown). These data suggested that receptor association with lipid rafts was not required for Fc{gamma}RIIA-mediated phagocytosis. To confirm this idea, the lipid raft marker ganglioside GM1 was labeled on RBL-2H3 cells with FITC-conjugated cholera toxin B subunit. Cells were then allowed to phagocytose IV.3-coated particles and observed by fluorescence microscopy. Cells phagocytosing via WT Fc{gamma}RIIA showed a clear accumulation of GM1 around phagocytic cups (Fig. 10B). In contrast, no accumulation of GM1 was observed around phagocytic cups formed in cells phagocytosing via the A224S mutant receptor, which does not associate with lipid rafts (Fig. 10C). Phagocytosis mediated by the transmembrane mutants VA231-2MM (Fig. 10D) and VVAL234-7GISF (Fig. 10E) also showed accumulation of GM1 at phagocytic cups. In addition, cholera toxin B-treated cells phagocytosed at the same level as unlabeled control cells. The phagocytic score was 70 ± 5 for treated cells vs 67 ± 8 for control cells (mean ± SEM of three experiments). These results demonstrate that although lipid rafts accumulate around ingested targets when the receptor is able to associate with lipid rafts, phagocytosis mediated by Fc{gamma}RIIA can be triggered outside lipid rafts.


Figure 10
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  		FIGURE 10. Fc{gamma}RIIA-mediated phagocytosis can occur independently of lipid raft association. A, Phagocytosis of IV.3-opsonized fluorescent latex particles by RBL-2H3 cells expressing WT Fc{gamma}RIIA, or the transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF. Cells were incubated at 37°C for various times to allow phagocytosis of the latex particles. Phagocytosis was then quantified by flow cytometry. Data are mean ± SEM of 12–16 determinations. *, Differences from control (WT Fc{gamma}RIIA) were statistically significant at p ≤ 0.006 using a one-way ANOVA test. B–E, Ganglioside GM1 in RBL-2H3 cells expressing WT Fc{gamma}RIIA (B), or the transmembrane mutants A224S (C), VA231-2MM (D), or VVAL234-7GISF (E), was labeled with FITC-conjugated cholera toxin B subunit. Cells were allowed to phagocytose non-fluorescent IV.3-opsonized latex particles for 20 min. Cells were immediately fixed, and analyzed by light (right panels) or fluorescence microscopy (left panels). Scale bar is 5 µm. Data are representative of three separate experiments.

 
To test the involvement of Syk in phagocytosis initiated inside or outside lipid rafts, we repeated the phagocytosis experiments in the presence of the Syk inhibitor piceatannol. Syk inhibition reduced the rate of phagocytosis promoted by WT Fc{gamma}RIIA and by all the transmembrane mutants (Fig. 11). These results indicated that phagocytosis promoted by Fc{gamma}RIIA inside or outside lipid rafts depends on the activity of Syk. The mutants VA231-2MM and VVAL234-7GISF mediated lower levels of phagocytosis than the WT receptor and the non-lipid raft-associated A224S mutant. One possible reason for these differences could be that the constitutively DRM-associated mutants had reduced avidity for their ligand. However, adhesion of IV.3-coated particles was the same for all Fc{gamma}RIIA variants at various target:cell ratios (Fig. 12). Thus, the reduced levels of phagocytosis mediated by the transmembrane mutants VA231-2MM and VVAL234-7GISF were not due to differences in receptor avidity.


Figure 11
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  		FIGURE 11. Phagocytosis promoted by Fc{gamma}RIIA inside or outside lipid rafts depends on the activity of Syk. Phagocytosis of IV.3-opsonized fluorescent latex particles by RBL-2H3 cells expressing WT Fc{gamma}RIIA, or the transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF. Some cell samples were treated with 50 µM piceatannol for 30 min before the phagocytosis assay. The phagocytic score of the cells in the absence of the Syk inhibitor (control) was considered 100%, and the change induced by Syk inhibition was plotted as a percentage of the control value. Data are mean ± SEM of 16–20 determinations. *, Differences from control were statistically significant at p ≤ 0.001.

 

Figure 12
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  		FIGURE 12. Fc{gamma}RIIA transmembrane mutants bind equivalent amounts of Ab-opsonized targets. RBL-2H3 cells expressing WT Fc{gamma}RIIA, or the transmembrane mutants A224S, VA231-2MM, and VVAL234-7GISF were mixed with IV.3-opsonized fluorescent latex particles at various target:cell ratios. Cells were incubated at 4°C for 30 min to allow binding of the latex particles. Adhesion was then quantified by flow cytometry. The adhesion score was calculated by multiplying the proportion of positive cells (cells whose fluorescence value was equal or higher than that of a single fluorescent latex particle) times the mean fluorescence value of the positive cell population. Data are mean ± SEM of 12 determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
An emerging feature of the signaling mechanisms used by various immunoreceptors is that they associate with DRM, or lipid rafts, upon ligand-induced cross-linking. Receptor-raft association appears to be a regulatory step for the signaling mechanisms used by many immunoreceptors (8, 9). Because the molecular requirements that regulate the association of immunoreceptors with DRM remain unclear, in this report we have investigated the role of various domains of Fc{gamma}RIIA in the regulation of receptor-lipid raft association. We found that the cytoplasmic tail of the receptor, including its palmitoylation site (30), was dispensable for DRM association, and that the TM of Fc{gamma}RIIA was responsible for regulating the inducible association of the receptor with DRM. Using mutants that differed in ability to associate with DRM, we found that Fc{gamma}RIIA-mediated responses, like NF-{kappa}B activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.

Several molecules including CD4 (23, 24) and CD20 (25) are reported to require their Cyt to interact with lipid rafts. None of the Fc{gamma}RIIA cytoplasmic mutants, however, showed any defects in their association with DRM, indicating that Fc{gamma}RIIA has no apparent functional motifs on its Cyt capable of mediating DRM association. Accordingly, the ITAM located on Fc{gamma}RIIA Cyt was not required for the receptor association with DRM after cross-linking. However, this ITAM is essential to initiate signaling events after Fc{gamma}RIIA receptor cross-linking (13, 42). The fact that the ITAM– Fc{gamma}RIIA mutant was still capable of associating with DRM supports the idea that receptor translocation to rafts occurs before receptor-initiated signaling events. This idea has already been suggested through the use of tyrosine kinase inhibitors (37). However, it was recently reported that ceramide production at the cell surface facilitated Fc{gamma}RIIA recruitment to lipid rafts (43). When Fc{gamma}RII was cross-linked acid sphingomyelinase was translocated from intracellular compartments to the cell surface and was rapidly activated leading to production of ceramide. How sphingomyelinase is activated by Fc{gamma}RIIA is not known, but it does not require the Fc{gamma}RIIA ITAM (44). Ceramide association with the Fc{gamma}RIIA TM may explain its role in mediating association with lipid rafts (43). Our data suggest that Fc{gamma}RIIA-triggered ITAM-dependent signaling events do not regulate receptor-DRM association, and that additional Fc{gamma}RIIA-initiated ITAM-independent signaling mechanisms exist that facilitate receptor-DRM association.

Our results differ from the conclusion of a recent study suggesting that palmitoylation of Fc{gamma}RIIA is important for lipid raft localization (30). In this report, a C(208)S mutation similar to ours prevented Fc{gamma}RIIA palmitoylation, and the mutant receptor expressed in a murine B cell lymphoma failed to redistribute to DRM fractions upon cross-linking, while in our experiments, no difference was found in the ability of this mutant to associate with DRM after cross-linking. This discrepancy may relate to the concentration of Triton X-100 used to isolate lipid rafts in the two studies. We used 0.05% Triton X-100 for cold extraction of Fc{gamma}RIIA, similarly to previous studies for Fc{epsilon}RI (28) and CD40 (45), while Barnes et al. (30), used 10 times as much detergent. It is important to note that under our conditions, naturally expressed Fc{gamma}RIIA in human THP-1 cells or the transfected Fc{gamma}RIIA in rat RBL-2H3 cells was entirely found in detergent soluble (non-lipid rafts) fractions during resting conditions, while upon cross-linking, Fc{gamma}RIIA completely redistributed to DRM fractions (Fig. 3). Under similar conditions, Barnes et al. (30) did not see a clean separation of Fc{gamma}RIIA expressed in the B cell line between detergent-soluble and -resistant fractions, raising the possibility that Fc{gamma}RIIA expressed in the B cell line behaves differently from the Fc{gamma}RIIA in other cells. We believe that palmitoylation may enhance or stabilize raft association of Fc{gamma}RIIA, making it more resistant to solubilization in the higher detergent concentration used by Barnes et al. (30), but it is clear from our data that TM sequences are absolutely required for lipid raft association.

For other membrane proteins, such as neuraminidase (38), T cell AgR (39), and HA (21), sequences within the TM have been reported to be responsible for association of these molecules with lipid rafts. In the case of Fc{gamma}RIIA, we identified several mutations in the TM resulting in abnormal association of the receptor with DRM. One transmembrane mutant, A224S, failed to associate with DRM when the receptor was cross-linked, and two transmembrane mutants, A231-2MM and VVAL234-7GISF, showed increased DRM association even in the absence of receptor cross-linking. The fact that specific mutations in Fc{gamma}RIIA TM result in altered DRM association indicates that this part of the molecule is responsible for the capacity of the receptor to associate with lipid rafts. Supporting this idea is the recent finding of a transmembrane polymorphism in another Fc{gamma}RII family member, the Fc{gamma}RIIB inhibitory receptor (46, 47). This polymorphism, associated with systemic lupus erythematosus, decreased the affinity of the receptor for lipid rafts, and involved the substitution of isoleucine 232 for threonine (I232T). In an alignment of the Fc{gamma}RIIB and Fc{gamma}RIIA TMs, this isoleucine would correspond to position 223 in the TM of Fc{gamma}RIIA (Fig. 1C). It is interesting to note that this isoleucine is right next to the alanine we have shown to be required for ligand-induced DRM association. Thus, this recent finding (47) and our present report suggest together that the amino acids isoleucine 223 and alanine 224 in the TM of Fc{gamma}RIIA are critical for regulating the association of the receptor with lipid rafts.

How changes at amino acid level affect the ability of Fc{gamma}RIIA to associate with lipid rafts is not known. Transmembrane domains are predicted to possess an {alpha}-helix structure. However, their precise three-dimensional structure in a lipidic environment is unknown, simply because protein crystallography is performed on aqueous phases. However, it has been suggested that transmembrane proteins associate with lipid rafts through direct interactions of transmembrane amino acids with cholesterol-sphingolipid complexes (4) and that this interaction may influence the tendency of the protein to associate with lipid rafts. It may be that the A224S transmembrane mutation modifies the three-dimensional structure of Fc{gamma}RIIA in such a way that its affinity for DRM components is reduced. This idea is further supported by the fact that mutations affecting lipid raft association of Fc{gamma}RIIB (I232T) (47) and Fc{gamma}RIIA (A224S) (this report) involve changes from a nonpolar amino acid to a polar one, in contiguous transmembrane positions. By the same token, there are proteins that permanently reside in lipid rafts because of their transmembrane sequences. One such protein is the HA of influenza virus (21). Our Fc{gamma}RIIA transmembrane mutants A231-2MM and VVAL234-7GISF strongly resemble the carboxyl end of the TM of HA, and showed increased association of the receptor with DRM. It may be then that these mutations generate in the Fc{gamma}RIIA a three-dimensional structure with higher affinity for lipid rafts components. Further mutations that would make the C-terminal end of the TM of Fc{gamma}RIIA identical with the transmembrane region of HA could confirm this idea.

Lipid rafts are also considered to have important roles in assembling cell-signaling complexes on the cytosolic face of the plasma membrane, especially for immunoreceptors (8). In this model, signaling molecules associate within lipid rafts to initiate intracellular signaling. This idea implies that only receptor molecules localized within rafts can initiate intracellular signaling. The validity of this idea is, however, a matter of strong debate (5). We looked at the signaling capacity of the various Fc{gamma}RIIA mutants that showed different types of association to DRM. The WT receptor and all the transmembrane mutants activated the tyrosine kinase Syk upon cross-linking. Syk is known to be one of the first signaling events after FcR engagement and requires tyrosine phosphorylation of the receptor (15, 48). The fact that the transmembrane mutant A224S, which fails to associate with lipid rafts, was tyrosine phosphorylated upon receptor cross-linking with a similar kinetics as the WT receptor (Fig. 7), suggests that, at least for initial signaling, Fc{gamma}RIIA does not need to associate with lipid rafts. However, this association may still be necessary for the receptor to convey further intracellular signaling. In accord with this hypothesis, activation of the transcription factor NF-{kappa}B upon Fc{gamma}RIIA cross-linking was completely dependent on DRM association of the receptor (Fig. 8). Moreover, the level of NF-{kappa}B activation was higher for the VA231-2MM and VVAL234-7GISF transmembrane mutants that associate more strongly to lipid rafts (Fig. 8). One possibility for this result is that while the A224S transmembrane mutant (which could not associate with DRM) did not activate ERK, the constitutively DRM-associated mutants, VA231-2MM and VVAL234-7GISF, induced a stronger activation of ERK (Fig. 9), and it is known that Fc{gamma}R-mediated activation of NF-{kappa}B depends on ERK activity (32). Thus, these data indicated that only lipid raft-associated Fc{gamma}RIIA variants were capable of inducing strong NF-{kappa}B activation through better activation of ERK.

Phagocytosis is another important cell response mediated by Fc{gamma}RIIA. We found that cells expressing the WT receptor presented efficient phagocytosis. Surprisingly, cells expressing the Fc{gamma}RIIA transmembrane mutant A224S, which does not associate with DRM, also presented efficient phagocytosis with no difference in kinetics or extent, compared with WT Fc{gamma}RIIA. This data strongly suggested that phagocytosis could proceed without the need for receptor association with lipid rafts. Supporting this idea is the fact that no accumulation of ganglioside GM1 could be found at phagocytic cups formed in RBL-2H3 cells expressing the A224S mutant (Fig. 10). However, we cannot rule out the possibility that a minimal association of A224S with lipid rafts, not detected by our methods, was still sufficient for phagocytosis, but not sufficient for NF-{kappa}B activation. Our data suggest that Fc{gamma}RIIA cross-linking can induce Syk activation and signaling to the rest of the phagocytic machinery, even when the receptor stays outside of DRM. A possible explanation for this result would be that Fc{gamma}RIIA is expressed at such high levels that cross-linking it outside DRM is enough for signaling. In contrast, it could still be possible that association of the receptor with DRM at very low levels is enough for phagocytosis signaling.

Compared with phagocytosis by the WT Fc{gamma}RIIA, phagocytosis by the constitutively lipid rafts-associated mutants VA231-2MM and VVAL234-7GISF occurred at a lower level. This apparent defect in the phagocytic process was not due to decreased avidity of these transmembrane receptor mutants (Fig. 12). The differences in phagocytic efficiency are likely the result of differential use of signaling enzymes for phagocytosis (49), for changes in the way the receptor associates with lipid rafts may result in assembly of different signaling complexes at the membrane.

In conclusion, this report identifies the TM of Fc{gamma}RIIA as responsible for mediating cross-linking-dependent association of the receptor with lipid rafts. Our data support the notion that immunoreceptor signaling can occur in the absence of lipid raft association to regulate some cell responses such as phagocytosis. Data also suggest that, in addition to being permissive for intracellular signaling leading to particular cell responses, lipid rafts can act as signaling modulators, depending on whether receptor-lipid raft association is inducible or constitutive.


   Acknowledgment
 
We thank Nancy Mora for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant 36407-M form Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico and by a grant from UC-MEXUS-CONACyT. Back

2 Address correspondence and reprint requests to Dr. Carlos Rosales, Department of Immunology, Instituto de Investigaciones Biomédicas-Universidad Nacional Autónoma de México, Apartado Postal 70228, Ciudad Universitaria, México D.F.-04510, Mexico. E-mail address: carosal{at}servidor.unam.mx Back

3 Abbreviations used in this paper: DRM, detergent-resistant membrane domain; TM, transmembrane domain; Cyt, cytoplasmic domain; WT, wild type; HA, hemagglutinin A; PVDF, polyvinylidene fluoride; MFI, mean fluorescence intensity; EC, extracellular domain. Back

Received for publication August 1, 2006. Accepted for publication December 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Fullekrug, J., K. Simons. 2004. Lipid rafts and apical membrane traffic. Ann. NY Acad. Sci. 1014: 164-169. [Medline]
  2. Rajendran, L., K. Simons. 2005. Lipid rafts and membrane dynamics. J. Cell Sci. 118: 1099-1102. [Free Full Text]
  3. Simons, K., E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387: 569-572. [Medline]
  4. Anderson, R. G., K. Jacobson. 2002. A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296: 1821-1825. [Abstract/Free Full Text]
  5. Munro, S.. 2003. Lipid rafts: elusive or illusive?. Cell 115: 377-388. [Medline]
  6. Pike, L. J.. 2004. Lipid rafts: heterogeneity on the high seas. Biochem. J. 378: 281-292. [Medline]
  7. Golub, T., S. Wacha, P. Caroni. 2004. Spatial and temporal control of signaling through lipid rafts. Curr. Opin. Neurobiol. 14: 542-550. [Medline]
  8. Taner, S. B., B. Onfelt, N. J. Pirinen, F. E. McCann, A. I. Magee, D. M. Davis. 2004. Control of immune responses by trafficking cell surface proteins, vesicles and lipid rafts to and from the immunological synapse. Traffic 5: 651-661. [Medline]
  9. Matko, J., J. Szollosi. 2002. Landing of immune receptors and signal proteins on lipid rafts: a safe way to be spatio-temporally coordinated?. Immunol. Lett. 82: 3-15. [Medline]
  10. Brown, D. A., E. London. 1998. Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 14: 111-136. [Medline]
  11. Bodin, S., C. Viala, A. Ragab, B. Payrastre. 2003. A critical role of lipid rafts in the organization of a key Fc{gamma}RIIa-mediated signaling pathway in human platelets. Thromb. Haemost. 89: 318-330. [Medline]
  12. Holowka, D., B. Baird. 2001. Fc{epsilon}RI as a paradigm for a lipid raft-dependent receptor in hematopoietic cells. Semin. Immunol. 13: 99-105. [Medline]
  13. Ravetch, J. V., S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19: 275-290. [Medline]
  14. Daeron, M.. 1997. Fc receptor biology. Annu. Rev. Immunol. 15: 203-234. [Medline]
  15. Garcia-Garcia, E., C. Rosales. 2002. Signal transduction during Fc receptor-mediated phagocytosis. J. Leukocyte Biol. 72: 1092-1108. [Abstract/Free Full Text]
  16. May, R. C., L. M. Machesky. 2001. Phagocytosis and the actin cytoskeleton. J. Cell Sci. 114: 1061-1077. [Abstract]
  17. Swanson, J. A., A. D. Hoppe. 2004. The coordination of signaling during Fc receptor-mediated phagocytosis. J. Leukocyte Biol. 76: 1093-1103. [Abstract/Free Full Text]
  18. Katsumata, O., M. Hara-Yokoyama, C. Sautes-Fridman, Y. Nagatsuka, T. Katada, Y. Hirabayashi, K. Shimizu, J. Fujita-Yoshigaki, H. Sugiya, S. Furuyama. 2001. Association of Fc{gamma}RII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation. J. Immunol. 167: 5814-5823. [Abstract/Free Full Text]
  19. Kwiatkowska, K., A. Sobota. 2001. The clustered Fc{gamma} receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner. Eur. J. Immunol. 31: 989-998. [Medline]
  20. Strzelecka-Kiliszek, A., M. Korzeniowski, K. Kwiatkowska, K. Mrozinska, A. Sobota. 2004. Activated Fc{gamma}RII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets. Mol. Membr. Biol. 21: 101-108. [Medline]
  21. Scheiffele, P., M. G. Roth, K. Simons. 1997. Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. EMBO J. 16: 5501-5508. [Medline]
  22. Zhang, J., A. Pekosz, R. A. Lamb. 2000. Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins. J. Virol. 74: 4634-4644. [Abstract/Free Full Text]
  23. Balamuth, F., J. L. Brogdon, K. Bottomly. 2004. CD4 raft association and signaling regulate molecular clustering at the immunological synapse site. J. Immunol. 172: 5887-5892. [Abstract/Free Full Text]
  24. Popik, W., T. M. Alce. 2004. CD4 receptor localized to non-raft membrane microdomains supports HIV-1 entry: identification of a novel raft localization marker in CD4. J. Biol. Chem. 279: 704-712. [Abstract/Free Full Text]
  25. Polyak, M. J., S. H. Tailor, J. P. Deans. 1998. Identification of a cytoplasmic region of CD20 required for its redistribution to a detergent-insoluble membrane compartment. J. Immunol. 161: 3242-3248. [Abstract/Free Full Text]
  26. Yamabhai, M., R. G. Anderson. 2002. Second cysteine-rich region of epidermal growth factor receptor contains targeting information for caveolae/rafts. J. Biol. Chem. 277: 24843-24846. [Abstract/Free Full Text]
  27. Walmsley, A. R., F. Zeng, N. M. Hooper. 2003. The N-terminal region of the prion protein ectodomain contains a lipid raft targeting determinant. J. Biol. Chem. 278: 37241-37248. [Abstract/Free Full Text]
  28. Field, K. A., D. Holowka, B. Baird. 1997. Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains. J. Biol. Chem. 272: 4276-4280. [Abstract/Free Full Text]
  29. Korzeniowski, M., K. Kwiatkowska, A. Sobota. 2003. Insights into the association of Fc{gamma}RII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution. Biochemistry 42: 5358-5367. [Medline]
  30. Barnes, N. C., M. S. Powell, H. M. Trist, A. L. Gavin, B. D. Wines, P. M. Hogarth. 2006. Raft localisation of Fc{gamma}RIIa and efficient signaling are dependent on palmitoylation of cysteine 208. Immunol. Lett. 104: 118-123. [Medline]
  31. Reyes-Reyes, M., N. Mora, A. Zentella, C. Rosales. 2001. Phosphatidylinositol 3-kinase mediates integrin-dependent NF-{kappa}B and MAPK activation through separate signaling pathways. J. Cell Sci. 114: 1579-1589. [Abstract]
  32. Sanchez-Mejorada, G., C. Rosales. 1998. Fc{gamma} receptor-mediated mitogen-activated protein kinase activation in monocytes is independent of Ras. J. Biol. Chem. 273: 27610-27619. [Abstract/Free Full Text]
  33. Melkonian, K. A., A. G. Ostermeyer, J. Z. Chen, M. G. Roth, D. A. Brown. 1999. Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts: many raft proteins are acylated, while few are prenylated. J. Biol. Chem. 274: 3910-3917. [Abstract/Free Full Text]
  34. Barker, S. A., K. K. Caldwell, A. Hall, A. M. Martinez, J. R. Pfeiffer, J. M. Oliver, B. S. Wilson. 1995. Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc{epsilon}R1 cross-linking. Mol. Biol. Cell 6: 1145-1158. [Abstract]
  35. Dbaibo, G. S., L. M. Obeid, Y. A. Hannun. 1993. Tumor necrosis factor-{alpha} (TNF-{alpha}) signal transduction through ceramide: dissociation of growth inhibitory effects of TNF-{alpha} from activation of nuclear factor-{kappa}B. J. Biol. Chem. 268: 17762-17766. [Abstract/Free Full Text]
  36. Cabedo, H., C. Luna, A. M. Fernandez, J. Gallar, A. Ferrer-Montiel. 2002. Molecular determinants of the sensory and motor neuron-derived factor insertion into plasma membrane. J. Biol. Chem. 277: 19905-19912. [Abstract/Free Full Text]
  37. Kono, H., T. Suzuki, K. Yamamoto, M. Okada, T. Yamamoto, Z. Honda. 2002. Spatial raft coalescence represents an initial step in Fc{gamma}R signaling. J. Immunol. 169: 193-203. [Abstract/Free Full Text]
  38. Barman, S., D. P. Nayak. 2000. Analysis of the transmembrane domain of influenza virus neuraminidase, a type II transmembrane glycoprotein, for apical sorting and raft association. J. Virol. 74: 6538-6545. [Abstract/Free Full Text]
  39. Gosse, J. A., A. Wagenknecht-Wiesner, D. Holowka, B. Baird. 2005. Transmembrane sequences are determinants of immunoreceptor signaling. J. Immunol. 175: 2123-2131. [Abstract/Free Full Text]
  40. Berton, G., A. Mocsai, C. A. Lowell. 2005. Src and Syk kinases: key regulators of phagocytic cell activation. Trends Immunol. 26: 208-214. [Medline]
  41. Turner, M., E. Schweighoffer, F. Colucci, J. P. Di Santo, V. L. Tybulewicz. 2000. Tyrosine kinase SYK: essential functions for immunoreceptor signalling. Immunol. Today 21: 148-154. [Medline]
  42. Strzelecka, A., K. Kwiatkowska, A. Sobota. 1997. Tyrosine phosphorylation and Fc{gamma} receptor-mediated phagocytosis. FEBS Lett. 400: 11-14. [Medline]
  43. Abdel Shakor, A. B., K. Kwiatkowska, A. Sobota. 2004. Cell surface ceramide generation precedes and controls Fc{gamma}RII clustering and phosphorylation in rafts. J. Biol. Chem. 279: 36778-36787. [Abstract/Free Full Text]
  44. Mansfield, P. J., V. Hinkovska-Galcheva, M. S. Borofsky, J. A. Shayman, L. A. Boxer. 2005. Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with Fc{gamma}RIIA. Biochem. Biophys. Res. Commun. 331: 132-138. [Medline]
  45. Hostager, B. S., I. M. Catlett, G. A. Bishop. 2000. Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling. J. Biol. Chem. 275: 15392-15398. [Abstract/Free Full Text]
  46. Floto, R. A., M. R. Clatworthy, K. R. Heilbronn, D. R. Rosner, P. A. MacAry, A. Rankin, P. J. Lehner, W. H. Ouwehand, J. M. Allen, N. A. Watkins, K. G. Smith. 2005. Loss of function of a lupus-associated Fc{gamma}RIIb polymorphism through exclusion from lipid rafts. Nat. Med. 11: 1056-1058. [Medline]
  47. Kono, H., C. Kyogoku, T. Suzuki, N. Tsuchiya, H. Honda, K. Yamamoto, K. Tokunaga, Z. Honda. 2005. Fc{gamma}RIIB Ile232Thr transmembrane polymorphism associated with human systemic lupus erythematosus decreases affinity to lipid rafts and attenuates inhibitory effects on B cell receptor signaling. Hum. Mol. Genet. 14: 2881-2892. [Abstract/Free Full Text]
  48. Sanchez-Mejorada, G., C. Rosales. 1998. Signal transduction by immunoglobulin Fc receptors. J. Leukocyte Biol. 63: 521-533. [Abstract]
  49. Garcia-Garcia, E., R. Rosales, C. Rosales. 2002. Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation. J. Leukocyte Biol. 72: 107-114. [Abstract/Free Full Text]
  50. Slimane, T. A., C. Lenoir, V. Bello, J. L. Delaunay, J. W. Goding, S. Chwetzoff, M. Maurice, J. A. Fransen, G. Trugnan. 2001. The cytoplasmic/transmembrane domain of dipeptidyl peptidase IV, a type II glycoprotein, contains an apical targeting signal that does not specifically interact with lipid rafts. Exp. Cell Res. 270: 45-55. [Medline]
  51. Zhang, M., M. Moran, J. Round, T. A. Low, V. P. Patel, T. Tomassian, J. D. Hernandez, M. C. Miceli. 2005. CD45 signals outside of lipid rafts to promote ERK activation, synaptic raft clustering, and IL-2 production. J. Immunol. 174: 1479-1490. [Abstract/Free Full Text]
  52. Dunbar, L. A., P. Aronson, M. J. Caplan. 2000. A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase. J. Cell Biol. 148: 769-778. [Abstract/Free Full Text]
  53. Meerson, N. R., V. Bello, J. L. Delaunay, T. A. Slimane, D. Delautier, C. Lenoir, G. Trugnan, M. Maurice. 2000. Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation. J. Cell Sci. 113: (Pt. 23):4193-4202. [Abstract]
  54. Zheng, X., D. Lu, J. E. Sadler. 1999. Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts. J. Biol. Chem. 274: 1596-1605. [Abstract/Free Full Text]
  55. Harder, T.. 2004. Lipid raft domains and protein networks in T-cell receptor signal transduction. Curr. Opin. Immunol. 16: 353-359. [Medline]
  56. Marzolo, M. P., M. I. Yuseff, C. Retamal, M. Donoso, F. Ezquer, P. Farfan, Y. Li, G. Bu. 2003. Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains. Traffic 4: 273-288. [Medline]
  57. Schubert, A. L., W. Schubert, D. C. Spray, M. P. Lisanti. 2002. Connexin family members target to lipid raft domains and interact with caveolin-1. Biochemistry 41: 5754-5764. [Medline]
  58. Bravo, A., I. Gomez, J. Conde, C. Munoz-Garay, J. Sanchez, R. Miranda, M. Zhuang, S. S. Gill, M. Soberon. 2004. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim. Biophys. Acta 1667: 38-46. [Medline]



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