The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, P. C.
Right arrow Articles by Pierce, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, P. C.
Right arrow Articles by Pierce, S. K.
The Journal of Immunology, 2001, 166: 3693-3701.
Copyright © 2001 by The American Association of Immunologists

Translocation of the B Cell Antigen Receptor into Lipid Rafts Reveals a Novel Step in Signaling1

Paul C. Cheng*, Bruce K. Brown{dagger}, Wenxia Song{dagger} and Susan K. Pierce2,{ddagger}

* Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208; {dagger} Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; and {ddagger} Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cross-linking of the B cell Ag receptor (BCR) leads to the initiation of a signal transduction cascade in which the earliest events involve the phosphorylation of the immunoreceptor tyrosine-based activation motifs of Ig{alpha} and Ig{beta} by the Src family kinase Lyn and association of the BCR with the actin cytoskeleton. However, the mechanism by which BCR cross-linking initiates the cascade remains obscure. In this study, using various A20-transfected cell lines, biochemical and genetic evidence is provided that BCR cross-linking leads to the translocation of the BCR into cholesterol- and sphingolipid-rich lipid rafts in a process that is independent of the initiation of BCR signaling and does not require the actin cytoskeleton. Translocation of the BCR into lipid rafts did not require the Ig{alpha}/Ig{beta} signaling complex, was not dependent on engagement of the FcR, and was not blocked by the Src family kinase inhibitor PP2 or the actin-depolymerizing agents cytochalasin D or latrunculin. Thus, cross-linking or oligomerization of the BCR induces the BCR translocation into lipid rafts, defining an event in B cell activation that precedes receptor phosphorylation and association with the actin cytoskeleton.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The initiation of Ab responses requires the binding of multivalent Ag to Ag-specific B cells. The B cell Ag receptor (BCR)3 on the cell surface acts as a sensor for Ag, discerning both the affinity and valency of the B cell for the Ag and setting thresholds for activation to the Ag (1, 2). The BCR is comprised of a membrane-bound surface Ig molecule that binds Ag. For IgM, the cytoplasmic tail is only three amino acids long, precluding the ability of Ig alone to initiate cytoplasmic signaling (3). To form a signaling receptor, Ig noncovalently associates with the heterodimer, Ig{alpha}/Ig{beta}, which contains immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domain (4). Upon Ag binding and receptor cross-linking, the BCR initiates a signal transduction cascade ending with the expression of a variety of genes associated with B cell activation (5). Subsequently, the BCR rapidly delivers Ag to intracellular compartments in which the Ag is proteolytically cleaved and the resulting peptides bound to MHC class II molecules for presentation to Th cells (6). The accelerated Ag-targeting function of the BCR is signaling dependent (7).

Although a great deal of the BCR signal transduction cascade has been detailed in recent years (3, 8), at present the molecular mechanisms underlying the initiation of BCR signaling and the relationship to Ag targeting are not well defined. One of the earliest events in BCR-mediated signal transduction is the activation of Lyn, a 53-/56-kDa signaling regulator that belongs to the Src family of protein tyrosine kinases (9). Lyn is constitutively acylated and consequently localizes to the plasma membrane. Lyn is responsible for the initial phosphorylation of the ITAMs of Ig{alpha} and Ig{beta} cytoplasmic domains. The kinase activity of Lyn is regulated by the phosphorylation of a carboxyl-terminal regulatory tyrosine by the kinase Csk (10) and dephosphorylation by the phosphatase CD45 (11, 12). Lyn is also activated by substrate binding to its Src homology 2 domain. Recent evidence indicates that Csk is recruited to the plasma membrane by a transmembrane phosphoprotein Cbp (Csk-binding protein) that binds specifically to the Src homology 2 domain of Csk (13). Syk kinase is recruited to the phosphorylated ITAMs of Ig{alpha} and Ig{beta}, and an activation loop tyrosine of Syk becomes phosphorylated either by Lyn or through autophosphorylation (14, 15). The activation of Syk then results in the induction of the calcium pathway by the recruitment of the B cell linker protein (BLNK) and the activation of phospholipase C-{gamma}2. Although these events are well documented to occur immediately upon BCR cross-linking and oligomerization, the primary initiating event in the signaling cascade triggered by receptor oligomerization is not known (3). At present, it is unclear whether signaling is induced by the activity of an as of yet unidentified signal transducer or simply the result of BCR oligomerization induced by multivalent Ag binding.

A previously unappreciated step in BCR-mediated signal transduction was recently described (16, 17, 18). Namely, upon Ag binding and receptor cross-linking, the BCR rapidly translocates from detergent-soluble plasma membrane, where the phosphatase CD45R is restricted, into detergent-insoluble cholesterol- and sphingolipid-rich lipid rafts, where Lyn is concentrated and Ig{alpha}/Ig{beta} are phosphorylated. From the rafts, the BCR delivers the Ag in an accelerated manner to degradative compartments for processing and presentation. Thus, cross-linking or oligomerizing the BCR results in a change in the BCR such that it preferentially localizes to the cholesterol- and sphingolipid-rich microdomains of the plasma membrane. Recent evidence indicates that BCR entry into lipid rafts is regulated during B cell development (19) and by viral infection by EBV (20), suggesting that rafts play important physiological roles as platforms for B cell signaling.

An appreciation of this early translocation event focuses attention on the transmembrane regions of the BCR Ig and Ig{alpha}/Ig{beta}, which initially reside in the detergent-soluble plasma membrane and following translocation must be accommodated by the specialized environment of the lipid rafts. The importance of the Ig transmembrane domain for BCR function, including Ig{alpha}/Ig{beta} association, signal transduction, and Ag presentation, was demonstrated by a series of heavy chain transmembrane mutants (21, 22, 23, 24). Mutations were introduced into adjacent tyrosine and serine residues in the transmembrane domain of a human µ-chain, which are predicted to face the lower leaflet of the lipid bilayer. A mutant with a Y587->F change associated with Ig{alpha}/Ig{beta} and resulted in a signaling-competent BCR that could be internalized, yet the Y/F mutant BCR could not deliver Ag for MHC class II processing and presentation. Because correct Ag targeting for processing is dependent on BCR signaling (7), it may be assumed that the qualitative or quantitative nature of the signal initiated from the Y/F BCR may not be identical to that of the WT BCR. Additionally, a mutant Ig with a Y587S588->VV change no longer associated with Ig{alpha}/Ig{beta} and was signaling incompetent, but the mutant could still be internalized upon Ag binding. However, like the Y/F BCR, the YS/VV mutant could not deliver Ag for presentation. These transmembrane mutants provided an opportunity to assess the requirement of the Ig{alpha}/Ig{beta} signaling complex in translocation of the BCR into lipid rafts. The results presented in this work provide evidence that the initial event in BCR signaling is the translocation of the BCR into lipid rafts, and that this event is not dependent on phosphorylation of Ig{alpha}/Ig{beta} or the attachment of the BCR to the actin cytoskeleton.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

Cell lines of the mouse B cell lymphoma A20 expressing an IgG2a mouse BCR (H-2d, Fc{gamma}RIIB1+) stably transfected with human µ-chain either with a wild-type transmembrane region (WT) or containing transmembrane mutations Y587->F (Y/F) or Y587S588->VV (YS/VV) (21) were maintained in supplemented DMEM and 15% FCS (15% complete medium (CM)) (25) containing 600 µg/ml G418. Expression of the human µ-chain by the mutant cell lines was confirmed by both genomic PCR sequencing and flow cytometry using FITC-labeled goat anti-human µ-chain. The mean fluorescence intensity for WT was 400, for Y/F was 300, and for YS/VV was 200. A20IIA1.6 cells that lack FcR were provided by Dr. John Cambier (National Jewish Hospital, Denver, CO). Hen egg lysozyme and p-aminophenylphosphorylcholine were purchased from Sigma (St. Louis, MO), and chemically conjugated, as previously described (21). Whole and F(ab')2 rabbit Abs specific for human Fc5 µ (anti-µ); whole and F(ab')2 rabbit Abs specific for mouse IgG and IgM heavy and light chains (anti-mouse Ig); and HRP-labeled goat Abs specific for human Fc5 µ (HRP anti-µ) and rabbit IgG (HRP anti-rabbit Ig) were purchased from Jackson ImmunoResearch (West Grove, PA). RC20H phosphotyrosine-specific recombinant mAb conjugated to HRP was purchased from Transduction Laboratories (San Diego, CA). Actin-depolymerizing agent cytochalasin D was purchased from Sigma, and the Src family kinase inhibitor PP2 from Calbiochem (La Jolla, CA).

Pervanadate treatment

Cells were stimulated with pervanadate, as previously described (26). Briefly, cells (1 x 108) were preincubated for 30 min with DMEM-BSA at 4°C and treated with a final concentration of 750 µM sodium orthovanadate and 0.075% hydrogen peroxide (Sigma) for 1 min. Cells were lysed and lipid rafts were isolated, as described below.

Isolation of lipid rafts

Lipid rafts were isolated by lysis of cells in 1% Triton X-100 at 4°C and flotation on a discontinuous sucrose gradient, as previously described (16). Briefly, 1 x 108 cells per sample were washed with ice-cold PBS and lysed for 30 min on ice in 1% Triton X-100 in TNEV (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA; CLAP, 2.5 mg/ml each of chymostatin, leupeptin, antipain, and pepstatin A in DMSO; and 1 mM sodium orthovanadate). Nuclei and cellular debris were pelleted by centrifugation at 900 x g for 10 min, and 1 ml of cleared supernatant was mixed with 1 ml of 85% sucrose in a Beckman 14 x 89-mm centrifuge tube (Fullerton, CA). The sample was overlaid with 6 ml 35% sucrose in TNEV and finally 3.5 ml 5% sucrose in TNEV. The samples were centrifuged in a SW41 rotor at 200,000 x g for 16–20 h at 4°C, and 1-ml fractions were collected from the top of the gradient. Fractions 3–5 were pooled as the raft-containing fractions, and fractions 10–12 were pooled as the soluble fractions. Isolation of lipid raft membranes was confirmed by immunoblotting for GM1, Lyn, and CD45R, as described previously (16).

Actin depolymerization

Cells were incubated with 10 µM cytochalasin D (Sigma, St. Louis, MO) at 4°C, or latrunculin B (Calbiochem, San Diego, CA) (27, 28) for 30 min at 37°C to disrupt actin filaments. Cells were then chilled on ice for 10 min, and incubated with rabbit anti-human IgM, Fc5 µ (Jackson ImmunoResearch) for 30 min, and Cy3-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) for an additional 30 min. An aliquot of the cells was stained for trypan blue to ensure viability greater than 90%, and the samples were processed for lipid raft isolation, as described above.

For immunofluorescence microscopy, the cells were then chilled on ice for 10 min, and incubated with rabbit anti-human IgM, Fc5 µ (Jackson ImmunoResearch) for 30 min, and Cy3-conjugated goat anti-rabbit IgG (H+L; Jackson ImmunoResearch) for an additional 30 min. The cells were then fixed with 4% paraformaldehyde for 20 min at room temperature. After fixation, cells were incubated with permeabilization buffer (1% gelatin, 0.05% saponin, 10 mM glycine, 10 mM HEPES) for 15 min. The cells were incubated with FITC-conjugated phalloidin (FL-phalloidin) (Molecular Probes, Eugene, OR) in permeabilization buffer for 30 min, washed, and mounted with Gel/mount (Biomeda, Foster City, CA) for viewing. Imaging was conducted on a scanning laser confocal microscope (Bio-Rad, Richmond, CA; model MRC-1024). Images were acquired using a x60 objective, and cropped using Photoshop (Adobe, Mountain View, CA).

Measurement of HRP activity

An aliquot (100 µl) of each pooled fraction from the discontinuous sucrose gradient was incubated with 100 µl of substrate solution (5 mg/ml 2, 2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid in 0.1 M citrate-phosphate buffer, pH 4, with 0.015% H2O2). The absorbance was measured at 405 nm on an ELISA plate reader.

Immunoblotting

Samples were subjected to 10% SDS-PAGE and transferred onto Millipore Immobilon polyvinylidene difluoride membrane (Bedford, MA). The membranes were blocked for 1 h at 25°C in PBS/0.1% Tween 20 and 1% BSA and then probed with RC20H at a 1/2500 dilution in the same blocking buffer to detect phosphotyrosine-containing proteins. The blots were washed in PBS/0.1% Tween-20 (wash buffer) and visualized with ECL (Amersham-Pharmacia, Piscataway, NJ). The membranes were then rinsed in wash buffer and placed into BLOTTO containing 0.02% sodium azide for 30 min at 25°C, which inactivates HRP activity. The blots were then washed to eliminate the azide, reprobed with HRP goat anti-human µ, and visualized with ECL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The signaling and translocation properties of the Ig transmembrane mutants Y/F and YS/VV

Previous studies showed that following cross-linking, the BCR rapidly translocates into lipid rafts, an event that is accompanied by initiation of protein tyrosine phosphorylation of raft-associated proteins (16). To determine the requirement of BCR signaling for raft translocation, two BCRs were analyzed that contain mutations in the transmembrane regions: Y/F, a signaling-competent Ig that associates with Ig{alpha}/Ig{beta} but fails to target Ag for processing, and YS/VV, a signaling-incompetent Ig that fails to associate with Ig{alpha}/Ig{beta} and also fails to target Ag. The behavior of the mutants Y/F and YS/VV and the WT BCR following Ag binding were compared in terms of their induction of protein tyrosine phosphorylation and translocation into lipid rafts.

Cells were incubated on ice with whole rabbit anti-µ for 30 min, washed, and then incubated with HRP anti-rabbit Ig for an additional 30 min. The HRP anti-Ig served as a secondary cross-linking reagent for the BCR and allowed detection of the cross-linked BCR by assay for HRP enzyme activity. The cells were solubilized in 1% Triton X-100 lysis buffer, and the rafts were isolated on sucrose density gradients. Cross-linking the BCR resulted in the induction of protein tyrosine phosphorylation in the lipid rafts in both the Y/F and WT cells (Fig. 1GoA). Moreover, the protein phosphotyrosine pattern in the Y/F rafts closely resembled that of the WT cells. The protein phosphotyrosine patterns of the Y/F and WT cells were also similar in the soluble fractions. In contrast, the cross-linking of YS/VV did not result in significant protein phosphorylation.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. The mutant Y/F is translocation competent. WT, YS/VV, and Y/F cells were incubated with media alone (cross-linking, -) or with rabbit anti-human µ-chain (20 µg/ml; cross-linking, +) for 30 min at 4°C, washed, and then incubated with HRP-labeled goat anti-rabbit Ig (20 µg/ml) for an additional 30 min at 4°C. Cells were lysed, and detergent-insoluble membranes were isolated on a sucrose density gradient. A, Fractions were subjected to SDS-PAGE and immunoblotting probing for tyrosine-phosphorylated proteins. No bands were observed in immunoblots treated with ECL alone in the absence of phosphotyrosine-specific Ab probe. The fractions containing lipid rafts were confirmed by immunoblotting for GM1, Lyn, and CD45R (data not shown). B, To determine the location of the BCR, the blots were reprobed for human µ-chain, and the bands representing the mature Ig form were quantified from three independent experiments. C, The location of the HRP anti-Ig was determined by enzyme assays for HRP activity from three independent experiments.

 
The location of the ligated BCR in the rafts was determined both by immunoblotting the raft fractions probing for the BCR µ-chain and by colorimetric assay for the presence of HRP activity (Fig. 1Go, B and C). Two µ bands were resolved in the immunoblots, which most likely represent two different glycosylated forms of the µ-chain that we refer to as the mature and immature forms. Both µ-chains have been previously shown to be present on the B cell surface by surface biotinylation (29). Cross-linking the BCR resulted in significant translocation of the upper µ band of the BCR into the lipid rafts in both the Y/F and WT cells, as shown by immunoblotting of the raft fractions (Fig. 1GoB). However, there was no detectable translocation of the BCR in YS/VV cells into the lipid rafts. The inability to detect translocation of the BCR into raft in the YS/VV cells is not due to differences in the amount of BCR expressed on YS/VV cells, as all three cell lines express similar amounts of BCR detected by immunoblotting of the soluble fractions and flow cytometry (data not shown). The lower m.w. µ band appears to be constitutively present in rafts to varying degrees, and cross-linking the BCR does not induce changes in its location. The identity and potential function of this band remain to be elucidated. The translocation of the BCR in both the WT and Y/F cells following cross-linking was also observed by measuring the HRP activity of the ligated BCR (Fig. 1GoC). In contrast, no translocation of the BCR in the YS/VV cells was detected.

The temperature dependence of BCR-induced tyrosine phosphorylation and raft translocation

The results presented above indicated that the BCR in Y/F and WT cells, but not in YS/VV, was both signaling and translocation competent. It was of interest to determine whether signaling or translocation was affected by temperature, which would affect the fluidity of the membrane. Cells were incubated with the BCR cross-linking Abs at 4°C, washed, and warmed for 2 min to 37°C. Isolation and immunoblot comparison of the raft and detergent-soluble fractions from WT and Y/F cells revealed that maximal tyrosine phosphorylation within the rafts and BCR raft translocation occurs at 4°C, and that even 2 min following warming to 37°C, less BCR was detected in the rafts and protein tyrosine phosphorylation within the rafts was reduced (Fig. 2Go). Thus, maximal translocation and stable residency within the rafts occurred at 4°C for the WT and Y/F BCR.



View larger version (74K):
[in this window]
[in a new window]
  		FIGURE 2. Protein tyrosine phosphorylation and BCR raft translocation occur maximally at 4°C in WT and Y/F cells. WT and Y/F cells were incubated in media alone (0-) or whole rabbit anti-human µ (20 µg/ml) for 30 min at 4°C, washed, and incubated with HRP-labeled goat anti-rabbit Ig (20 µg/ml) for an additional 30 min at 4°C. The cells were washed and resuspended in either ice-cold media (0+) or media prewarmed to 37°C for 2 min (2+). The cells were pelleted at 4°C and lysed in Triton X-100 lysis buffer, and the raft fractions were isolated on a discontinuous sucrose gradient. The fractions were subjected to SDS-PAGE and immunoblotting, sequentially probing for phosphotyrosine (upper) and human µ-chain (lower).

 
The effect of temperature on the behavior of the YS/VV BCR was also analyzed. The YS/VV cells were incubated with the BCR cross-linking Abs at 4°C and warmed to 37°C for up to 10 min. At the end of each time point, the cells were lysed, and the raft and soluble fractions isolated and analyzed for tyrosine-phosphorylated proteins and the BCR. The YS/VV rafts showed insignificant increases in the levels of phosphoproteins upon warming to 37°C, and a constitutively phosphorylated band at 80–90 kDa decreased with time (Fig. 3GoA). The YS/VV mature µ-chain, however, was detected by immunoblotting in the lipid rafts at 2 min after warming and remained in the raft fractions even at 10 min (Fig. 3GoB). The translocation of the YS/VV BCR into the rafts was also detected by measuring the HRP activity of the HRP anti-Ig. The HRP activity increased 50% in raft fractions and plateaued upon warming for 2–10 min (Fig. 3GoC). To determine the temperature for maximal raft translocation of the YS/VV BCR, the cells were incubated at 4°C with the BCR cross-linking Abs and warmed to various temperatures up to 37°C for 2 min. By both immunoblotting and HRP activity, translocation of the YS/VV into rafts was detected at 20°C and reached maximal levels at 30°C (Fig. 3Go, E and F). Significantly, the translocation of the YS/VV into the rafts was not accompanied by tyrosine phosphorylation in the raft region at any temperature (Fig. 3GoD). These results indicate that BCR signaling and Ig{alpha}/Ig{beta} association are not required for translocation into lipid rafts. However, in the absence of the associated Ig{alpha}/Ig{beta} signal transduction complex, the BCR requires higher temperatures and presumably a more fluid bilayer to translocate into the rafts.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3. Raft translocation of the YS/VV mutant is temperature dependent and signaling independent. YS/VV cells were incubated with media alone (-) or whole rabbit anti-human µ-chain (20 µg/ml) (+) for 30 min at 4°C, washed, and incubated with HRP-labeled goat anti-rabbit Ig (20 µg/ml) for an additional 30 min at 4°C. Cells were washed and then resuspended at 37°C in DME-BSA for either 0, 2, 5, or 10 min (A, B, and C), or the cells were resuspended for 2 min in media prewarmed to 4, 10, 20, 30, or 37°C (D, E, and F). Cells were lysed in 1% Triton X-100 lysis buffer, and lipid rafts were isolated on discontinuous sucrose density gradients. The gradient fractions were subjected to SDS-PAGE and immunoblotting probing for phosphotyrosine (A and D). To determine the location of the BCR, the immunoblots were reprobed for human µ-chain, and the bands representing the mature Ig were quantified from two independent experiments (B and E). The location of the HRP anti-Ig was determined by assays for HRP enzyme activity (C and F).

 
BCR translocation into rafts is independent of the activity of Src kinases and the actin cytoskeleton

The observation that YS/VV translocates into lipid rafts following cross-linking at 37°C strongly suggests that the level of protein tyrosine phosphorylation in the raft regions that accompanies translocation of the WT and Y/F BCR into lipid rafts is not required for translocation. To further explore the requirement for protein phosphorylation, the effect of the Src family kinase inhibitor PP2 (30) on the translocation of the WT and the Y/F BCR into lipid rafts was assessed. Initial titration experiments determined that 50 µM PP2 reduced WT BCR-induced protein tyrosine phosphorylation to undetectable levels by immunoblot probing with RC20H (data not shown). Thus, WT and Y/F cells were incubated with 50 µM PP2 from 30 min before the addition of BCR cross-linking Abs. Cells were solubilized in Triton X-100 detergent, and the rafts were isolated on sucrose density gradients. Incubation of both WT and Y/F cells with PP2 abolished tyrosine phosphorylation in the raft and detergent-soluble fractions (Fig. 4GoA). Significantly, the presence of PP2 did not block the translocation of the WT or Y/F BCR into the lipid rafts as measured by immunoblotting for the µ-chain (Fig. 4GoB) and by HRP activity (Fig. 4GoC), indicating that tyrosine phosphorylation of proteins in the raft was not essential for translocation. Treatment with PP2 decreases the amount of BCR stably associating with the rafts, which may indicate a need for Src family kinase in retaining the translocated BCR in the rafts. As shown above, oligomerization of the YS/VV BCR yielded insignificant levels of tyrosine phosphorylation. Upon treatment with PP2, no detectable phosphoproteins were observed following BCR cross-linking in YS/VV cells (Fig. 4GoA), even upon long exposures to film (data not shown). However, the YS/VV BCR still translocated into the lipid rafts at 37°C even in the presence of PP2 (Fig. 4Go, B and C). Thus, signaling as measured by protein tyrosine phosphorylation was not necessary for translocation of the BCR into lipid rafts following oligomerization. Moreover, induction of tyrosine phosphorylation of the Ig{alpha}/Ig{beta} ITAMs and other phosphoproteins by treatment of WT, Y/F, and YS/VV cells with pervanadate was not sufficient to result in BCR translocation in the absence of BCR cross-linking (Fig. 4GoC).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. Translocation of the BCR into lipid rafts does not require Src family kinase activity. WT, Y/F, and YS/VV cells were incubated in media alone (-), or whole rabbit anti-human µ-chain (20 µg/ml) (+) for 30 min at 4°C, washed, and incubated with HRP-labeled goat anti-rabbit Ig (20 µg/ml) for an additional 30 min at 4°C. YS/VV cells were warmed to 37°C for 2 min (2+). Cells were also treated with the kinase inhibitor PP2 (50 µM) 30 min before the addition of anti-Ig (P), or treated with pervanadate (750 µM Na3VO4 and 0.075% H2O2) for 1 min (V). Cells were lysed in 1% Triton X-100 lysis buffer, and lipid rafts were isolated on discontinuous sucrose density gradients. A, The gradient fractions were subjected to SDS-PAGE and immunoblotting probing for phosphotyrosine. To determine the location of the BCR and HRP anti-Ig, the immunoblots were reprobed for human µ-chain (B), and HRP activity was determined for three independent experiments (C).

 
Cross-linking the BCR results in rapid association with the actin cytoskeleton (31). Thus, it was of interest to determine whether association with the actin cytoskeleton was necessary for translocation into lipid rafts. WT, Y/F, and YS/VV cells were pretreated with the actin-depolymerizing drug, cytochalasin D, at 4°C for 30 min before BCR cross-linking. Treatment with cytochalasin D had no effect on translocation of the BCR into lipid rafts (Fig. 5GoA) even with pretreatment at 37°C for 30 min (data not shown). Cytochalasin D treatment causes disorganization of the actin cytoskeleton, but does not result in dispersion of all actin filaments. To further explore the requirement for actin in BCR translocation into rafts, a second inhibitor that causes depolymerization of actin filaments, latrunculin (27, 28), was used to treat cells and compared with the effect of cytochalasin D on BCR translocation. The WT, Y/F, and YS/VV cells were incubated at 37°C with latrunculin, the BCR was cross-linked, and the position of the BCR in the raft regions of the plasma membrane was determined. Latrunculin was unable to block BCR translocation into lipid rafts for WT, Y/F, or YS/VV (Fig. 5GoB). Cytochalasin D treatment had no detectable effect on the association of the BCR with the rafts, while latrunculin treatment reduced somewhat the amount of BCR stably associated with the rafts. Latrunculin treatment resulted in a complete dispersion of the actin cytoskeleton, as measured by fluorescent microscopy using FITC-labeled phalloidin (Fig. 5GoC). Thus, the complete dissociation of the actin cytoskeleton did not block BCR translocation into lipid rafts, although the stable association of the BCR with lipid rafts may be affected.



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 5. Dispersion of the actin cytoskeleton does not block translocation of the BCR into lipid rafts. A WT, Y/F, and YS/VV cells were incubated in media alone (-), or whole rabbit anti-human µ-chain (20 µg/ml) for 30 min at 4°C, washed, and incubated with HRP-labeled goat anti-rabbit Ig (20 µg/ml) for an additional 30 min at 4°C (+). YS/VV cells were then warmed to 37°C for 2 min (2+). Cells treated to cross-link the BCR were also treated with the inhibitor cytochalasin D (c) (10 µM) at 4°C for 30 min before cross-linking the BCR, which causes the depolymerization of the actin cytoskeleton. Cells were lysed in 1% Triton X-100 lysis buffer, and lipid rafts were isolated on discontinuous sucrose density gradients. The fractions from the gradient were subjected to SDS-PAGE and immunoblotting probing for human µ-chain. B The WT, Y/F, and YS/VV cells were either untreated (-) or treated to cross-link the BCR (+) as in A. The experiments were repeated with the actin-depolymerizing agent latrunculin (L) at 37°C for 30 min. C The effect of treatment with latrunculin on the actin cytoskeleton was determined by fluorescent microscopy staining for actin using FL-phalloidin and for the BCR using rhodamine anti-Ig. Shown are fluorescent micrographs of WT, Y/F, and YS/VV cells untreated (-LA) or latrunculin treated (+LA).

 
Translocation of the BCR into lipid rafts is independent of Fc{gamma}RIIB

The results presented above indicate that the BCR in Y/F and YS/VV cells have the potential to translocate into lipid rafts upon cross-linking. The cross-linking was accomplished using rabbit Abs that have the ability to interact with the Fc{gamma}RIIB on the cells. To ensure that BCR translocation following cross-linking was not dependent on FcR function, translocation of an endogenous mouse BCR (IgG2a) was analyzed in A20IIA1.6 cells lacking the Fc{gamma}RIIB. Cells were incubated on ice with either whole or F(ab')2 rabbit anti-mouse Ig, washed, and incubated with goat anti-rabbit Ig. Both F(ab')2 and whole Ig used as primary cross-linking reagents resulted in similar levels of tyrosine phosphorylation of proteins in the rafts and soluble membranes (Fig. 6GoA). The whole Ig was, however, more effective in inducing translocation as compared with the F(ab')2, presumably because it allowed more extensive cross-linking of the BCR (Fig. 6GoB). These results indicate that translocation of the BCR occurs independently of the Fc{gamma}RIIB. To directly rule out an effect of the Fc{gamma}RIIB on the translocation of the WT and Y/F BCR into lipid rafts, cells were preincubated with the FcR-specific mAb 2.4G2 that blocks Fc binding to Fc{gamma}RIIB before the addition of HRP F(ab')2 anti-Ig or HRP anti-Ig. The presence of the 2.4G2 mAb before cross-linking had no significant effect on the raft translocation of the WT or Y/F BCR (Fig. 6GoC).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 6. Translocation of endogenous mouse BCR into lipid rafts occurs independently of the Fc{gamma}RIIB. A20IIA1.6 cells that lack the Fc{gamma}RIIB were incubated in media alone (-) or in media containing F(ab')2 rabbit anti-mouse Ig (10 µg/ml; F) or whole rabbit anti-mouse Ig (20 µg/ml; W) for 30 min at 4°C, washed, and incubated with HRP-labeled goat anti-rabbit Ig (20 µg/ml) for an additional 30 min. Cells were lysed, and detergent-insoluble membranes were isolated on a sucrose density gradient. A, The fractions were subjected to SDS-PAGE and immunoblotting probing for phosphotyrosines. B, To determine the location of the mouse BCR and Ag, the immunoblots were reprobed for mouse {gamma}-chain, and the level of HRP activity was determined for the gradient fractions. C, In addition, WT and Y/F cells were incubated with mAb 2.4G2, which blocks the Fc{gamma}RIIB for 30 min before cross-linking, and the HRP activity of the gradient fractions was measured.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Upon cross-linking by multivalent Ag, the BCR complex functions to initiate transcription of genes involved in B cell activation. It was recently shown that Ag binding and oligomerization of the BCR result in rapid translocation of the BCR into detergent-insoluble membrane microdomains enriched in cholesterol and sphingolipids, termed lipid rafts (16, 17, 18). The lipid rafts are thought to be platforms for signaling, as Lyn kinase is concentrated within raft regions, while the phosphatase CD45 is excluded. Consistent with a role for lipid rafts in signal transduction, our recent results (16) and that of others (17, 18, 32) showed that both the BCR and Lyn within lipid rafts are phosphorylated in cells in which the BCR has been cross-linked. Translocation of the BCR into lipid rafts has also been shown to result in the recruitment of several components of the BCR signal cascade, including BLNK, phosphatidylinositol 3 kinase, VAV, and Btk (17, 18, 32). Evidence that the rafts play important physiological roles in B cell activation has been provided by the recent observations that BCR access to rafts is controlled during B cell development (19), by viral infection (20), and by the essential B cell coreceptor CD19 (Cherukuri, Sohn, and Pierce, unpublished observations). Thus, rafts appear to play key roles in the initiation and regulation of BCR signaling.

At present, Lyn phosphorylation of the Ig{alpha}/Ig{beta} ITAMs is the earliest signaling event known. However, it is unclear how the cross-linking of the BCR results in phosphorylation of the Ig{alpha}/Ig{beta} complex and the initiation of the signal transduction cascade. An intriguing possibility proposed for the high affinity IgE receptor (Fc{epsilon}R) (33) is that Fc{epsilon}R translocation into lipid rafts is the initiating event in the Fc{epsilon}R signal transduction cascade. The results presented in this work show that BCR translocation into lipid rafts occurs when protein tyrosine phosphorylation is inhibited and does not require the association of Ig with Ig{alpha}/Ig{beta}. Thus, BCR translocation occurs independently of signaling or biochemical or conformational changes in Ig{alpha}/Ig{beta}. These results are consistent with the model for the initiation of BCR signaling in which signaling is a repercussion of BCR translocation into a signaling platform enriched with Lyn, where the Ig{alpha}/Ig{beta} ITAMs become phosphorylated and Syk is recruited to the phosphorylated ITAMs to continue the signaling cascade.

The model predicts that Ag binding by the BCR would initiate oligomerization of the BCR, resulting in a conformation of the oligomerized BCR that prefers the lipid environment of the rafts. Thus, the initiation of the BCR signaling pathway would be solely dependent on the ability of Ag binding by the BCR to achieve the appropriate conformation of the oligomer that would lead to BCR translocation. Presumably, the key factors in achieving this conformation would be the affinity of the BCR for the Ag and the valency of the Ag as well as the spatial array of the BCR antigenic determinants on the Ag. In this regard, the initiating event in the BCR signaling pathway would be simply an Ag-sensing event. Indeed, recent unpublished results showed that the translocation of the BCR into lipid rafts was sensitive to the degree of cross-linking achieved is consistent with this model.

Our earlier results (16) and those presented in this work suggest that the initiation of the kinase-dependent BCR signaling cascade is restricted to lipid rafts. However, it is not known whether encounter of the BCR with Lyn outside the microdomains might also initiate signaling. While CD45 phosphatase is localized in the detergent-soluble membrane fractions and is believed to play a role in the activation of Lyn by dephosphorylating the carboxyl phosphotyrosine of Lyn, the paucity of Lyn in the soluble fractions suggests that the initiation of signal transduction is more likely to occur within lipid rafts. It has been found recently that a key Lyn-regulatory protein, Cbp, exists in lipid rafts as an acylated transmembrane phosphoprotein (13). The phosphorylation of the cytoplasmic Y314 of Cbp results in Csk binding, thereby recruiting Csk to the lipid rafts. Csk is responsible for the suppression of Lyn activity by phosphorylating the carboxyl tyrosine of Lyn (10, 11). Under steady state conditions, Cbp is basally phosphorylated, suggesting a low level of Csk is localized to the rafts to keep Lyn quiescent. Upon BCR cross-linking, the Ig and Ig{alpha}/Ig{beta} enter into the lipid rafts, providing the ITAMs as substrates for kinase binding and phosphorylation. Full activation of the Src family kinases results in further phosphorylation of Cbp, which recruits more Csk to the rafts, thus providing a mechanism by which signaling can be terminated. Taken together, these findings suggest that all the machinery necessary for the initiation and termination of BCR signaling may be concentrated in the lipid rafts.

Although the molecular forces driving raft localization of integral membrane proteins are unclear, previous studies of influenza membrane protein hemagglutinin (HA) show that the transmembrane region of HA, particularly the hydrophobic amino acid residues in the exoplasmic half of the plasma membrane, is crucial for its ability to localize in lipid rafts (34). HA is constitutively localized in raft regions; however, the BCR and other immune receptors translocate into rafts only upon aggregation, suggesting that the HA transmembrane domain inherently prefers the raft lipid environment, while the BCR must undergo an oligomerization-dependent change to allow stable association with the rafts. Presumably, the nature of the BCR transmembrane domain dictates whether the appropriate conformation of the oligomer can be achieved such that translocation occurs. Clearly, not all integral membrane proteins when cross-linked translocate into lipid rafts. Studies using domain swapping in chimeric receptors revealed that the transmembrane regions of the Fc{epsilon}R {alpha}-chain and the IL-2R allowed for raft translocation upon aggregation, although the transmembrane region of the IL-1R did not (35). To date, analysis of the transmembrane amino acid sequences of the molecules capable of localizing to raft domains has not revealed any motifs or patterns that would predict raft translocation. A conformational change that occurs upon ligand binding, or summation of the partition capabilities of individual transmembrane regions upon oligomerization are other possible mechanisms of raft translocation (36).

In this study, we used Ig transmembrane mutants to demonstrate that raft translocation depended on the Ig transmembrane region and not on Ig{alpha}/Ig{beta} association or their tyrosine phosphorylation. The Ig mutants contained amino acid substitutions facing the lower leaflet of the lipid bilayer that differed in their association with Ig{alpha}/Ig{beta} and signaling (21, 22, 23). A YS to VV double substitution resulted in a BCR lacking Ig{alpha}/Ig{beta} that was essentially incapable of raft translocation under conditions in which the WT and Y/F BCR were induced to translocate. The failure to translocate was overcome for the YS/VV BCR by increasing the temperature. How the change in temperature allowed translocation of the YS/VV BCR into lipid rafts remains to be determined.

Previous biochemical studies showed that within 30 s of cross-linking at 37°C, nearly 100% of the YS/VV BCR expressed in J558L plasmacytoma cells became cytoskeletally attached, as defined by insolubility in Nonidet P-40 detergent (31). The results presented in this work reveal that the integrity of the actin cytoskeleton does not affect BCR translocation into detergent-insoluble lipid domains. Pretreatment of cells with cytochalasin D or latrunculin and presence of the inhibition throughout the experiment did not block BCR raft translocation. It was recently demonstrated that for the Fc{epsilon}R, aggregation at 4°C and actin disruption with cytochalasin D at 22°C resulted in increased colocalization of the receptor with Lyn, as determined by immunofluorescence microscopy, and increased protein tyrosine phosphorylation (37). Thus, the available evidence suggests BCR translocation into lipid rafts precedes any involvement of the actin cytoskeleton. Our previous studies showed that actin is associated with B cell lipid rafts (16). Lipid rafts may facilitate the attachment of the BCR to the actin cytoskeleton and the subsequent internalization and rapid delivery of the BCR to the MHC class II peptide-loading compartment, as previously described (29, 38, 39).


   Acknowledgments
 
We thank Peter Yoon and Michael R. Chen for their technical assistance.


   Footnotes
 
1 This work is supported by grants from the National Institute of Allergy and Infectious Diseases (AI 27957, AI 18939, and AI 40309, to S.K.P). W.S. is supported by National Institute of Allergy and Infectious Diseases Grant AI42093. P.C.C. is supported by a National Institute of General Medical Sciences Medical Scientist Training Program Fellowship GM08152. Back

2 Address correspondence and reprint requests to Dr. Susan K. Pierce, National Institute of Allergy and Infectious Diseases/National Institutes of Health/Twinbrook II, 12441 Parklawn Drive, Room 200B, MSC 8180, Rockville, MD 20852-8180. Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; Cbp, Csk-binding protein; HA, hemagglutinin; ITAM, immunoreceptor tyrosine-based activation motif; WT, wild type. Back

Received for publication June 28, 2000. Accepted for publication January 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kouskoff, V., S. Famiglietti, G. Lacaud, P. Lang, J. E. Rider, B. K. Kay, J. C. Cambier, D. Nemazee. 2000. Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses. J. Exp. Med. 188:1453.[Abstract/Free Full Text]
  2. Batista, F. D., M. S. Neuberger. 1998. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8:751.[Medline]
  3. Reth, M., J. Wienands. 1997. Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15:453.[Medline]
  4. Cambier, J. C., C. M. Pleiman, M. R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12:457.[Medline]
  5. DeFranco, A. L.. 1997. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9:296.[Medline]
  6. Watts, C.. 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev. Immunol. 15:821.[Medline]
  7. Wagle, N. M., P. Cheng, J. Kim, T. W. Sproul, K. D. Kausch, S. K. Pierce. 1999. B lymphocyte signaling receptors and the control of class II antigen processing. Curr. Top. Microbiol. Immunol. 245:106.
  8. Kurosaki, T.. 1999. Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17:555.[Medline]
  9. Saouaf, S. J., S. Mahajan, R. B. Rowley, S. A. Kut, J. Fargnoli, A. L. Burkhardt, S. Tsukada, O. N. Witte, J. B. Bolen. 1994. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement. Proc. Natl. Acad. Sci. USA 91:9524.[Abstract/Free Full Text]
  10. Hata, A., H. Sabe, T. Kurosaki, M. Takata, H. Hanafusa. 1994. Functional analysis of Csk in signal transduction through the B-cell antigen receptor. Mol. Cell. Biol. 14:7306.[Abstract/Free Full Text]
  11. Yanagi, S., H. Sugawara, M. Kurosaki, H. Sabe, H. Yamamura, T. Kurosaki. 1996. CD45 modulates phosphorylation of both autophosphorylation and negative regulatory tyrosines of Lyn in B cells. J. Biol. Chem. 271:30487.[Abstract/Free Full Text]
  12. Pao, L. I., J. C. Cambier. 1997. Syk, but not Lyn, recruitment of B cell antigen receptor and activation following stimulation of CD45- B cells. J. Immunol. 158:2663.[Abstract]
  13. Kawabuchi, M., Y. Satomi, T. Takao, Y. Shimonishi, S. Nada, K. Nagai, A. Tarakhovsky, M. Okada. 2000. Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature 404:999.[Medline]
  14. Kurosaki, T., M. Takata, Y. Yamanashi, T. Inazu, T. Taniguchi, T. Yamamoto, H. Yamamura. 1994. Syk activation by the Src-family tyrosine kinase in B cell receptor signaling. J. Exp. Med. 179:1725.[Abstract/Free Full Text]
  15. Kurosaki, T., S. A. Johnson, L. Pao, K. Sada, H. Yamamura, J. C. Cambier. 1995. Role of the Syk autophosphorylation site and SH2 domains in B cell receptor signaling. J. Exp. Med. 182:1815.[Abstract/Free Full Text]
  16. Cheng, P. C., M. L. Dykstra, R. N. Mitchell, S. K. Pierce. 1999. A role for lipid rafts in BCR signaling and antigen targeting. J. Exp. Med. 190:1549.[Abstract/Free Full Text]
  17. Aman, M. J., K. S. Ravichandran. 2000. A requirement for lipid rafts in B cell receptor induced Ca2+ flux. Curr. Biol. 10:393.[Medline]
  18. Petrie, R. J., P. P. Schnetkamp, K. D. Patel, M. Awasthi-Kalia, J. P. Deans. 2000. Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation. J. Immunol. 165:1220.[Abstract/Free Full Text]
  19. Sproul, T. W., S. Malapati, S. K. Pierce. 2000. Cutting edge: B cell antigen receptor signaling occurs outside lipid rafts in immature B cells. J. Immunol. 165:6020.[Abstract/Free Full Text]
  20. Dykstra, M. L., R. Longnecker, and S. K. Pierce. 2001. Epstein Barr virus co-opts lipid rafts to block the signaling and antigen transport functions of the BCR. Immunity. In press.
  21. Shaw, A. C., R. N. Mitchell, Y. K. Weaver, J. Campos-Torres, A. K. Abbas, P. Leder. 1990. Mutations of immunoglobulin transmembrane and cytoplasmic domains: effect on intracellular signalling and antigen presentation. Cell 63:381.[Medline]
  22. Mitchell, R. N., K. A. Barnes, S. A. Grupp, M. Sanchez, Z. Misulovin, C. Nussenzweig, A. K. Abbas. 1995. Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes. J. Exp. Med. 181:1705.[Abstract/Free Full Text]
  23. Grupp, S. A., K. Campbell, R. N. Mitchell, J. C. Cambier, and A. K. Abbas. 1993. Signaling-defective mutants of the B lymphocyte antigen receptor fail to associate with Ig-{alpha} and Ig-{beta}/{gamma}. J. Biol. Chem. 268:25,776.
  24. Barnes, K. A., R. N. Mitchell. 1995. Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing. J. Exp. Med. 181:1715.[Abstract/Free Full Text]
  25. Jelachich, M. L., M. J. Grusby, D. Clark, D. Tasch, E. Margoliash, S. K. Pierce. 1984. Synergistic effects of antigen and soluble T-cell factors in B-lymphocyte activation. Proc. Natl. Acad. Sci. USA 81:5537.[Abstract/Free Full Text]
  26. Baumann, G., D. Maier, F. Freuler, C. Tschopp, K. Baudisch, J. Wienands. 1994. In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells. Eur. J. Immunol. 24:1799.[Medline]
  27. Coue, M., S. L. Brenner, I. Spector, E. D. Korn. 1987. Inhibition of actin polymerization by latrunculin A. FEBS Lett. 213:316.[Medline]
  28. Spector, I., N. R. Shochet, Y. Kashman, A. Groweiss. 1983. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219:493.[Abstract/Free Full Text]
  29. Song, W., H. Cho, P. Cheng, S. K. Pierce. 1995. Entry of the B-cell antigen receptor and antigen into the class II peptide-loading compartment is independent of receptor cross-linking. J. Immunol. 155:4255.[Abstract]
  30. Hanke, J. H., J. P. Gardner, R. L. Dow, P. S. Changelian, W. H. Brissette, E. J. Weringer, B. A. Pollock, P. A. Connelly. 1996. Discovery of a novel, potent, and Src family-sensitive tyrosine kinase inhibitor: study of Lck- and FynT-dependent T cell activation. J. Biol. Chem. 271:695.[Abstract/Free Full Text]
  31. Hartwig, J. H., L. S. Jugloff, N. J. DeGroot, S. A. Grupp, J. Jongstra-Bilen. 1995. The ligand-induced membrane IgM association with the cytoskeletal matrix of B cells is not mediated through the Ig {alpha}{beta} heterodimer. J. Immunol. 155:3769.[Abstract]
  32. Guo, B., R. M. Kato, M. Garcia-Lloret, M. I. Wahl, D. J. Rawlings. 2000. Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Immunity 13:243.[Medline]
  33. 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.[Abstract/Free Full Text]
  34. 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.[Medline]
  35. Field, K. A., D. Holowka, B. Baird. 1999. Structural aspects of the association of Fc{epsilon}RI with detergent-resistant membranes. J. Biol. Chem. 274:1753.[Abstract/Free Full Text]
  36. Harder, T., P. Scheiffele, P. Verkade, K. Simons. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141:929.[Abstract/Free Full Text]
  37. Holowka, D., E. D. Sheets, B. Baird. 2000. Interactions between Fc{epsilon}RI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113:1009.[Abstract]
  38. Aluvihare, V. R., A. A. Khamlichi, G. T. Williams, L. Adorini, M. S. Neuberger. 1997. Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen-antibody interaction. EMBO J. 16:3553.[Medline]
  39. Cheng, P. C., C. R. Steele, L. Gu, W. Song, S. K. Pierce. 1999. MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens. J. Immunol. 162:7171.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
A. Aucher, E. Magdeleine, E. Joly, and D. Hudrisier
Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in T cells but not in B cells
Blood, June 15, 2008; 111(12): 5621 - 5628.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. W. Sohn, S. K. Pierce, and S.-J. Tzeng
Live Cell Imaging Reveals that the Inhibitory Fc{gamma}RIIB Destabilizes B Cell Receptor Membrane-Lipid Interactions and Blocks Immune Synapse Formation
J. Immunol., January 15, 2008; 180(2): 793 - 799.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
H. T. T. Nguyen, L. Charrier-Hisamuddin, G. Dalmasso, A. Hiol, S. Sitaraman, and D. Merlin
Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells
Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1155 - G1165.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
J. H. Kim, J. A. Rutan, and B. J. Vilen
The transmembrane tyrosine of {micro}-heavy chain is required for BCR destabilization and entry of antigen into clathrin-coated vesicles
Int. Immunol., December 1, 2007; 19(12): 1403 - 1412.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
D. Le Roux, D. Lankar, M.-I. Yuseff, F. Vascotto, T. Yokozeki, G. Faure-Andre, E. Mougneau, N. Glaichenhaus, B. Manoury, C. Bonnerot, et al.
Syk-dependent Actin Dynamics Regulate Endocytic Trafficking and Processing of Antigens Internalized through the B-Cell Receptor
Mol. Biol. Cell, September 1, 2007; 18(9): 3451 - 3462.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Reyes-Moreno, E. Sharif-Askari, J. Girouard, C. Leveille, M. Jundi, A. Akoum, R. Lapointe, A. Darveau, and W. Mourad
Requirement of Oxidation-dependent CD40 Homodimers for CD154/CD40 Bidirectional Signaling
J. Biol. Chem., July 6, 2007; 282(27): 19473 - 19480.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Hudrisier, A. Aucher, A.-L. Puaux, C. Bordier, and E. Joly
Capture of Target Cell Membrane Components via Trogocytosis Is Triggered by a Selected Set of Surface Molecules on T or B Cells
J. Immunol., March 15, 2007; 178(6): 3637 - 3647.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. Rovedo and R. Longnecker
Epstein-Barr Virus Latent Membrane Protein 2B (LMP2B) Modulates LMP2A Activity
J. Virol., January 1, 2007; 81(1): 84 - 94.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
D. A. Brown
Lipid Rafts, Detergent-Resistant Membranes, and Raft Targeting Signals.
Physiology, December 1, 2006; 21(6): 430 - 439.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. W. Sohn, P. Tolar, T. Jin, and S. K. Pierce
Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling
PNAS, May 23, 2006; 103(21): 8143 - 8148.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
H. Kono, C. Kyogoku, T. Suzuki, N. Tsuchiya, H. Honda, K. Yamamoto, K. Tokunaga, and Z.-I. Honda
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., October 1, 2005; 14(19): 2881 - 2892.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. J. Allsup, A. S. Kamiguti, K. Lin, P. D. Sherrington, Z. Matrai, J. R. Slupsky, J. C. Cawley, and M. Zuzel
B-Cell Receptor Translocation to Lipid Rafts and Associated Signaling Differ between Prognostically Important Subgroups of Chronic Lymphocytic Leukemia
Cancer Res., August 15, 2005; 65(16): 7328 - 7337.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Mielenz, C. Vettermann, M. Hampel, C. Lang, A. Avramidou, M. Karas, and H.-M. Jack
Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition
J. Immunol., March 15, 2005; 174(6): 3508 - 3517.
[Abstract] [Full Text] [PDF]