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Originally published In Press as doi:10.1074/jbc.M404410200 on May 25, 2004

J. Biol. Chem., Vol. 279, Issue 30, 31973-31982, July 23, 2004
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B Cell Signaling Is Regulated by Induced Palmitoylation of CD81*

Anu Cherukuri{ddagger}, Robert H. Carter§, Stephen Brooks§, William Bornmann||**, Ronald Finn||, Cynthia S. Dowd{ddagger}, and Susan K. Pierce{ddagger}{ddagger}{ddagger}

From the {ddagger}Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852, the §Department of Medicine and Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, and the ||Memorial Sloan Kettering Cancer Center, Radiochemistry Core Facility, New York, New York 10021

Received for publication, April 21, 2004 , and in revised form, May 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signaling through the B cell antigen receptor (BCR) is amplified and prolonged by coligation of the BCR to the CD19/CD21/CD81 coreceptor complex. Coligation is induced during immune responses by the simultaneous binding of complement-tagged antigens to the complement receptor, CD21, and to the BCR. Enhanced signaling is due in part to the ability of the CD19/CD21/CD81 complex to stabilize the BCR in sphingolipid- and cholesterol-rich membrane microdomains termed lipid rafts. The tetraspanin CD81 is essential for the raft-stabilizing function of the coreceptor. Here we show that coligation of the BCR and the CD19/CD21/CD81 complex leads to selective, rapid, and reversible palmitoylation of CD81 and that palmitoylation is necessary for the raft stabilizing function of the CD19/CD21/CD81 complex. Inducible palmitoylation may represent a novel mechanism by which tetraspanins function to facilitate lipid raft-dependent receptor signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts, appear to play a role in the initiation of B cell receptor (BCR)1 signaling (1). In resting B cells, lipid rafts concentrate the dually acylated Src family kinase, Lyn, and exclude the BCR. Oligomerization of the BCR by antigen binding results in the partitioning of the BCR into lipid rafts where the receptor is phosphorylated and signaling is initiated (2–4). Partitioning of the BCR into lipid rafts does not require a signaling competent BCR nor association of the BCR with the actin cytoskeleton (5). However, if BCR signaling or association with the cytoskeleton are blocked, the association of the BCR with rafts is highly transient suggesting that BCR signaling and attachment to the actin cytoskeleton promote raft stability. The B cell coreceptor complex, CD19/CD21/CD81, when coligated to the BCR through the binding of complement-tagged antigens greatly enhances signaling through the BCR resulting in a significantly reduced threshold for B cell activation (6). The CD19/CD21/CD81 complex functions, in part, by prolonging the residency of the BCR in lipid rafts (7). In resting B cells both the BCR and the CD19/CD21/CD81 complex are excluded from lipid rafts. Coligation of the BCR and the CD19/CD21/CD81 complex results in the partitioning of the coligated complexes into lipid rafts. As compared with cross-linking the BCR alone, coligation of the BCR to the CD19/CD21/CD81 complex results in prolonged residency in and signaling from rafts.

Recently, evidence was provided that the tetraspanin CD81 was essential for the ability of the CD19/CD21/CD81 complex to facilitate the association of the coligated BCR with lipid rafts (8). Tetraspanins are an evolutionarily conserved family of proteins containing four membrane spanning domains, two extracellular domains and short N- and C-terminal cytoplasmic domains (9). Members of the tetraspanin family associate with a variety of different membrane protein complexes, including signaling receptors and integrins, and have been hypothesized to facilitate or promote the activities of such proteins (10). However, the mechanism by which tetraspanins function is not completely understood. Recently, we showed that in B cells from CD81-deficient mice, coligated BCR and CD19/CD21/CD81 complexes failed to associate with lipid rafts and failed to signal (8). In addition, a CD19/CD21 complex that contained a chimeric CD19 protein, the ectodomain of which had been replaced by that of CD4 and consequently associated weakly if at all with CD81 (11), failed to promote BCR raft association and signaling (8). The observation that CD81 is required for the stable association of coligated BCR-CD19/CD21/CD81 complexes with lipid rafts suggested that tetraspanins may function as molecular facilitators by promoting raft association.

Protein palmitoylation has been shown to be critical for both the partitioning of proteins into lipid rafts and for their function (12, 13). For example, the T cell adaptor protein LAT requires constitutive palmitoylation to associate with rafts and mutant LAT proteins that are not palmitoylated fail to be phosphorylated upon T cell antigen receptor engagement and thus fail to function (14). In a second example, the Src family kinases Fyn and Lck that play an essential role in T cell receptor signaling are targeted to lipid rafts due to their constitutive dual acylation with myristate and palmitate (15, 16). Blocking acylation of Fyn, using the inhibitor 2-bromopalmitate, blocked both raft association and T cell receptor signaling (17). Palmitoylation is a readily reversible posttranslational protein modification that occurs primarily through cysteine residues (18). The reversibility of palmitoylation suggests the possibility that palmitoylation is a regulated modification of proteins similar to protein phosphorylation. While protein phosphorylation serves to regulate protein-protein interactions, palmitoylation functions to regulate protein-lipid interactions (19). Palmitoylation could function to stabilize the association of CD81 with lipid rafts and consequently the interaction of the CD19/CD21/CD81 complex with components of the BCR signaling cascade. Several members of the tetraspanin family, including CD81, contain cysteine residues in their cytoplasmic loops, a portion of which appears to be constitutively palmitoylated (20, 21). The palmitoylation of the tetraspanin CD151 has been shown to be important for its interactions with other tetraspanins and with the {alpha}3{beta}1 integrin (21, 22). Here we provide evidence that CD81 is inducibly palmitoylated upon coligation of the BCR and CD19/CD21/CD81 complex and that palmitoylation is necessary for the stable association of BCR-CD19/CD21/CD81 complexes with rafts and signaling from rafts. The inducible palmitoylation of CD81 may represent a novel mechanism by which the tetraspanins influence receptor signaling by promoting raft association.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—Daudi cells were stably transfected with a chimeric CD19 construct (CD4/19) in which the ectodomain of CD19 was replaced with that of CD4 as described previously (11). Wild type Daudi cells were cultured in IMDM (ATCC, Manassas, VA) supplemented with 10% fetal calf serum (Invitrogen) and penicillin (100 units/ml) and streptomycin (100 µg/ml) (Sigma). CD4/19-transfected cells were cultured in the same medium containing 2 µg/ml G-418 (Invitrogen).

Antigens and Antibodies—Biotinylated Fab of mouse mAb specific for human IgM (DA4.4), biotinylated (Fab)'2 mouse mAb specific for human CD19 (ADF4.2), and polyclonal rabbit Abs specific for the cytoplasmic domain of CD19 are as described (23). A mAb specific for human CD81 (5A6) was generated and characterized as described (24, 25) and was a kind gift of Dr. Shoshana Levy (Stanford University). Biotinylated Abs specific for human CD4 were purchased from Caltag (Burlingame, CA). HRP-conjugated goat Abs specific for human µchain were purchased from BIOSOURCE International (Camarillo, CA). A mAb specific for human CD45 was purchased from Transduction Laboratories (San Diego, CA). Rabbit polyclonal Abs specific for Lyn kinase, PLC{gamma}2, and Vav were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated goat Abs specific for rabbit and mouse IgG and avidin were purchased from Jackson ImmunoResearch (West Grove, PA). HM57 a mouse mAb specific for human Ig{alpha} was generated and characterized as described (26). The HRP-conjugated phosphotyrosine-specific mAbs, PY20H and RC20H, were purchased from Transduction Laboratories. Methyl-{beta}-cyclodextrin (M{beta}CD), 16-bromohexadecanoic acid, 2-bromohexadecanoic acid, sodium iodide, chloroform, acetone, bovine serum albumin (BSA), hydroxylamine hydrochloride, and fatty acid-free (defatted) BSA were obtained from Sigma. The Src family kinase inhibitor PP2 was purchased from Calbiochem. The HRP-based chemiluminescene ECL kit was purchased from Amersham Biosciences (Buckinghamshire, UK).

Receptor Cross-linking and Lipid Raft Isolation—The BCR on Daudi cells was cross-linked by incubating the cells (1 x 108 in 1 ml) at 4 °C for 30 min with biotinylated Fab DA4.4 (10 µg/ml) followed by avidin (5 µg/ml). The BCR was coligated to the CD19/CD21 complex using biotinylated Fab DA4.4 and biotinylated (Fab)'2 ADF4.2 at 4 °C for 30 min followed by avidin. CD4 cross-linking in the cells expressing the chimeric CD4/19 was similarly done using biotinylated Abs specific for human CD4 followed by avidin. Cells were washed three times in cold HBSS containing 3% BSA and chased, where indicated, in culture medium at 37 °C in the absence of Ab. All cells were lysed in 1% Triton X-100 (Sigma)-containing lysis buffer and lipid rafts isolated from the lysates as described previously (3).

Immunoprecipitation and Immunoblotting—CD81, Lyn, CD45, Vav, and PLC{gamma}2 were immunoprecipitated from either whole cell lysates or sucrose density gradient fractions solubilized in 500 µl of 5x RIPA (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.5) lysis buffer by first preclearing with protein A-Sepharose (Amersham Biosciences, Uppsala, Sweden) and for Lyn, Vav, and PLC{gamma}2 immunoprecipitates by preclearing with protein A-Sepharose and normal rabbit serum (Sigma). Lysates were then incubated with 5 µg/ml amounts of either specific rabbit Ab, rabbit IgG isotype control, and protein A-Sepharose or mouse mAbs, mouse IgG isotype control, and protein G-Sepharose at 4 °C with rotation overnight. Immunoprecipitates or gradient fractions were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Co., Bedford, MA). CD81 was resolved on non-reducing, denaturing SDS-PAGE as the CD81-specific mAb recognizes epitopes that are sensitive to reduction. Membranes were probed using Abs specific for Igµ, Ig{alpha}, CD19, CD81, Lyn, and CD45 followed by incubation with HRP-conjugated goat Abs specific for the primary Abs. HRP-conjugated PY20H was used to detect tyrosine-phosphorylated proteins. Immunoblots were developed using ECL (Amersham Biosciences, Buckinghamshire, UK) and all films quantified by densitometry using Scion Image software (National Institutes of Health, Bethesda, MD).

Detection of Palmitoylated Proteins—Protein palmitoylation was measured by the incorporation of 125I-labeled 16-iodohexadecanoic acid (125IC16). IC16 was synthesized from 16-bromohexadecanoic acid as described previously (27) and generously provided by Dr. Marilyn Resh at the Memorial Sloan-Kettering Cancer Center (New York). IC16 was radiolabeled with 125I as described previously (28). Alternatively, the method developed by Knapp et al. (29) was used to prepare IC16. Briefly, a solution of 16-bromohexadecanoic acid in acetone was stirred at room temperature with three equivalents of sodium iodide for 19 h. The acetone was evaporated under reduced pressure, and the residue was extracted between water and chloroform. Drying over MgSO4, filtration, and concentration yielded 90% of the crude product. Recrystallization of IC16 from petroleum ether gave a white powder, which matched literature values for melting point and NMR of the desired material. Amersham Biosciences (Woburn, MA) was furnished with IC16 and performed the radiolabeling. Cells to be labeled with 125IC16 were cultured overnight in IMDM containing 2.5% dialyzed serum and 0.25% defatted BSA. Prior to labeling, ~1 x 108 cells were starved for 1 h in 3 ml of IMDM containing 2% dialyzed fetal bovine serum and then incubated for 2 h with 25–40 µCi/ml 125IC16 in IMDM containing 2.5% dialyzed fetal bovine serum and 0.5% defatted BSA. When indicated, cells were preincubated with 100 µM 2-bromopalmitate for 15 min prior to the addition of 125IC16. For cholesterol depletion, 10 mM M{beta}CD was added to the cells 15 min prior to the addition of 125IC16. An aliquot of M{beta}CD-treated cells was allowed to recover in cholesterol-containing medium for 3 h at 37 °C before washing in fatty acid-free medium and adding 125IC16 to the recovered cells. Alternatively, to address the role of Src kinases in palmitoylation, 100 µM PP2 was added to the cells at 37 °C for 30 min prior to the addition of 125IC16. Abs to cross-link the BCR or coligate the BCR and CD19 in Wild type cells or coligate the BCR and CD4 in transfected cells were added as described above followed by avidin treatment. 125IC16 associated with the immunoprecipitates from whole cell lysates or the pooled sucrose gradient fractions was detected using a CobraTM II Auto {gamma}-Counter (PerkinElmer Life Sciences) and expressed as counts per minute (cpm). Where indicated CD81, isotype-matched control and Lyn immunoprecipitates were incubated in 1 M hydroxylamine, pH 7.0, for 24 h at 4 °C followed by washing and detection of the associated 125IC16 by {gamma}-counting. 125IC16-containing proteins resolved by SDS-PAGE and transferred to PVDF were detected following exposure to a phosphorimager screen for 1–7 days using a Typhoon 8600 Variable Mode Imager (Amersham Biosciences, Piscataway, NJ). Intensities of the bands obtained from phosphorimager analyses were quantified by densitometry using the NIH Scion Image Software (Bethesda, MD).

Measurement of Intracellular Free Ca2+—Daudi cells were incubated at 30 °C for 30 min in buffer containing 2 µg/ml Fluo-4 AM and 5 µg/ml Fura Red and according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Cells were briefly warmed to 37 °C and analyzed by flow cytometry using a FACSCalibur (BD Biosciences) to measure changes in Fluo-4 and Fura Red fluorescence intensities. After a 30-s analysis to establish a base line, Abs to cross-link the BCR or coligate the BCR and CD19 were added, cells briefly vortexed, and data collection continued until 60 s when avidin was added to the cells. When indicated, cells were preincubated with 100 µM 2-bromopalmitate at 37 °C for 15 min prior to start of data acquisition. Data were acquired for a total of 10 min and displayed as a ratio of Fluo-4 (520 nm) to Fura Red (650 nm) intensities versus time.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Coligation of the BCR and the CD19/CD21/CD81 Complex Results in Raft Localization and Palmitoylation of CD81— Coligation of the BCR and CD19/CD21 complex on the human Daudi B cell line resulted in the partitioning of both the BCR and the CD19/CD21 complex into lipid rafts and prolonged signaling from rafts as compared with BCR cross-linking alone (Figs. 1 and 2) as described previously for other mouse and human B cell lines (7, 8). Daudi B cells were incubated for 30 min at 4 °C in medium alone or in medium containing biotinylated Abs specific for the BCR alone (BCR-cross-linked) or Abs specific for the BCR and for CD19 (BCR-CD19 coligated), washed, and incubated with avidin to ligate the receptors. The cells were either lysed immediately or warmed to 37 °C for up to 60 min and then lysed. Lysis was at 4 °C in 1% Triton X-100 detergent, conditions under which lipid rafts are insoluble, and the soluble and insoluble membranes were separated on discontinuous sucrose density gradients. The gradient fractions were analyzed by SDS-PAGE and immunoblotting to detect the BCR Igµ and Ig{alpha} chains, CD19 and CD81, respectively. In untreated cells the insoluble membranes, fractions 3–6, contained Lyn but excluded CD45 that was present in the soluble membrane fractions 9–12, thus, operationally identifying fractions 3–6 as containing rafts. In untreated cells, the BCR Igµ and Ig{alpha}, CD19 and CD81, respectively, were found almost exclusively in the soluble fractions 9–12 (Fig. 1). CD21 was also detected in the soluble fractions (data not shown). Cross-linking the BCR alone led to the immediate and transient association of the BCR Igµ and Ig{alpha} chains with the raft fraction. By 60 min after cross-linking the BCR was no longer raft associated. BCR cross-linking had no effect on the position of either CD19 or CD81 in the plasma membrane. In contrast, the coligation of the BCR and CD19/CD21/CD81 complex resulted in the partitioning of nearly all the CD19, CD81, and BCR Igµ and Ig{alpha} chains into the insoluble raft membranes (Fig. 1). The partitioning was highly stable such that 60 min following coligation the BCR and the CD19/CD21/CD81 complex were still raft-associated.



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  		FIG. 1.
Coligation of the BCR and the CD19/CD21/CD81 complex on Daudi B cells induces the stable partitioning of the coligated complex into lipid rafts in a cholesterol-dependent fashion. Daudi B cells were incubated for 30 min at 4 °C either in medium alone (untreated) or media containing biotinylated Abs specific for Igµ alone (BCR-cross-linked) or with Abs specific for Igµ and CD19 (BCR-CD19 coligated), washed, and incubated with avidin for 30 min at 4 °C. The cells were lysed either immediately at 4 °C in 1% Triton X-100 detergent (0 m) or warmed to 37 °C for 60 min and then lysed (60 m). The soluble and insoluble membranes were separated on discontinuous sucrose density gradients, and the resulting gradient fractions were analyzed by SDS-PAGE and immunoblotting probing for CD19, CD81, Igµ, Ig{alpha}, Lyn, and CD45. To determine the cholesterol dependence of the partitioning of the CD19/CD21 complex into detergent insoluble membranes, Daudi cells were pretreated with M{beta}CD (10 mM) for 15 min at 37 °C to remove membrane cholesterol before coligating the BCR and CD19/CD21/CD81 complex. To determine the reversibility of the M{beta}CD treatment, M{beta}CD-treated cells were incubated in cholesterol-containing medium for 3 h at 37 °C prior to coligation.

 



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  		FIG. 2.
Partitioning of the BCR-CD19/CD21/CD81 complexes into lipid rafts correlates with signaling from rafts. Daudi B cells were either untreated or treated as described for Fig. 1 to cross-link the BCR or to coligate the BCR and the CD19/CD21/CD81 complex. Cells were lysed in cold 1% Triton X-100 and the raft and soluble fractions separated on a discontinuous sucrose density gradient. Gradient fractions were analyzed by SDS-PAGE and immunoblotting probing for tyrosine-phosphorylated proteins. Alternatively Vav and PLC{gamma}2 were immunoprecipitated from lysates, and the immunoprecipitates were analyzed by SDS-PAGE and immunoblotting probing with a phosphotyrosine-specific Ab.

 
The integrity of lipid rafts is dependent on cholesterol and extraction of cholesterol from the Daudi cells by treatment with M{beta}CD prior to coligation of CD19, and the BCR resulted in the failure of CD19 and CD81 and the BCR to associate with detergent-insoluble membranes (Fig. 1 and data not shown). The association of CD81 with insoluble membranes, although significantly reduced by M{beta}CD treatment, was somewhat more resistant to cholesterol extraction as compared with CD19, which may indicate a difference in the nature of the raft association for CD19 and CD81. The effect of M{beta}CD was reversible such that in M{beta}CD-treated cells that were subsequently incubated in cholesterol-containing medium, CD19 and CD81 were isolated in the raft regions of the membrane following coligation (Fig. 1).

The partitioning of the coligated BCR-CD19/CD21/CD81 complex in lipid rafts correlated with the induced appearance of tyrosine phosphorylated CD19, Lyn, and Ig{alpha} in rafts and in the assembly of a signaling complex in rafts that contained phosphorylated Vav and PLC{gamma}2 (Fig. 2). BCR cross-linking alone resulted in phosphorylation of Lyn and Ig{alpha} in rafts. Although CD19, Vav, and PLC{gamma}2 were phosphorylated upon BCR cross-linking, these did not form a stable signaling complex in rafts (Fig. 2).

Coligation of the BCR and the CD19/CD21/CD81 Complex Induces the Palmitoylation of CD81—To determine whether coligation of the BCR and the CD19/CD21/CD81 complex altered the degree of palmitoylation of CD81, cells were incubated with 125IC16 for 2 h at 37 °C prior to treating the cells with Abs at 4 °C for 30 min to coligate the BCR and the CD19/CD21/CD81 complex as described in the legend to Fig. 1. The cells were lysed in 1% Triton X-100, and CD81 was immunoprecipitated from the lysates in RIPA lysis buffer. The amount of 125IC16 associated with CD81 immunoprecipitates was determined using a {gamma}-counter. The amount of 125IC16 associated with CD81 doubled in cells following coligation of the BCR and the CD19/CD21/CD81 complex (Fig. 3A). The CD81 immunoprecipitates were subjected to SDS-PAGE, and the proteins in the gel were transferred to PVDF. Phosphorimaging of the membrane showed only one major band that appeared at the appropriate molecular weight of CD81 (Fig. 3B). Subsequent probing of the membrane with CD81-specific antibody identified the band as CD81 (Fig. 3C). Quantification of the immunoblot showed that similar amounts of CD81 protein were immunoprecipitated from untreated and coligated cells (Fig. 3C). Quantification of the bands on the phosphorscreen showed ~2-fold more 125IC16 associated with the band corresponding to CD81 in the immunoprecipitates from the lysates of coligated as compared with untreated B cells (Fig. 3C). Thus, the increase in the 125IC16 associated with the CD81 immunoprecipitate in the coligated cells as compared with untreated cells could be accounted for as an increase in the 125IC16 bound to CD81. The 125IC16 incorporated into CD81 in untreated cells presumably represents constitutively palmitoylated CD81 as described by Yang et al. (21). There was no significant radioactivity associated with the isotype-matched control immunoprecipitates from either untreated or coligated cells.



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  		FIG. 3.
Coligation of the BCR and the CD19/CD21/CD81 complex on Daudi B cells induced the palmitoylation of CD81. Daudi B cells were serum-starved for 1 h and then incubated in fatty acid-free medium (5 x 107 cells in 1 ml) containing 125IC16 (25–40 µCi) for 2 h at 37 °C. The cells were cooled to 4 °C, and Abs specific for CD19 and Igµ were added to coligate the CD19/CD21/CD81 complex and the BCR as described for Fig. 1. The cells were immediately lysed in 1% Triton X-100 at 4 °C, and CD81 was immunoprecipitated from an aliquot of the lysates in RIPA lysis buffer. Isotype-matched, nonspecific Abs were used for control immunoprecipitation (IC). A, the cpm associated with the CD81 or IC immunoprecipitates are given. B, the CD81 and IC immunoprecipitates were subjected to SDS-PAGE and the proteins transferred to PVDF. The membranes were analyzed by phosphorimaging as described under "Experimental Procedures." The region of the phosphorimage corresponding to the CD81 bands is shown. The 125IC16 associated with the bands was quantified by densitometry, and the mean intensity values are given. C, the membrane in B was probed with CD81-specific Ab. The CD81 bands were quantified by densitometry and the values given. Shown in A–C is a representative analysis from one of five separate experiments. D, CD81 and Lyn were immunoprecipitated from the 1% Triton X-100 lysates prepared from untreated or coligated cells in RIPA buffer using specific Abs. The 125IC16 associated with the immunoprecipitates was determined. The immunoprecipitates were soaked overnight in 1 M hydroxylamine (+OH), pH 7.0 at 4 °C, followed by washing, and the 125IC16 associated with the treated immunoprecipitates was determined. Shown are the average values for three separate experiments.

 
The 125IC16 associated with CD81 in the immunoprecipitates was removed by treatment of the immunoprecipitate with hydroxylamine that hydrolyzes thioester bonds at neutral pH (30) indicating that the palmitoylation was through cysteine residues (Fig. 3D). CD81 appeared to be selectively palmitoylated as there was no significant increase in the amount of 125IC16 in Lyn immunoprecipitates following BCR-CD19/CD21/CD81 coligation (Fig. 3D). Lyn is constitutively palmitoylated, and its palmitoylation is necessary for its association with lipid rafts. However, in contrast to CD81, the amount of 125IC16 associated with Lyn did not change significantly following coligation of the BCR and the CD19/CD21/CD81 complex. The treatment of the Lyn immunoprecipitates with hydroxylamine reduced the amount of associated 125IC16 (Fig. 3D) indicating that the 125IC16 in the immunoprecipitate was incorporated into cysteine residues in Lyn.

CD81 Is Reversibly Palmitoylated in Rafts—The palmitoylation of CD81 did not occur to any significant extent following BCR cross-linking alone, and BCR cross-linking did not induce the palmitoylation of Lyn (Fig. 4A). In additional control immunoprecipitates, the amount of 125IC16 associated with CD45, which is excluded from rafts and is not known to be palmitoylated, did not increase following BCR cross-linking alone or BCR-CD19/CD21/CD81 coligation. Thus, the inducible palmitoylation of CD81 requires coligation of the BCR and the CD19/CD21/CD81 complex and is not simply a repercussion of cell activation.



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  		FIG. 4.
The coligation-induced palmitoylation of CD81 is selective and transient and occurs in rafts. The Daudi cells were serumstarved for 1 h and incubated with 125IC16 for 2 h at 37 °C as described for Fig. 3. A, Daudi B cells were either untreated, treated to coligate the BCR and the CD19/CD21/CD81 complex (COL) as described for Fig. 3, or treated with biotinylated Abs specific for Igµ and avidin to cross-link the BCR alone (BCR-XL). The cells were lysed in Triton X-100 at 4 °C and CD81, Lyn, and CD45 immunoprecipitated from the lysates. The 125IC16 associated with the immunoprecipitates was determined. Shown are average values for three separate experiments. B, Daudi B cells were either untreated or treated to coligate the BCR and CD19/CD21/CD81 complex as described for Fig. 3 and lysed either immediately following ligation (COL (0h)) or incubated at 37 °C for 1 h (COL (1h)). The cells were lysed in 1% Triton X-100 at 4 °C and the lysates subjected to discontinuous sucrose gradient centrifugation. CD81 was immunoprecipitated from pooled gradient fractions in RIPA buffer and the 125IC16 associated with the resulting CD81 immunoprecipitates determined. An aliquot of the pooled gradient fractions 3–6 in RIPA buffer was immunoprecipitated using an isotype-matched control Ab (IC). Shown are results from one representative experiment of two. C, Daudi B cells were either untreated or treated to coligate the BCR and the CD19/CD21/CD81 complex, washed, and lysed either immediately (0 h) or after 1- or 3-h incubation in complete medium at 37 °C. The cells were lysed in Triton X-100 buffer at 4 °C, and CD81 was immunoprecipitated from the lysates in RIPA buffer. The 125IC16 associated with the CD81 immunoprecipitates was determined. Shown are the average values for three separate experiments. D, Daudi B cells expressing both an endogenous CD19 and a transfected CD4/19 chimeric protein were serum-starved for 1 h and incubated with 125IC16 for 2 h at 37 °C as described for Fig. 3. The cells were either untreated, treated to coligate the endogenous CD19/CD21 complex and the BCR as described for Fig. 3 (CD19 COL), or treated to coligate the chimeric CD4/19 complex to the BCR by incubating cells with biotinylated antibodies specific for CD4 and the BCR followed by incubation with avidin (CD4 COL). The cells were lysed in 1% Triton X-100 and CD81, Lyn, and CD45 immunoprecipitated from aliquots of the lysates in RIPA buffer. The 125IC16 associated with the immunoprecipitates was determined and shown as an average fold increase over the cpm of the untreated controls in four separate experiments.

 
To determine where in the membrane the newly palmitoylated CD81 resided, Triton X-100 lysates from cells incubated with 125IC16 and either untreated or treated with Abs to coligate the BCR and the CD19/CD21/CD81 complex were applied to discontinuous sucrose gradients. CD81 was immunoprecipitated from the gradient fractions in RIPA buffer and the amount of 125IC16 associated with the immunoprecipitates determined. In controls, isotype-matched nonspecific Abs were used for immunoprecipitates of the raft fractions 3–6. Immediately following coligation the amount of 125IC16 associated with the CD81 immunoprecipitates increased over 3-fold, and this increase was entirely within the insoluble membrane fractions 3–6 (Fig. 4B). By 1 h following coligation the amount of 125IC16 associated with the CD81 immunoprecipitate in the raft fractions had decreased significantly indicating the reversible nature of the 125IC16 modification (Fig. 4B).

The time course of palmitoylation of CD81 was investigated further (Fig. 4C). Cells were incubated with 125IC16 for 2 h at 37 °C, the BCR and the CD19/CD21/CD81 complex coligated at 4 °C, and the cells washed and chased in complete medium at 37 °C for increasing periods of time up to 16 h (Fig. 4C and data not shown). The cells were lysed and CD81 immunoprecipitated from the lysates. Coligation of the BCR and the CD19/CD21/CD81 complex resulted in a 2-fold increase in the incorporation of 125IC16 into CD81 as compared with untreated cells, and the increase was observed immediately upon coligation. By 1 h following coligation the basal level of 125IC16 incorporation into CD81 had not changed, but the coligation-induced level of palmitoylation had decreased significantly. Thus, the half-life of the induced palmitoylation appeared shorter than the half-life of the constitutive palmitoylation. By 3 h after the initiation of the experiment the basal level of 125IC16 associated with CD81 in untreated cells decreased likely reflecting the half-life of the palmitate that has been reported for other proteins to be ~2 h (31). Similarly, by 3 h the level of coligation-induced 125IC16 associated with CD81 further decreased. Additional time points taken up to 16 h after coligation showed little 125IC16 in the CD81 immunoprecipitates from either untreated or coligated cells (data not shown).

CD81 is expressed on B cell surfaces independently and in association with other tetraspanins and integrin complexes (10). To determine which pool of CD81 becomes palmitoylated following coligation of the BCR and CD19/CD21 complex, the effect of coligating the BCR to a chimeric CD19 that associates weakly if at all with CD81 was tested. CD81 associates with CD19 primarily through the extracellular domain of CD19 (11, 32). Consequently, a chimeric CD19 protein in which the extracellular domain of CD19 was replaced with the extracellular domain of CD4 (CD4/19) associates only weakly if at all with CD81 (11). Previous studies showed that Daudi B cells transfected with the CD4/19 chimera express the CD4/19 chimera at levels similar to the endogenous wild type CD19 and express normal levels of CD21 and CD81 (8, 23). However, when coligated to the BCR, CD4/19 failed to promote the stable association of the BCR with rafts (8). Coligation of CD4/19 to the BCR using antibodies specific for CD4 and the BCR showed only a small increase in the palmitoylation of CD81 as compared with the 2-fold increase in palmitoylation induced by coligating the endogenous wild type CD19/CD21/CD81 complex to the BCR (Fig. 4D). In control immunoprecipitates, coligating CD4/19 to the BCR had no effect on the incorporation of 125IC16 into Lyn or CD45. Thus, it appears that coligation of the CD19/CD21/CD81 complex to the BCR results primarily in the palmitoylation of the CD81 associated with the coreceptor complex.

Palmitoylation of CD81 Is Dependent on the Integrity of Lipid Rafts but Not on BCR-CD19/CD21 Signaling—To determine whether the palmitoylation of CD81 induced by coligation of the BCR and the CD19/CD21/CD81 complex was dependent on the integrity of lipid rafts, Daudi B cells were treated with M{beta}CD to remove cholesterol from the membrane as described for Fig. 1 prior to BCR-CD19/CD21/CD81 coligation. Following cholesterol depletion the cells were incubated with 125IC16, and the BCR and the CD19/CD21/CD81 complex were coligated. The cells were immediately lysed, and CD81 was immunoprecipitated from the lysates. In untreated cells coligation resulted in an increase in the 125IC16 associated with the CD81 immunoprecipitates (Fig. 5). In controls, there was no increase in the amount of 125IC16 associated with immunoprecipitates of Lyn or CD45. M{beta}CD treatment blocked the increase in the amount of 125IC16 associated with the CD81 immunoprecipitates following coligation. The effect of M{beta}CD was reversible with the addition of cholesterol back into the medium. Thus, the induced palmitoylation of CD81 was dependent on the integrity of lipid rafts. In contrast, pretreating cells with the Src family kinase inhibitor PP2 had little effect on the colligation-induced palmitoylation of CD81 (Fig. 5) at a concentration of PP2 that inhibited the coligation-induced tyrosine phosphorylation of Daudi proteins (data not shown). Thus, the partitioning of the coligated BCR-CD19/CD21/CD81 complex into rafts appears to be sufficient to induce the palmitoylation of CD81 even in the absence of signaling.



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  		FIG. 5.
Palmitoylation of CD81 is dependent on the integrity of lipid rafts but not on receptor signaling. Daudi B cells were serumstarved for 1 h and either untreated or treated with M{beta}CD for 15 min to remove cholesterol prior to labeling with 125IC16 or with the Src family kinase inhibitor, PP2, for 30 min at 37 °C. In controls the cells were treated with M{beta}CD and then incubated in cholesterol-containing medium for 3 h at 37 °C prior to labeling with 125IC16. The BCR and CD19/CD21 complex were then either coligated or not. Cells were lysed in Triton X-100, and CD81, Lyn, and CD45 were immunoprecipitated from the lysates in RIPA buffer. The 125IC16 associated with the immunoprecipitates was determined. Shown are the average values for two separate experiments.

 
The Palmitoylation Inhibitor, 2-Bromopalmitate, Blocks CD81 Palmitoylation and Raft Partitioning of Coligated BCR-CD19/CD21/CD81 Complexes—To determine whether the observed palmitoylation of CD81 was necessary for its function in promoting the stabilization of coligated BCR-CD19/CD21/CD81 complexes in lipid rafts, cells were treated with the palmitoylation inhibitor, 2-bromopalmitate (17), just prior to incubation with 125IC16 and coligation. Lysates were prepared from the cells in 1% Triton X-100 and the insoluble and soluble membranes separated on a sucrose density gradient. CD81 was immunoprecipitated from the resulting gradient fractions in RIPA buffer and the immunoprecipitates counted. Additional immunoprecipitates using isotype-matched nonspecific Abs were carried out for raft fractions 3–6. The presence of 2-bromopalmitate blocked the coligation-induced increase in the 125IC16 counts associated with the CD81 immunoprecipitates in the raft fractions (Fig. 6A). The 2-bromopalmitate did not affect the basal level of 125IC16 incorporated into CD81 (Fig. 6A) nor did 2-bromopalmitate affect the amount of 125IC16 associated with Lyn immunoprecipitates in the insoluble raft membranes (Fig. 6B). The observation that hydroxylamine treatment resulted in the complete loss of 125IC16 from the CD81 and Lyn immunoprecipitates (Fig. 3D) indicated that the basal level of incorporation of 125IC16 was into CD81 and Lyn cysteines. The observation that the basal incorporation is not blocked by 2-bromopalmitate suggests that either the inhibitor was ineffective in blocking enzyme-dependent constitutive palmitoylation or that basal palmitoylation was non-enzymatic as has been documented to occur in vitro (19). The 2-bromopalmitate treatment of Daudi cells that had been incubated with 125IC16 for longer periods of time (24–36 h) to more completely label Lyn significantly reduced the amount of 125IC16 incorporated into Lyn (data not shown) indicating that the palmitoylation of Lyn could be blocked over time by 2-bromopalmitate, as shown recently by others for Fyn (17, 33).



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  		FIG. 6.
The palmitoylation inhibitor, 2-bromopalmitate, blocks the coligation-induced palmitoylation of CD81. Daudi B cells were serum-starved for 1 h at 37 °C and then incubated in fatty acid-free medium in the absence or presence of 2-bromopalmitate (+2BP) for 15 min at 37 °C. 125IC16 was added to the medium and the incubation continued at 37 °C for 2 h. The temperature was reduced to 4 °C, and the cells were treated as described for Fig. 2 to coligate the BCR and the CD19/CD21/CD81 complex. The cells were lysed in Triton X-100 and the lysates subjected to discontinuous sucrose density gradient centrifugation. CD81 (A) and Lyn (B) were immunoprecipitated from the resulting pooled gradient fractions in RIPA buffer and the 125IC16 associated with the immunoprecipitates measured. Shown are the average values for three separate experiments.

 
To determine whether the failure to inducibly palmitoylate CD81 influenced the ability of coligated BCR-CD19/CD21/CD81 complexes to be stabilized in lipid rafts, Daudi cells were pretreated with 2-bromopalmitate for 15 min prior to either cross-linking the BCR alone or coligating the BCR and the CD19/CD21/CD81 complex. The cells were lysed in 1% Triton X-100 and the soluble and insoluble membranes separated on a discontinuous sucrose gradient. Fractions from the gradient were analyzed by SDS-PAGE and immunoblotting, probing the blots for Igµ, CD19, CD81, Lyn, and CD45. Cross-linking the BCR in untreated cells resulted in the association with the Igµ chain of BCR with rafts and the tyrosine phosphorylation of Vav and PLC{gamma}2, although these did not stably associate in the rafts (Fig. 7A). BCR cross-linking did not affect the position of CD19 or CD81 in the plasma membrane. The presence of 2-bromopalmitate had no significant effect on the partitioning of BCR into rafts or on the induced phosphorylation of Vav and PLC{gamma}2 (Fig. 7A). The 2-bromopalmitate partially blocked B cell activation as measured by a decrease in the intracellular Ca2+ flux indicating that a step in the downstream signaling pathway was partially dependent on a palmitoylation event (Fig. 7B). In contrast, 2-bromopalmitate completely inhibited the coligation-induced association of the BCR and the CD19/CD21/CD81 complex with rafts (Fig. 8A). Thus, the failure of CD81 to be palmitoylated following coligation of the BCR and the CD19/CD21/CD81 complex correlated with a failure of the coligated complexes to stably associate with lipid rafts. Neither Vav nor PLC{gamma}2 was significantly phosphorylated in cells treated with 2-bromopalmitate (Fig. 8A) indicating a block in BCR signaling as well. The inhibition of tyrosine phosphorylation of Vav and PLC{gamma}2 was reflected in an inhibition of Ca2+ mobilization upon coligating the BCR and CD19/CD21/CD81 complex following pretreatment of cells for 15 min with 2-bromopalmitate (Fig. 8B). Taken together these results indicate that inhibition of palmitoylation selectively blocked the assembly of coligation-induced signaling complexes in the rafts and had little or no effect on the signaling function of the BCR alone.



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  		FIG. 7.
The inhibitor, 2-bromopalmitate, does not affect the partitioning of cross-linked BCR into rafts. Daudi B cells were incubated in fatty acid-free medium alone or in medium containing 2-bromopalmitate for 15 min at 37 °C (as described for Fig. 6) and then treated to cross-link the BCR as described for Fig. 4B. The cells were lysed in Triton X-100, subjected to discontinuous sucrose density gradient centrifugation, and the resulting fractions analyzed by SDS-PAGE and immunoblotting probing for Igµ, CD19, CD81, and Lyn. Phosphorylated Vav (Vav-P) and PLC{gamma}2 (PLC{gamma}2-P) were detected in pooled gradient fractions by SDS-PAGE and immunoblotting of Vav and PLC{gamma}2 immunoprecipitates, probing for phosphorylated proteins using a phosphotyrosine-specific Ab. One of three representative experiments is shown. B, intracellular free Ca2+ was measured after loading cells with the dyes Fluo-4 and Fura Red for 30 min at 30 °C followed by treatment in the absence (BCR-crosslinked) or presence (2-BP+BCR-crosslinked) of 100 µM 2-bromopalmitate for 15 min at 37 °C. Abs to cross-link the BCR were added at 30 s after data acquisition on the flow cytometer was started followed by the avidin addition at 60 s. Data were acquired for a total of 10 min and expressed as a ratio of Fluo-4/Fura Red fluorescence intensities versus time. One of four representative experiments is shown.

 



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  		FIG. 8.
The inhibitor 2-bromopalmitate blocks the stabilization and signaling of the coligated BCR-CD19/CD21/CD81 complexes in rafts. Daudi B cells were incubated in fatty acid-free media alone or in media containing 2-bromopalmitate for 15 min at 37 °C and then treated to coligate the BCR and the CD19/CD21/CD81 complex as described for Fig. 5. The cells were lysed in Triton X-100, subjected to discontinuous sucrose density gradient centrifugation, and the resulting fractions analyzed by SDS-PAGE and immunoblotting probing for Igµ, CD19, CD81, Lyn, and CD45. Phosphorylated Vav (Vav-P) and PLC{gamma}2 (PLC{gamma}2-P) were detected in pooled gradient fractions by SDS-PAGE and immunoblotting of Vav and PLC{gamma}2 immunoprecipitates from the fractions, probing for phosphorylated proteins using a phosphotyrosine-specific Ab. One of three representative experiments is shown. B, intracellular free Ca2+ was measured after loading cells with the dyes Fluo-4 and Fura Red for 30 min at 30 °C followed by treatment in the absence (Coligated) or presence (2-BP+Coligated) of 100 µM 2-bromopalmitate for 15 min at 37 °C. Abs to coligate the BCR and the CD19/CD21/CD81 complex were added at 30 s after data acquisition on the flow cytometer was started followed by the avidin addition at 60 s. Data were acquired for a total of 10 min and expressed as a ratio of Fluo-4/Fura Red fluorescence intensities versus time. One of four representative experiments is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
One of the earliest events in BCR signaling following multivalent antigen binding is the phosphorylation of the Ig{alpha} and Ig{beta} chains of BCR by the Src family kinase Lyn (34, 35). At present, the mechanism by which antigen binding, and presumably receptor oligomerization, results in the association of the BCR with Lyn is not fully understood. Recently, evidence was provided that lipid rafts play a role in the initial events in BCR signaling (reviewed in Ref. 1). Lipid rafts are formed by the partitioning of sphingolipids out of the unsaturated phospholipid bilayer due to the saturated nature of the sphingolipid acyl chains. Cholesterol preferentially binds to the sphingolipids resulting in liquid-ordered domains that define rafts. Recent evidence suggests that in resting cells rafts are dynamic, submicroscopic domains containing only thousands of lipids and several proteins (36, 37). In immune cells a significant portion of the membrane (~40%) has raft-like character (38) suggesting that the cell surface resembles a fine mosaic of raft and non-raft domains. Earlier, we and others (2–4) provided evidence that in resting cells the BCR is excluded from rafts that contain Lyn and that upon BCR oligomerization following the binding of multivalent antigens the BCR partitioned into rafts where it was phosphorylated. The partitioning of the BCR into rafts did not require a signaling-competent BCR or association of the BCR with the actin cytoskeleton (5, 39). However, the association of BCR with rafts in the absence of signaling and actin cytoskeleton association was highly transient. These observations suggested a model in which antigen binding resulted in the formation of a BCR oligomer, the transmembrane domains of which preferred the ordered lipid environment of rafts and consequently partitioned into rafts in a signaling and actin cytoskeleton-independent fashion (40). Partitioning allowed the phosphorylation of the BCR and the initiation of the formation of signaling complexes. The model predicted that as a consequence of multivalent antigen binding and the signaling-dependent recruitment of molecular adaptors rafts would cluster to form larger more stable signaling domains. The ligand-induced clustering of elemental rafts has been proposed by others to be a key step in promoting the signaling process in a variety of cell types (41). A further prediction of the model is that factors that affect the outcome of BCR signaling, such as the involvement of coreceptors, would influence raft clustering and stability. Evidence was provided that the BCR coreceptor, the CD19/CD21/CD81 complex, which functions to enhance BCR signaling, when coligated to the BCR did indeed result in the prolonged residency of the BCR in lipid rafts and signaling from rafts (7). Recently, the tetraspanin CD81 was shown to be necessary for the raft-stabilizing function of the CD19/CD21/CD81 complex (8). Here evidence is provided that inducible palmitoylation of CD81 is an important mechanism in promoting raft clustering and stabilization.

Palmitoylation is the reversible addition of palmitate to cysteine residues within proteins through a thioester linkage (reviewed in Refs. 18 and 19). Recently palmitoylation through N-terminal amide linkages to either cysteine or glycine residues has also been described (42). The cellular proteins that undergo constitutive palmitoylation are diverse and include both transmembrane proteins, for example, in lymphocytes the molecular adaptors LAT and PAG, and peripherally associated proteins, such as the Src family kinases, Lyn, Fyn, and Lck (14, 43–45). For many raft-associated proteins palmitoylation appears to be essential both for their association with rafts and for their function. For example, blocking palmitoylation of the T cell adaptor, LAT, or the kinases Lck and Fyn either by mutation of cysteine residues in the cytoplasmic domains (14) or by treatment with the palmitoylation inhibitor, 2-bromopalmitate (17), blocked both raft localization and T cell signaling. CD81 itself is constitutively palmitoylated and palmitoylation of the tetraspanins appears to be essential for the ability of tetraspanins to form a network of molecular interactions termed the tetraspanin web (10). Although many proteins such as CD81, LAT, and the Src family kinases are constitutively palmitoylated, the reversibility of palmitoylation suggests that it, like phosphorylation, may play a regulatory role. The recent identification of 2-bromopalmitate as a selective and potent inhibitor of palmitoyl acyltransferase (PAT) activity has provided a powerful tool to investigate this possibility. Indeed, using 2-bromopalmitate evidence was recently provided that in neurons synaptic strength is regulated by turnover of palmitate on the postsynaptic density protein, PSD-95, as a consequence of glutamate receptor signaling (46). The results presented here provide evidence that palmitoylation of CD81 is induced by coligation of the BCR and the CD19/CD21/CD81 complex, and the palmitoylation affects the interaction of CD81 with membrane lipids inducing the stabilization of the BCR-CD19/CD21/CD81 complexes in lipid rafts. Thus, the inducible palmitoylation of CD81 may serve to alter the position of the coligated BCR-CD19/CD21/CD81 complex in the membrane and as a consequence alters its interaction with components of the B cell signaling cascade. Recently, the palmitoylation state of CD81 was shown to regulate its interaction with the serine/threonine-binding signaling protein, 14-3-3 (47). Blocking palmitoylation by either oxidative stress or mutation of the five intracellular cysteines shown previously to be palmitoylated (21, 22) resulted in the constitutive association of CD81 with the adaptor, 14-3-3. Thus, it is possible that palmitoylation, in addition to altering the location of the CD19/CD21/CD81 complex in the plasma membrane, directly influences the ability of CD81 to be linked to the signaling apparatus of the cell.

The molecular mechanisms by which proteins are palmitoylated remain to be elucidated. Here we show that coligation of the BCR and the CD19/CD21/CD81 complex results in a two to 3-fold increase in the incorporation of 125I-palmitate into CD81. Even though the induced increase in palmitoylation is relatively modest, blocking palmitoylation by the inhibitor 2-bromopalmitate completely blocked the raft-stabilizing function of CD81. Indeed, the inability of the coligated BCR-CD19/CD21/CD81 complex to stably partition into and signal from rafts in 2-bromopalmitate-treated wild type cells is similar to that of the previously reported CD81-deficient B cells (8). CD81 has five cysteines in its cytoplasmic domains, and we do not yet know if all cysteines are targets of the induced palmitoylation. If only one cysteine residue is inducibly palmitoylated in CD81 the observed 2–3-fold increase in 125I-palmitate associated with CD81 would be highly significant. It is also not known if the palmitoylation of CD81 is through a free cysteine or whether a palmitoylated cysteine is depalmitoylated and then repalmitoylated. Both enzymatic and nonenzymatic mechanisms of S-acylation have been proposed (reviewed in Refs. 18 and 19). The palmitoylating enzymes have not been identified in mammals, although recently genes encoding PATs have been identified in yeast and in Drosophila (48–50). The results presented here suggest that the palmitoylation of CD81 occurs in the B cell rafts. The induced palmitoylation required intact rafts as shown by the failure to palmitoylate CD81 in cells treated with M{beta}CD that disrupts rafts. PAT activity has been shown in the rafts of KBC cells using G-protein {alpha} subunit as a substrate (51). Our preliminary results indicate PAT activity in the rafts of coligated Daudi B cells,2 further suggesting the rafts as sights of CD81 palmitoylation. Palmitoylation of CD81 was shown here to be independent of Src family kinase signaling analogous to the Src-independent initial partitioning of the BCR into rafts following oligomerization (5). Thus, for the BCR, phosphorylation of Ig{alpha} may be the repercussion of the partitioning of the BCR oligomer into Lyn-containing rafts. Similarly, the independence of CD81 palmitoylation on signaling suggests that palmitoylation could simply be the repercussion of the partitioning of CD81 into rafts that contain PAT activity. The results presented here also indicated that the induced palmitoylation is reversible by 1 h after coligation. Cytosolic palmitoylthioesterases that remove palmitoyl moieties preferentially from membrane-associated proteins have been described (18) but whether these play a role in the case of CD81 is not known.

The evidence provided here suggests that the tetraspanin CD81 plays an essential role in facilitating the function of the CD19/CD21/CD81 coreceptor complex by promoting its association with lipid rafts through a mechanism involving reversible palmitoylation of CD81. Whether inducible palmitoylation plays a role in the function of other members of the tetraspanin family remains to be determined and will be of interest. The tetraspanins have been described to physically associate in functional arrays termed tetraspanin-enriched microdomains or TEMs (10). These tetraspanin complexes appear to be distinct from lipid raft sphingolipid- and cholesterol-rich membrane microdomains. Our results indicate that CD81 in resting cells is not in lipid rafts but is induced to partition into lipid rafts upon BCR-CD19/CD21 coligation. The relationship between TEMs and lipid rafts has not been explored here, and it is possible that the TEMs reflect tetraspanin interactions that exist independently of lipid rafts. It will be of interest to determine the relationship between TEMs, lipid rafts, and the palmitoylation state of CD81 in resting and activated cells.


   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. Back

Current address: Molecular Inflammation Section, NIAMS, NIH, Bethesda, MD 20892. Back

** Current address: M. D. Anderson Cancer Center, Houston, TX 77030. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Laboratory of Immunogenetics, NIAID/NIH/Twinbrook II, 12441 Parklawn Dr., Rm. 200B, MSC 8180, Rockville, MD 20852. Tel.: 301-496-9589; Fax: 301-402-0259; E-mail: spierce{at}nih.gov.

1 The abbreviations used are: BCR, B cell receptor; Ab, antibody; mAb, monoclonal antibody; M{beta}CD, methyl-{beta}-cyclodextrin; PAT, palmitoyl acyltransferase; PVDF, polyvinylidene fluoride; IMDM, Iscove's modified Dulbecco's medium; HRP, horseradish peroxidase; PLC, phospholipase C; 125IC16, 125I-labeled 16-iodohexadecanoic acid; TEM, tetraspanin-enriched microdomain. Back

2 J. Swarthout, A. Cherukuri, S. K. Pierce, and M. E. Linder, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Marilyn Resh and Dr. Xiquan Liang for generously providing us the IC16. We also thank Dr. Shoshana Levy and Dr. Tsipi Shoham for providing the CD81 specific mAb, 5A6.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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