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Originally published In Press as doi:10.1074/jbc.M800784200 on May 12, 2008

J. Biol. Chem., Vol. 283, Issue 27, 18545-18552, July 4, 2008
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CD20 Homo-oligomers Physically Associate with the B Cell Antigen Receptor

DISSOCIATION UPON RECEPTOR ENGAGEMENT AND RECRUITMENT OF PHOSPHOPROTEINS AND CALMODULIN-BINDING PROTEINS*

Maria J. Polyak1, Haidong Li2, Neda Shariat, and Julie P. Deans3

From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, January 30, 2008 , and in revised form, April 17, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
B cell antigen receptor (BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation, and activation of B lymphocytes. CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. Using coprecipitation of native CD20 with tagged or truncated forms of the molecule, we provide here direct evidence of CD20 homo-oligomerization into tetramers. Additionally, the function of CD20 was explored by examining its association with surface-labeled and intracellular proteins before and after BCR signaling. Two major surface-labeled proteins that coprecipitated with CD20 were identified as the heavy and light chains of cell surface IgM, the antigen-binding components of the BCR. After activation, BCR-CD20 complexes dissociated, and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. These data provide new evidence of the involvement of CD20 in signaling downstream of the BCR and, together with the previously described involvement of CD20 in calcium influx, the first evidence of physical coupling of the BCR to a calcium entry pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signaling from the BCR,4 which is composed of antigen-binding immunoglobulin (Ig) and signaling Ig{alpha}/Igβ subunits, is necessary for the development, differentiation, and activation of B lymphocytes. Tyrosine kinase-dependent activation of phospholipase C-{gamma}2, one of many intracellular signaling events that follow BCR ligation, leads to rapid release of calcium from the lumen of the endoplasmic reticulum into the cytoplasm. Depletion of the intracellular calcium stores triggers a second phase of calcium mobilization, characterized by sustained calcium entry into the cell via plasma membrane-associated "store-operated" calcium entry (SOCE) channels (1, 2). The amplitude and duration of the calcium response is translated into differential gene expression by calcium-sensitive proteins, determining the cellular response to receptor engagement (3–5).

Key molecular components of SOCE have recently been identified with the discovery and characterization of the endoplasmic reticulum calcium sensor, STIM1, and the pore-forming subunit of the Icrac channel, Orai1 (2, 4, 6). A point mutation in Orai1 causes a form of hereditary severe combined immune deficiency syndrome, emphasizing the importance of intracellular calcium in lymphocyte physiology. However, although SOCE is absent in T lymphocytes from these patients, SOCE in B lymphocytes is reduced but not abolished, indicating additional SOCE pathways in the B cell lineage (1, 7).

Increasing evidence has indicated that CD20, a B cell-restricted member of the MS4A (multispanning 4A) family of tetraspanins, functions in calcium entry. Intracellular calcium levels were increased in non-B cells ectopically expressing CD20 by transfection, and a calcium-mediated current, similar to that observed in B cells, was detected by whole cell patch clamp analysis (8, 9). BCR-activated calcium entry was reduced after siRNA-mediated down-regulation of CD20 in a human B cell line (10) and in CD20-deficient murine B cells (11). A large increase in calcium influx was observed in thapsigargin-treated Chinese hamster ovary cells expressing CD20, demonstrating that CD20 is involved in SOCE (10). The accumulated evidence thus firmly points to a role for CD20 in BCR-activated SOCE, although it is not yet clear whether CD20 is a channel, a component of a channel, or if it regulates SOCE in some other way. Although there is no direct evidence that CD20 forms an ion channel, it does assemble into multimeric molecular complexes. After chemical cross-linking of proximal cell surface proteins, CD20 immunoprecipitates in 70-kDa and 140-kDa complexes, in addition to 33–35-kDa forms of monomeric CD20, suggestive of homo-oligomerization (8). Subsequently, we found that large CD20 complexes (>200 kDa) were preserved in digitonin lysates (12), allowing analysis without the need for exogenous cross-linkers. Here, by coprecipitation of native CD20 with tagged or truncated forms of the protein, we provide direct evidence that CD20 monomers are indeed assembled into homo-tetrameric complexes.

We showed previously by immunofluorescence that CD20 and the BCR colocalize in the plasma membrane of resting B cells (13). In this report, CD20 is shown to be expressed in constitutive physical association with cell surface immunoglobulin (sIgM), the antigen-binding component of the BCR. After BCR ligation, the CD20 oligomer dissociates from sIgM and becomes transiently associated with phosphoproteins and calmodulin-binding proteins. These data provide new evidence of the involvement of CD20 in signaling downstream of the BCR and, together with the previously described involvement of CD20 in SOCE, the first evidence of physical coupling of the BCR to a calcium entry pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—BJAB and Ramos lymphoma B cell lines were maintained in RPMI 1640 medium and 7.5% fetal bovine serum. BJAB cells expressing CD20-GFP and Molt4 T cells expressing either wild-type CD20 (CD20WT) or CD20 truncated at residue 278 (CD20TR) were previously described and were grown in RPMI 1640 medium and 10% fetal bovine serum with 0.4 mg/ml Geneticin (Invitrogen) (14, 15). CD20WT and CD20TR cDNA constructs were subcloned into the pCDM8 mammalian expression vector (Invitrogen) and transfected into HEK293 cells using the calcium phosphate method as described (12).

Antibodies and Reagents—Polyclonal rabbit antisera directed against intracellular epitopes at or near the N or C terminus of CD20 (anti-CD20N or anti-CD20C, respectively) were described previously (13, 16). L26 (anti-CD20) monoclonal antibody was purchased from Abcam (Cambridge, MA). Anti-GFP was from Eusera (Edmonton, Alberta, Canada). For immunoprecipitation of MHCII and CD45, we used HB10 and 9.4, respectively, from Dr. J. Ledbetter (Seattle, WA). For detection of CD45 by immunoblotting, we used anti-CD45 from BD Transduction Laboratories (Lexington, KY). Goat anti-human Igµ, F(ab')2, and Fab, unlabeled and conjugated to biotin, Cy3, or horseradish peroxidase, were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Cy3-conjugated streptavidin, Cy3-conjugated goat anti-rabbit IgG (H + L), and Cy3-conjugated F(ab')2 goat anti-mouse IgG (H + L) were also from Jackson ImmunoResearch Laboratories. Anti-phosphotyrosine (4G10) and anti-phosphoserine/threonine were from Millipore (Billerica, MA) and BD Transduction Laboratories, respectively. Affinity-purified goat anti-rabbit IgG (H + L) conjugated to IR dye 700 DX was from Rockland (Gilbertsville, PA). Streptavidin conjugated to IR dye 800 CW was from Li-Cor Biosciences (Lincoln, NE). Cholera toxin B subunit conjugated to biotin was purchased from Sigma, and biotinylated calmodulin was from Calbiochem. Protein A-horseradish peroxidase, neutralite avidin-horseradish peroxidase, and goat anti-mouse IgG (H + L)-horseradish peroxidase were obtained from Bio-Rad, Southern Biotech (Birmingham, AL), and Chemicon International, Inc (Temecula, CA), respectively. For cell surface biotinylation, EZ-Link sulfo-NHS-LC-biotin was purchased from Pierce and used according to the manufacturer's directions.

Immunoprecipitation—Ramos or BJAB B cells were lysed in buffer containing 1% digitonin (Calbiochem) and protease and phosphatase inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM NaVO4, 1 mM NaMoO4, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA). Microcystin-LR (Sigma) (1 mM) was included in the lysis buffer for experiments examining serine/threonine-phosphorylated proteins. For cholesterol depletion, Ramos cells (107 cells/ml) were treated before lysis with 10 mM methyl-β-cyclodextrin (Sigma) for 15 min at room temperature. After lysis, the detergent-insoluble material was pelleted at 13,000 x g for 15 min at 4 °C. Residual detergent-insoluble material was cleared at 100,000 x g for 1 h at 4 °C in some experiments. The soluble lysates were transferred to clean chilled tubes and incubated with antibodies for 2 h on ice followed by the addition of protein A-Sepharose (Repligen, Cambridge, MA), protein G-Sepharose (Santa Cruz Biotechnology), or avidin-agarose (Pierce) depending upon the immunoprecipitating antibody. Samples were rotated at 4 °C for 2 h, pelleted, washed with lysis buffer, alternating between high salt-containing (500 mM NaCl) and low salt-containing (150 mM NaCl) buffers, and finally washed once with PBS; all wash buffers contained the inhibitors listed above. In experiments where CD20 dephosphorylation was required, immunoprecipitates were resuspended in 50 µl of 10 mM Tris (pH 6.8) and treated with 4 units of alkaline phosphatase (Amersham Biosciences) at 37 °C for 1 h. Immunoprecipitated proteins were eluted and solubilized in 2x SDS sample buffer.

Cell Stimulation—Cells were incubated with F(ab')2 anti-human Igµ (50 µg of antibody/108 cells) at the temperatures and times indicated. Cell stimulation was halted with the addition of an equal volume of 2x lysis buffer. After at least 15 min on ice, detergent-insoluble matter was pelleted, and the soluble lysate was transferred to clean microfuge tubes.

Western Blotting and Calmodulin Overlay Assays—Proteins were separated by SDS-PAGE under reducing conditions and transferred to Immobilon-P membranes (Millipore). Membranes were blocked in 5% bovine serum albumin and blotted using the reagents indicated in the figure legends. Proteins were visualized using enhanced chemiluminescence (Pierce) and recorded using a Fluor-S MAX imager (Bio-Rad).

Samples for calmodulin overlay were prepared essentially as for Western blot analysis except that 1 mM CaCl2, Tris-buffered saline (20 mM Tris (pH 7.5), and 150 mM NaCl) buffer was used in all steps. Samples were run on SDS-PAGE reducing gels and transferred to nitrocellulose (Bio-Rad) at 50 V for 1.5 h and then at 100 V for 30 min. Membranes were washed in PBS, blocked for 1.5 h in 1 mM CaCl2/Odyssey infrared imaging system blocking buffer (Li-Cor Biosciences), and incubated for 1.5 h with 4 mg of biotinylated calmodulin/ml of blocking buffer containing 1 mM CaCl2 and 0.1% Tween. The membrane was then washed four times in 0.1% Tween 20 and PBS; incubated with streptavidin-800 CW, 0.1% Tween 20, and 1 mM CaCl2/blocking buffer for 1 h; washed with 0.1% Tween 20 and PBS; rinsed with PBS; and scanned using the Odyssey system.

Isolation of Detergent-resistant Membranes—Detergent-resistant membranes (DRMs) were isolated from 108 Ramos cells or 5 x 107 BJAB cells as described previously (13) using 1% Brij58. After sucrose gradient centrifugation, eight fractions (1.5 ml) were collected, and aliquots of each fraction were mixed with SDS sample buffer for Western blot analysis. For microscopy, fractions containing DRMs were diluted and pelleted as described (17). The DRMs were resuspended in 200 µl of Tris buffer (10 mM Tris and 150 mM NaCl), fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA), and stained as described (13, 17) using the reagents indicated in the figure legends.

Isolation of Detergent-free Raft Membranes—Detergent-free raft membranes were isolated as described (17, 18). Briefly, cells were suspended in ice-cold 250 mM sucrose, 1 mM CaCl2, 1 mM MgCl2, and 20 mM Tris (pH 7.8) with the inhibitors listed above and then sheared by repeated passage through a 22G 3-inch needle. Samples were centrifuged at 1,000 x g for 10 min at 4 °C, and the shearing/centrifugation steps were repeated on the cell pellets. Supernatants from each round were combined, mixed with an equal volume of 50% iodixanol (Optiprep, Axis-Shield PoCAs, Oslo, Norway), 250 mM sucrose, and 120 mM Tris (pH 7.8), and overlayered with a step gradient consisting of 20 and 5% iodixanol in the same buffer. Following centrifugation, the membranes at the 5–20% interface were collected and treated as described above for DRMs.

Immunofluorescence Microscopy—Preparation of samples for immunofluorescence microscopy and analysis of colocalization were described in detail previously (13, 17).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CD20 Homo-oligomerization—To examine homo-oligomerization of CD20 without using chemical cross-linking, we tested its ability to coprecipitate with tagged or truncated forms of CD20 that could be distinguished by size from native CD20. We first used BJAB cells expressing GFP-conjugated CD20 (CD20-GFP) to ask whether CD20-GFP formed mixed oligomers with endogenous CD20. In these cells, CD20-GFP is localized to the plasma membrane with no detectable intracellular pool and colocalizes and co-caps with endogenous CD20, as assessed by immunofluorescence microscopy (data not shown). Anti-GFP or anti-CD20 antibodies were used to immunoprecipitate CD20-GFP from digitonin lysates, and the immunoprecipitates were probed by anti-CD20 immunoblotting. As shown in Fig. 1A, endogenous CD20 was readily detected in association with CD20-GFP.

To assess the number of CD20 monomers in each oligomeric complex, we used a truncated form of CD20 (CD20TR) that lacked 20 amino acids at the C terminus. An antibody raised against a C-terminal peptide (anti-CD20C) was used to specifically immunoprecipitate CD20WT, whereas both CD20WT and CD20TR could be detected on immunoblots using an antibody raised against a peptide near the N terminus (anti-CD20N) (Fig. 1B). When CD20WT and CD20TR were co-expressed with one construct at a limiting concentration, the ratio of their association was expected to predict the number of CD20 molecules in each complex. Titrated amounts of CD20WT cDNA were cotransfected into HEK293 cells with a fixed amount of CD20TR cDNA to determine concentrations that would result in high expression of CD20TR and low expression of CD20WT. Under these conditions (Fig. 1C, fourth lane), we found that the ratio of coprecipitation of CD20TR with CD20WT was 1:3 (Fig. 1C, duplicate samples shown in second and third lanes), consistent with homo-tetramers.


Figure 1
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  		FIGURE 1.
Homo-tetrameric configuration of CD20. A, endogenous CD20 coprecipitates with CD20-GFP. Lysates from BJAB cells expressing CD20-GFP were subjected to immunoprecipitation (IP) with anti-GFP or anti-CD20, followed by CD20 immunoblotting. n = 4. CT, non-immune rabbit IgG. B, a specificity control experiment for C is shown. Lysates from Molt4 cells expressing CD20WT or CD20 truncated at the C terminus (CD20TR) were subjected to immunoprecipitation using anti-CD20N, which was generated against a peptide near the N terminus, or anti-CD20C, which was generated against a C-terminal peptide. CD20WT and CD20TR were detected on immunoblots using anti-CD20N. Anti-CD20C immunoprecipitated CD20WT but not CD20TR. C, lysates from HEK293 cells cotransfected with 5 µg of CD20WT cDNA and 15 µg of CD20TR cDNA were subjected to immunoprecipitation with anti-CD20C and immunoblotting using anti-CD20N (duplicate samples shown). The relative density of the bands is shown below the lanes, as a ratio of CD20WT:CD20TR. n = 2. D, specificity control for E is shown. Lysates from HEK293 cells expressing HA-tagged or untagged CD20 were subjected to immunoprecipitation with anti-CD20 or anti-HA, followed by CD20 immunoblotting. Note that anti-HA is equally effective at immunoprecipitating CD20-HA and that there is no nonspecific immunoprecipitation of untagged CD20. CD20 migrates as a doublet, and the position of CD20-HA on the gels is close to the upper band of CD20WT; therefore, immunoprecipitated samples were subjected to alkaline phosphatase (AP) treatment to eliminate the slower migrating form of CD20WT that would otherwise interfere with the detection of CD20-HA (lower panel). E, lysates from HEK293 cells cotransfected with 15 µg of CD20WT cDNA or 5 µg of CD20-HA cDNA, or both, were subjected to immunoprecipitation with anti-CD20 or anti-HA, followed by alkaline phosphatase treatment and CD20 immunoblotting. The relative density of the bands is shown as a ratio of CD20-HA:CD20WT. n = 2. In all experiments, cells were lysed in 1% digitonin.

 
Similar experiments were performed using CD20WT cotransfected with a limiting concentration of HA-tagged CD20. Fig. 1D demonstrates the efficiency and specificity of immunoprecipitation of CD20-HA using anti-HA. CD20 typically migrates as a doublet after SDS-PAGE, because of differential phosphorylation (19), and the position of CD20-HA on the gels is close to the upper band of CD20WT. Therefore, immunoprecipitated samples were subjected to alkaline phosphatase treatment to eliminate the slower migrating form of CD20WT that would otherwise interfere with the detection of CD20-HA (Fig. 1D, lower panel). For the experiments represented in Fig. 1E, cells were transfected with CD20WT, CD20-HA, or both with CD20-HA at limiting concentration. Immunoprecipitated samples were treated with alkaline phosphatase before SDS-PAGE and immunoblotting. The results showed that precipitation of CD20-HA with anti-HA coprecipitated CD20WT at an ~1:3 ratio, similar to results from the CD20WT/TR experiments represented in Fig. 1C, again consistent with tetrameric homo-oligomers.


Figure 2
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  		FIGURE 2.
Association of CD20 with cell surface immunoglobulin. A, CD20 was immunoprecipitated (IP) from lysates of surface biotinylated BJAB cells. Immunoprecipitates were analyzed by avidin blotting (upper panel), and then the membrane was stripped and reblotted for CD20 (lower panel). n > 25. B, lysates from biotinylated BJAB and Ramos cells were subjected to immunoprecipitation with anti-CD20 or anti-Igµ, followed by avidin blotting. Bands corresponding to immunoglobulin heavy and light chains are indicated (Igµ and IgL). n = 2. C, lysates from Ramos cells were subjected to immunoprecipitation using anti-CD20 or anti-Igµ. Samples were analyzed by anti-Igµ (upper panel; n = 5) or CD20 (lower panel; n = 2) immunoblotting. Lower exposure of the fourth lane (upper panel) shows the doublet of bands corresponding to the immature intracellular form of Igµ and the more heavily glycosylated, cell surface form (sIgµ). Only the upper band coprecipitated with CD20. Densitometry analysis indicated that 10.2% of total sIgµ coprecipitated with CD20, and 10.2% of total CD20 coprecipitated with sIgµ. D, lysates from biotinylated Ramos cells were subjected to immunoprecipitation with anti-CD45, anti-MHCII, or anti-CD20, followed by avidin or CD20 blotting as indicated. In all experiments, cells were lysed in 1% digitonin buffer. CT, preimmune serum control for anti-CD20, or avidin beads alone control for precipitation with biotin-conjugated F(ab')2 anti-Igµ.

 
CD20 Specifically Associates with Cell Surface Immunoglobulin—The size of the CD20 complex solubilized in digitonin (>200 kDa) exceeds the size of a tetramer (~140 kDa), indicating the inclusion of additional molecules (12). To examine whether the CD20 complex included other membrane proteins, BJAB cells were cell surface biotinylated, and then CD20 was immunoprecipitated for analysis by avidin blotting. Cell surface proteins of ~80, ~68, ~45, ~35, and ~29 kDa were detected in these experiments (Fig. 2A). The ~35-kDa protein comigrated with CD20, as shown by stripping and reblotting. The ~68-kDa protein was not always detected and was also sometimes observed in the control lane. The ~45-kDa protein was not identified (discussed below). The remaining two bands at 80 and 29 kDa comigrated with the heavy chains (sIgµ) and light chains (sIgL) of sIgM immunoprecipitated from BJAB lysates with anti-Igµ (Fig. 2B, left panel). Similar results were obtained using Ramos cells, except that sIgL from these cells migrated more slowly on SDS-PAGE gels (Fig. 2B, right panel).

To confirm the association of CD20 with sIgM, anti-Igµ Western blot analysis was performed on CD20 immunoprecipitates (Fig. 2C, upper panel). Total cellular IgM was immunoprecipitated for comparison; Igµ was detected as a doublet, with the upper band representing the mature glycoform at the plasma membrane and the lower band representing an intracellular, immature form (20, 21). CD20 coprecipitated only the upper band of Igµ, indicating its specific association with sIgM. In the reverse experiment, CD20 was detected in anti-Igµ immunoprecipitates (Fig. 2C, lower panel). Densitometry analysis estimated that 10.2% of total sIgM (measuring the upper band of Igµ only) coprecipitated with anti-CD20 and that the same proportion of total CD20 (10.2%) coprecipitated with anti-Igµ.

To further test the specificity of the association between CD20 and sIgM, we immunoprecipitated CD45 or MHCII from biotin-labeled cells. Avidin blot analysis did not reveal the presence of a band corresponding to sIgµ in these samples as it did in the immunoprecipitated CD20 sample from the same experiment (Fig. 2D).

CD20 Association with sIgM Is Cholesterol-independent—Previous studies have demonstrated that CD20 and a fraction of BCRs constitutively reside in lipid rafts (14, 22). As shown in Fig. 3A, only the upper band of Igµ (i.e. sIgM) was detected in the lipid raft fraction of lysates from unstimulated Ramos cells, and this was estimated by densitometry to represent 10.2% of total sIgM. A large proportion of CD20 also localized to the raft fraction, suggesting that the association of CD20 with sIgM may occur within lipid rafts; therefore, we examined whether the association was dependent upon cholesterol, an essential component of lipid rafts. Biotin-labeled Ramos B cells were untreated or treated with the cholesterol-depleting agent methyl-β-cyclodextrin using previously established conditions (23). The efficiency of cholesterol depletion was confirmed as described (23). Additionally, because digitonin binds to cholesterol, efficient lysis of methyl-β-cyclodextrin-treated cells in digitonin was tested and confirmed (data not shown). When CD20 was immunoprecipitated from untreated or cholesterol-depleted cells, there was no difference in the amount of coprecipitated sIgM (Fig. 3B), suggesting that the association between CD20 and sIgM was unlikely to be mediated by cholesterol or to be dependent on the integrity of rafts.

BCR Dissociates from CD20 upon Receptor Cross-linking—We had previously shown by immunofluorescence microscopy that sIgM and CD20, while colocalized in resting cells, laterally separate from one another on the cell surface within minutes of BCR ligation at 37 °C (13). This suggests that dissociation of BCR-CD20 complexes had occurred; however, because these complexes involve 10% of total sIgM, it was possible that the intact complex was retained as a minor subset of otherwise segregated receptors at the cell surface. Therefore, we next examined the effect of receptor cross-linking on the integrity of the isolated sIgM-CD20 complex. Biotin-labeled Ramos cells were incubated with or without F(ab')2 anti-Igµ before lysis and CD20 immunoprecipitation. After incubation with F(ab')2 anti-Igµ, the bands corresponding to sIgM were significantly reduced in intensity, even when BCR ligation was performed on ice (Fig. 4A). In cells that were stimulated over time at 37 °C, the dissociation was evident after 1 min and was complete by 5 min post-stimulation (data not shown). The dissociation of CD20 and sIgM was not a result of steric hindrance by the ligating antibody, as F(ab')2 anti-Igµ did not induce dissociation when it was added to the lysates (Fig. 4B), although it binds well to soluble sIgM, as evidenced by its use in immunoprecipitation (Fig. 2, B and C). Additionally, dissociation did not occur when the BCR was ligated using Fab anti-Igµ (Fig. 4C), indicating a requirement for cross-linking.


Figure 3
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  		FIGURE 3.
CD20-sIgM association is cholesterol-independent. A, Ramos cells were lysed in 1% Brij58, and lysates were subjected to sucrose density gradient centrifugation. Fractions 1–8 were collected from the top to the bottom of the gradients (P, pellet) and analyzed by immunoblotting as shown. G{alpha}i and Lyn blots identify the raft fraction as fraction 4; the absence of CD45 confirms a lack of contamination of this fraction with the non-raft membrane. The majority of CD20 and 10.2% of sIgµ (upper band, as seen more clearly in the lower exposure shown to the right of the figure) were found in fraction 4. B, biotinylated Ramos B cells were treated or untreated with 10 mM methyl-β-cyclodextrin before lysis. Lysates were subjected to immunoprecipitation (IP) using preimmune serum (CT) or anti-CD20 followed by avidin blotting (upper panel). Membranes were stripped and immunoblotted for CD20 (lower panel). n = 10.

 
After cross-linking, the BCR becomes largely insoluble in most detergents because of its translocation into lipid rafts and/or firm attachment to cytoskeletal elements. The solubility of CD20 in digitonin is not altered, as seen in Fig. 4, A–C. A trivial explanation for the results shown in Fig. 4, A–C, is that the dissociation occurred after lysis as a result of differential solubility of CD20 and sIgM. To examine this question using an assay where differential solubility is not an issue, we used a detergent-free lipid raft isolation procedure (18) that we had previously adapted and optimized for immunofluorescence microscopy (17). Cells expressing CD20-GFP were incubated on ice either with Fab anti-Igµ (Cy3) to label the BCR or with F(ab')2 anti-Igµ (Cy3) to both label and cross-link the BCR. The cells were then lysed without detergent and subjected to sucrose density gradient centrifugation. The buoyant, or light, membrane fragments were then imaged and analyzed using colocalization software. As shown in Fig. 4D, without BCR cross-linking there is a high frequency of CD20 colocalized with sIgM, similar to data obtained from detergent-resistant membranes (DRMs), whereas after BCR cross-linking the proportion of CD20 colocalized with sIgM drops significantly. These data demonstrate that rapid dissociation of sIgM-CD20 complexes occurs upon receptor engagement.

CD20 Associates with Phosphorylated and Calmodulin-binding Proteins after BCR Ligation—Phosphorylation of CD20 is enhanced at specific sites after BCR ligation (24), suggesting that associated signaling proteins could regulate its function. Rapid dissociation of sIgM-CD20 complexes made it possible to look for recruitment of signaling proteins specifically to CD20 after receptor engagement. To search for CD20-associated phosphoproteins, Western blot analysis of CD20 immunoprecipitates was performed using anti-phosphotyrosine and anti-phosphoserine/threonine antibodies. An ~60-kDa tyrosine-phosphorylated protein was reproducibly detected in the CD20 complex after 5 min of receptor stimulation at 37 °C (Fig. 5, A and B) but not at earlier or later time points (Fig. 5B). Serine- or threonine-phosphorylated proteins of ~100 kDa and >175 kDa were reproducibly detected after stimulation for 30 s and 1 min, respectively (Fig. 5C).

Given the involvement of CD20 in BCR-activated calcium influx, we next looked for recruitment of calcium-sensitive signaling molecules, using a calmodulin overlay assay with biotin-conjugated calmodulin (25). The calcium specificity of the overlay was first established by performing the calmodulin overlay on PAGE-resolved cell lysates in the presence or absence of calcium; numerous calmodulin-binding proteins were detected in the presence of calcium but not in EDTA-containing buffer (Fig. 6A). Calmodulin overlays were then performed on CD20 immunoprecipitates prepared from unstimulated or stimulated B cells. These experiments revealed transient association of CD20 with calmodulin-binding proteins of ~68 and ~83 kDa (Fig. 6B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The most important finding reported here is that the B cell antigen receptor associates with CD20 in unstimulated cells. The association is robust yet has not been reported previously despite intensive investigations into BCR-associated proteins. The BCR complex, composed of sIg and Ig{alpha}/β subunits, is preserved in digitonin and has been well characterized by immunoprecipitation of surface-labeled proteins from digitonin cell lysates using anti-IgM. In addition to Ig{alpha} and Igβ, a coprecipitating protein that appeared as an ~150-kDa band was identified as CD22 (26, 27). Failure to detect CD20 in these early experiments can be attributed to the size of the protein and to the very few residues available for cell surface labeling. In our experience, the success of biotin conjugation to surface CD20 is variable, and labeling is frequently poor. A relatively faint band in the 30–35-kDa region could well be obscured by sIgL or dismissed as a possible degradation product of Ig{alpha} or Igβ.


Figure 4
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  		FIGURE 4.
Dissociation of sIgM from CD20 upon receptor stimulation. A, biotinylated Ramos cells were incubated for 15 min on ice with or without F(ab')2 anti-Igµ and then lysed in digitonin. Lysates were then subjected to immunoprecipitation (IP) using either preimmune serum (CT) or anti-CD20, followed by avidin blotting (upper panel). Membranes were stripped and reblotted for CD20 (lower panel). B, as in A, except that F(ab')2 anti-Igµ was added either before (second lane) or after (fourth lane) lysis. n = 2. C, as in A, except that cells were incubated with either F(ab')2 anti-Igµ or Fab anti-Igµ. D, BJAB cells stably expressing CD20-GFP were incubated on ice either with Fab anti-Igµ (Cy3) to label the BCR, or with F(ab')2 anti-Igµ (Cy3) to both label and cross-link the BCR. Cells were either lysed in 1% Brij-58 for isolation of detergent-resistant membranes (DRM), or detergent-free raft "light" membranes (LM) were isolated as described under "Experimental Procedures." The mean percent of CD20 colocalized with sIgM was calculated from 6–10 image fields for each sample using CoLocalizer Pro software. The error bars represent S.E.M. The statistical significance was determined using an unpaired t test (p < 0.0001).

 
The Ig{alpha}/β heterodimer (48/39 kDa) was not clearly detected in CD20 immunoprecipitates from biotinylated Ramos or BJAB cells (see Fig. 2B); however, the presence of Igβ in both sIgM and CD20 immunoprecipitates was revealed using anti-Igβ immunoblots (data not shown). Therefore, it seems most likely that CD20 associates with the intact BCR signaling complex rather than with an unusual minor subset of sIgM that might exist at the cell surface without Ig{alpha}/β. The BCR and CD20 may in fact be coordinately expressed, as they appear at the cell surface at approximately the same time during B cell development (11). Furthermore, CD20 and sIgM are both characteristically expressed at diminished levels in malignant B cells from patients with chronic lymphocytic leukemia; in unusual chronic lymphocytic leukemia cases where the expression of sIgM is high, the expression of CD20 is also high (28). Coordinate expression of CD20 and the BCR, together with their colocalization on microvilli (14, 29, 30), their physical association as reported here, and the demonstrated involvement of CD20 in calcium influx downstream of BCR signaling (10, 11), suggests an important functional relationship that remains to be fully elucidated. Although CD20 has been linked to SOCE activated by BCR signals, humoral immune responses in CD20-deficient mice, at least to the antigens tested, appear to be normal (11, 31). Other members of the MS4A family are expressed in B cells, and compensation for the loss of CD20 by related proteins is possible (32, 33). Alternatively or in addition, the role of CD20 may be linked to specific antigen challenges that were not tested.

In addition to sIgM, a prominent cell surface protein of ~45 kDa (p45) was detected in association with CD20. This protein did not appear to be Ig{alpha} (see Fig. 2B). Indeed, Ig{alpha} and other candidate proteins such as CD23, CD40, or MHCII could be ruled out because p45 was also observed when CD20 was immunoprecipitated from transfected Molt4 T cells and Chinese hamster ovary cells (data not shown). We considered the possibility that p45 might be cell surface actin; however, actin immunoblots showed only the same trace amounts of actin in CD20 immunoprecipitates as in the control samples. We attempted to identify the protein by mass spectrometry, but actin and immunoglobulin degradation products obscured specific signals in this region of the gel. The 45-kDa protein therefore remains unidentified.

We also provide here the first direct evidence of CD20 homo-oligomerization into tetrameric complexes. Several well characterized ion channels are tetrameric, including the Orai1 Icrac channel (34). The size and topology of CD20 and Orai1, in addition to their tetrameric configuration, are remarkably similar. However, CD20 has no acidic residue near the position of the critical glutamate in the pore of the Orai1 channel (2) and on that basis seems unlikely to form a SOC channel itself. It is possible that CD20 constitutes another type of ion channel that indirectly regulates BCR-activated SOCE.

Experiments designed to determine the number of CD20 molecules per homo-oligomer yielded somewhat unexpected results. When CD20TR was overexpressed relative to CD20WT, we expected that immunoprecipitation of CD20WT would yield oligomers containing mostly CD20TR. Similarly, when CD20WT was overexpressed relative to CD20-HA, we expected that immunoprecipitation of CD20-HA would yield oligomers containing mostly CD20WT. In both cases the ratios of coprecipitation were 1:3 but in the reverse of the expected order, i.e. it was always the immunoprecipitated species that was most represented in the isolated oligomers, despite its lower abundance. In additional experiments, we found that as the immunoprecipitated species increased in abundance, it had a strong propensity to form homo-oligomers; only when it was present at very low relative levels could mixed oligomers be observed. We speculate that stable oligomers form immediately upon protein translation, with minimal opportunity for insertion of monomers produced from different ribosomes and that there is minimal subsequent mixing of components between fully formed oligomers.


Figure 5
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  		FIGURE 5.
CD20 associates with phosphorylated proteins after BCR stimulation. Ramos cells were unstimulated or stimulated with F(ab')2 anti-Igµ for the times and temperatures indicated. Cells were lysed in digitonin, and CD20 was immunoprecipitated from cleared lysates. Immunoprecipitates were probed using anti-phosphotyrosine (ptyr) (n = 4) (A) or anti-phosphoserine/threonine (pser/pthr) (n = 3) (B, upper panels). The membranes were stripped and reprobed for CD20 (B, lower panels).

 


Figure 6
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  		FIGURE 6.
Transient association of calmodulin-binding proteins with CD20 after BCR stimulation. A, Ramos cells were lysed in digitonin, and lysates were resolved and transferred to nitrocellulose. Calmodulin (CAM) overlays were performed in the presence of 1 mM calcium or 5 mM EDTA. This shows the calcium dependence and sensitivity of the overlay assay to detect calmodulin-binding proteins. B, Ramos cells were unstimulated or stimulated with F(ab')2 anti-Igµ antibody at 37 °C as indicated. Cells were lysed in digitonin, and CD20 was immunoprecipitated for analysis by calmodulin overlay assay (upper panel). Membranes were stripped and reblotted for CD20 (lower panel). n = 5.

 
We previously showed in immunofluorescence studies that sIgM and CD20 colocalize at the plasma membrane in resting cells and that sIgM ligation induces its lateral separation from CD20 prior to internalization (13). This is an active process requiring incubation at 37 °C. As shown here, dissociation of the sIgM-CD20 complex occurs rapidly after receptor ligation, even when performed on ice. These data are not in conflict with one another; although dissociation occurs rapidly it cannot be observed by immunofluorescence in whole cells until a few minutes at 37 °C has allowed sufficient lateral separation to overcome the limits of resolution of light microscopy.

The mechanism underlying the dissociation of ligated sIg from CD20 is unknown. It was not inhibited by potent pharmacological inhibition of Syk or Src family tyrosine kinases or of protein kinase C (data not shown), consistent with the data showing that dissociation occurs very rapidly and under cold conditions. Receptor signaling is therefore almost certainly not required. Dissociation more likely involves a conformational shift or realignment of transmembrane regions of the receptor in relation to those of CD20, induced by cross-linking of the receptor.

BCR engagement also destabilizes the sIg-Ig{alpha}/β complex; physical separation of the antigen-binding and signaling components of the BCR is thought to act as a mechanism for receptor desensitization in anergic B cells (35, 36), as well as to facilitate clathrin-mediated internalization of the receptor (37, 38). Most sIg-dissociated Ig{alpha}/β heterodimers remain on the cell surface (39, 40); however, our preliminary investigation indicated that Ig{alpha}/β did not remain in association with CD20. BCR dissociation from CD20 may be necessary to allow CD20 to remain at the cell surface while antigen-bound receptors are internalized, or it may regulate the function of CD20. Following BCR ligation, phosphoproteins and calmodulin-binding proteins were detected in CD20 complexes; it is possible that some of these proteins were present in the complex prior to receptor ligation, only becoming detectable by phosphoantibodies or calmodulin binding after ligation. However, these proteins are unlikely to be involved in dissociation of sIg-CD20 complexes, as signaling does not seem to be necessary. We speculate that they are involved in regulating the calcium channel or signaling functions of CD20 following activation and dissociation of the receptor. Identification of these proteins will be important in clarifying the role and regulation of CD20; however, the detected interactions were highly transient, and despite intensive efforts we have not yet been able to identify them.

We have demonstrated here that CD20 is organized at the cell surface as a tetramer in association with a subpopulation of sIg molecules, situating it with the receptor that triggers its involvement in calcium entry. sIgM-CD20 association was detected in primary (tonsil) B cells (data not shown), as well as in B cell lines, and it will be interesting in the future to determine whether CD20 forms complexes with other sIg isotypes. Among the many distinct cell surface proteins expressed by B cells, only two others, in addition to CD20 and CD22, have been reported to physically associate with the BCR (41, 42). Interestingly, one of these, CD19, has been functionally linked to CD20 (11). CD19 is a component of the CD21 complement receptor complex that amplifies signaling by the BCR when coligated by opsonized antigen. Reporting on the phenotype of CD20-knock-out mice, Uchida et al. (11) demonstrated that calcium influx activated by cross-linking CD19 was profoundly decreased in CD20-deficient B cells. CD20 and CD19/21 may function together in mediating calcium influx during complement-dependent BCR activation, an intriguing possibility that remains to be tested.


   FOOTNOTES
 
* This work was supported by an operating grant from the Canadian Institutes of Health Research. 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

1 Present address: Centre de recherche-Centre Hospitalier de I'Universitéde Montréal, Hopital Saint-Luc, Montreal, Quebec H2X 1P1, Canada. Back

2 Present address: Dept. of Biochemistry, Tianjin Medical University, Tianjin 300070, China. Back

3 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Calgary, Health Sciences Center, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada. Fax: 403-283-1267; E-mail: jdeans{at}ucalgary.ca.

4 The abbreviations used are: BCR, B cell antigen receptor; SOCE, store-operated calcium entry; sIg, cell surface immunoglobulin; GFP, green fluorescent protein; WT, wild-type; TR, truncated; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane; HA, hemagglutinin. Back


   ACKNOWLEDGMENTS
 
We thank Tammy Unruh for helpful comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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