CD20 Homo-oligomers Physically Associate with the B Cell Antigen Receptor
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.m800784200
ISSN1083-351X
AutoresMaria J. Polyak, Haidong Li, Neda Shariat, Julie P. Deans,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoB 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. 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. Signaling from the BCR, 4The 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. 4The 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. which is composed of antigen-binding immunoglobulin (Ig) and signaling Igα/Igβ subunits, is necessary for the development, differentiation, and activation of B lymphocytes. Tyrosine kinase-dependent activation of phospholipase C-γ2, 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 (1Engelke M. Engels N. Dittmann K. Stork B. Wienands J. Immunol. Rev. 2007; 218: 235-246Crossref PubMed Scopus (71) Google Scholar, 2Feske S. Nat. Rev. Immunol. 2007; 7: 690-702Crossref PubMed Scopus (764) Google Scholar). 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 (3Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1542) Google Scholar, 4Gwack Y. Feske S. Srikanth S. Hogan P.G. Rao A. Cell Calcium. 2007; 42: 145-156Crossref PubMed Scopus (242) Google Scholar, 5Scharenberg A.M. Humphries L.A. Rawlings D.J. Nat. Rev. Immunol. 2007; 7: 778-789Crossref PubMed Scopus (154) Google Scholar). 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 (2Feske S. Nat. Rev. Immunol. 2007; 7: 690-702Crossref PubMed Scopus (764) Google Scholar, 4Gwack Y. Feske S. Srikanth S. Hogan P.G. Rao A. Cell Calcium. 2007; 42: 145-156Crossref PubMed Scopus (242) Google Scholar, 6Luik R.M. Lewis R.S. Trends Mol. Med. 2007; 13: 103-107Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 (1Engelke M. Engels N. Dittmann K. Stork B. Wienands J. Immunol. Rev. 2007; 218: 235-246Crossref PubMed Scopus (71) Google Scholar, 7Feske S. Giltnane J. Dolmetsch R. Staudt L.M. Rao A. Nat. Immunol. 2001; 2: 316-324Crossref PubMed Scopus (492) Google Scholar). 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 (8Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (279) Google Scholar, 9Kanzaki M. Nie L. Shibata H. Kojima I. J. Biol. Chem. 1997; 272: 4964-4969Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). BCR-activated calcium entry was reduced after siRNA-mediated down-regulation of CD20 in a human B cell line (10Li H. Ayer L.M. Lytton J. Deans J.P. J. Biol. Chem. 2003; 278: 42427-42434Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and in CD20-deficient murine B cells (11Uchida J. Lee Y. Hasegawa M. Liang Y. Bradney A. Oliver J.A. Bowen K. Steeber D.A. Haas K.M. Poe J.C. Tedder T.F. Int. Immunol. 2004; 16: 119-129Crossref PubMed Scopus (198) Google Scholar). A large increase in calcium influx was observed in thapsigargin-treated Chinese hamster ovary cells expressing CD20, demonstrating that CD20 is involved in SOCE (10Li H. Ayer L.M. Lytton J. Deans J.P. J. Biol. Chem. 2003; 278: 42427-42434Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). 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 (8Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (279) Google Scholar). Subsequently, we found that large CD20 complexes (>200 kDa) were preserved in digitonin lysates (12Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (134) Google Scholar), 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 (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar). 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. 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) (14Li H. Ayer L.M. Polyak M.J. Mutch C.M. Petrie R.J. Gauthier L. Shariat N. Hendzel M.J. Shaw A.R. Patel K.D. Deans J.P. J. Biol. Chem. 2004; 279: 19893-19901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Deans J.P. Kalt L. Ledbetter J.A. Schieven G.L. Bolen J.B. Johnson P. J. Biol. Chem. 1995; 270: 22632-22638Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). 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 (12Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (134) Google Scholar). 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 (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar, 16Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar). 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 × g for 15 min at 4 °C. Residual detergent-insoluble material was cleared at 100,000 × 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 2× 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 2× 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 × 107 BJAB cells as described previously (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar) 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 (17Mutch C.M. Sanyal R. Unruh T.L. Grigoriou L. Zhu M. Zhang W. Deans J.P. Int. Immunol. 2007; 19: 19-30Crossref PubMed Scopus (21) Google Scholar). 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 (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar, 17Mutch C.M. Sanyal R. Unruh T.L. Grigoriou L. Zhu M. Zhang W. Deans J.P. Int. Immunol. 2007; 19: 19-30Crossref PubMed Scopus (21) Google Scholar) using the reagents indicated in the figure legends. Isolation of Detergent-free Raft Membranes—Detergent-free raft membranes were isolated as described (17Mutch C.M. Sanyal R. Unruh T.L. Grigoriou L. Zhu M. Zhang W. Deans J.P. Int. Immunol. 2007; 19: 19-30Crossref PubMed Scopus (21) Google Scholar, 18Macdonald J.L. Pike L.J. J. Lipid Res. 2005; 46: 1061-1067Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). 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 × 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 (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar, 17Mutch C.M. Sanyal R. Unruh T.L. Grigoriou L. Zhu M. Zhang W. Deans J.P. Int. Immunol. 2007; 19: 19-30Crossref PubMed Scopus (21) Google Scholar). 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. 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 (19Tedder T.F. Schlossman S.F. J. Biol. Chem. 1988; 263: 10009-10015Abstract Full Text PDF PubMed Google Scholar), 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. 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 (12Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (134) Google Scholar). 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 (20Bergman Y. Haimovich J. Eur. J. Immunol. 1978; 8: 876-880Crossref PubMed Scopus (39) Google Scholar, 21Melchers F. Biochemistry. 1972; 11: 2204-2208Crossref PubMed Scopus (34) Google Scholar). 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 (14Li H. Ayer L.M. Polyak M.J. Mutch C.M. Petrie R.J. Gauthier L. Shariat N. Hendzel M.J. Shaw A.R. Patel K.D. Deans J.P. J. Biol. Chem. 2004; 279: 19893-19901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22Pierce S.K. Nat. Rev. Immunol. 2002; 2: 96-105Crossref PubMed Scopus (270) Google Scholar). 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 (23Petrie R.J. Schnetkamp P.P. Patel K.D. Awasthi-Kalia M. Deans J.P. J. Immunol. 2000; 165: 1220-1227Crossref PubMed Scopus (128) Google Scholar). The efficiency of cholesterol depletion was confirmed as described (23Petrie R.J. Schnetkamp P.P. Patel K.D. Awasthi-Kalia M. Deans J.P. J. Immunol. 2000; 165: 1220-1227Crossref PubMed Scopus (128) Google Scholar). 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 (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar). 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. 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 (18Macdonald J.L. Pike L.J. J. Lipid Res. 2005; 46: 1061-1067Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) that we had previously adapted and optimized for immunofluorescence microscopy (17Mutch C.M. Sanyal R. Unruh T.L. Grigoriou L. Zhu M. Zhang W. Deans J.P. Int. Immunol. 2007; 19: 19-30Crossref PubMed Scopus (21) Google Scholar). 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 (24Valentine M.A. Meier K.E. Rossie S. Clark E.A. J. Biol. Chem. 1989; 264: 11282-11287Abstract Full Text PDF PubMed Google Scholar), 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 (25Billingsley M.L. Pennypacker K.R. Hoover C.G. Brigati D.J. Kincaid R.L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7585-7589Crossref PubMed Scopus (87) Google Scholar). The calcium specifici
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