CD20 Homo-oligomers Physically Associate with the B Cell Antigen Receptor

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. 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 and BD Transduction goat anti-rabbit IgG (H + L) conjugated to was from PA). conjugated to was from B subunit conjugated to was purchased from and was from peroxidase, peroxidase, and goat anti-mouse IgG (H + were from and For cell surface was purchased from and used to the or BJAB B cells were in digitonin and and and was in the for examining proteins. For Ramos cells were before with for at After the was at for at was at for at in some The lysates were to and with for by the addition of protein protein or the were at for with and and with the In CD20 was immunoprecipitates were in of and with of at for proteins were and in Cell were with F(ab′)2 anti-human of at the and Cell was with the addition of an of After at was and the was to and were by and to were in bovine serum and using the indicated in the were using and using a for were as for analysis that and was used in were and to at for and at for were in PBS, for in and for with of of and The membrane was in and with and for with and with and using the of were from Ramos cells or BJAB cells as described previously (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar) using After were and of were with for For were and as described C.M. R. L. M. Deans J.P. Int. Immunol. 2007; PubMed Scopus Google Scholar). The were in of and in and as described (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar, C.M. R. L. M. Deans J.P. Int. Immunol. 2007; PubMed Scopus Google Scholar) using the indicated in the of were as described C.M. R. L. M. Deans J.P. Int. Immunol. 2007; PubMed Scopus Google Scholar, L.J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in and with the and by a were at for at and the were the cell from were with an of and and with a of and in the the at the were and as described for of for immunofluorescence and analysis of were described in previously (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar, C.M. R. L. M. Deans J.P. Int. Immunol. 2007; PubMed Scopus Google Scholar). CD20 homo-oligomerization of CD20 without using chemical we its to with tagged or truncated forms of CD20 that be by from native CD20. We first used BJAB cells expressing CD20 to whether CD20-GFP with CD20. In these cells, CD20-GFP is to the plasma membrane with no intracellular and and with CD20, as by immunofluorescence not Anti-GFP or were used to CD20-GFP from digitonin and the immunoprecipitates were by shown in CD20 was detected in association with the of CD20 monomers in we used a truncated form of CD20 (CD20TR) that at the C antibody against a was used to CD20WT and CD20TR be detected using an antibody against a near the N terminus CD20WT and CD20TR were with one at a the of association was to the of CD20 in of CD20WT cDNA were into HEK293 cells with a of CD20TR cDNA to that in expression of CD20TR and expression of these we found that the of coprecipitation of CD20TR with CD20WT was shown in second and with were using CD20WT with a of CD20. the and of immunoprecipitation of using CD20 as a after of differential T.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the of the is to the of were to to the form of CD20WT that with the detection of For the in cells were transfected with or with at were with before and The showed that of with coprecipitated CD20WT at an similar to from the in with CD20 with Cell of the CD20 in digitonin (>200 kDa) the of a indicating the of additional (12Polyak M.J. Deans J.P. Blood. 2002; 99: 3256-3262Crossref PubMed Scopus (134) Google Scholar). whether the CD20 other membrane proteins, BJAB cells were cell surface and CD20 was for analysis by Cell surface proteins of and were detected in these The protein with CD20, as shown by and The protein was not detected and was also observed in the The protein was not identified The at and with the heavy chains and light chains of sIgM from BJAB lysates with were using Ramos cells, that from these cells the association of CD20 with analysis was CD20 immunoprecipitates cellular was for was detected as a with the the at the plasma membrane and the an form Y. J. J. Immunol. PubMed Scopus (39) Google Scholar, PubMed Scopus Google Scholar). CD20 coprecipitated the of Igμ, indicating its association with In the CD20 was detected in immunoprecipitates analysis that of sIgM the of coprecipitated with and that the of CD20 coprecipitated with the of the association CD20 and we CD45 or MHCII from analysis not the of a to in these as it in the CD20 from the CD20 with sIgM have that CD20 and a of in (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, Nat. Rev. Immunol. 2002; 2: PubMed Scopus Google Scholar). shown in the of was detected in the of lysates from Ramos cells, and this was by to of A large of CD20 also to the that the association of CD20 with sIgM we whether the association was an component of Ramos B cells were or with the using previously R.J. Patel K.D. M. Deans J.P. J. Immunol. PubMed Scopus Google Scholar). The of was as described R.J. Patel K.D. M. Deans J.P. J. Immunol. PubMed Scopus Google Scholar). Additionally, digitonin to of cells in digitonin was and not CD20 was from or cells, there was no in the of coprecipitated sIgM that the association CD20 and sIgM was to be by or to be the of BCR from CD20 previously shown by immunofluorescence that sIgM and CD20, in resting cells, from one the cell surface of BCR at (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar). that of BCR-CD20 complexes these complexes 10% of it was that the was as a of at the cell we the of receptor cross-linking the of the Ramos cells were with or without F(ab′)2 before and CD20 After with F(ab′)2 the to sIgM were reduced in BCR was In cells that were at the was after and was by not The of CD20 and sIgM was not a of by the as F(ab′)2 not it was to the lysates although it to as by its in immunoprecipitation B and Additionally, not the BCR was using indicating a for After the BCR becomes in of its into to The of CD20 in digitonin is not as in A for the shown in is that the after as a of differential of CD20 and this using an differential is not an we used a L.J. J. Full Text Full Text PDF PubMed Scopus Google Scholar) that we previously and for immunofluorescence C.M. R. L. M. Deans J.P. Int. Immunol. 2007; PubMed Scopus Google Scholar). expressing CD20-GFP were either with to the BCR or with F(ab′)2 to and the BCR. The cells were without and to The or membrane were and using shown in without BCR cross-linking there is a of CD20 with similar to data from detergent-resistant after BCR cross-linking the of CD20 with sIgM These data that rapid of complexes receptor CD20 with and after BCR of CD20 is at after BCR S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), that associated signaling proteins its of complexes it to for of signaling proteins to CD20 after receptor for analysis of CD20 immunoprecipitates was using and anti-phosphoserine/threonine protein was detected in the CD20 after of receptor at A and but not at or points or proteins of and were detected after for and the involvement of CD20 in BCR-activated calcium influx, we for of calcium-sensitive signaling using a with D.J. S. A. PubMed Scopus Google Scholar). The calcium of the was first by the cell lysates in the or of calmodulin-binding proteins were detected in the of calcium but not in were CD20 immunoprecipitates from or B These association of CD20 with calmodulin-binding proteins of and The here is that the B cell antigen receptor with CD20 in The association is yet has not been previously into proteins. The BCR composed of and subunits, is preserved in digitonin and has been characterized by immunoprecipitation of surface-labeled proteins from digitonin cell lysates using In addition to and a protein that as an was identified as Ledbetter J.A. S. A. 1993; PubMed Scopus Google Scholar, J. Immunol. 1993; PubMed Scopus Google Scholar). to CD20 in these be to the of the protein and to the for cell surface In the of to surface CD20 is and is A in the be by or as a of or The kDa) was not detected in CD20 immunoprecipitates from Ramos or BJAB cells the of in sIgM and CD20 immunoprecipitates was using not it that CD20 with the BCR signaling with an of sIgM that at the cell surface without The BCR and CD20 in be as at the cell surface at the B cell (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). CD20 and sIgM are expressed at levels in B cells from with in the expression of sIgM is the expression of CD20 is also A. M.J. J. PubMed Scopus Google Scholar). expression of CD20 and the BCR, together with (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, S. Nature. 272: PubMed Scopus Google Scholar, S. J. PubMed Scopus (39) Google Scholar), physical association as and the involvement of CD20 in calcium influx downstream of BCR signaling (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, 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), an that to be Although CD20 has been to SOCE by BCR immune in CD20-deficient at to the to be (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, 1998; PubMed Scopus Google Scholar). of the MS4A family are expressed in B cells, and for the of CD20 by proteins is K. M. S. M. 2001; PubMed Scopus Google Scholar, Y. Tedder T.F. 2001; PubMed Scopus Google Scholar). or in the role of CD20 be to antigen that were not In addition to a cell surface protein of was detected in association with CD20. protein not to be and other proteins as or MHCII be was also observed CD20 was from transfected Molt4 T cells and Chinese hamster ovary cells not We the that be cell surface showed the of in CD20 immunoprecipitates as in the We to the protein by but and immunoglobulin in this of the The protein We also provide here the first direct evidence of CD20 homo-oligomerization into complexes. characterized ion channels are the Orai1 Icrac J. PubMed Scopus Google Scholar). The and of CD20 and in addition to are However, CD20 has no residue near the of the in the of the Orai1 (2Feske S. Nat. Rev. Immunol. 2007; 7: 690-702Crossref PubMed Scopus (764) Google Scholar) and that to form a is that CD20 of ion that regulates BCR-activated to the of CD20 CD20TR was to we that immunoprecipitation of CD20WT CD20WT was to we that immunoprecipitation of In the of coprecipitation were but in the of the it was the that was in the its In additional we found that as the increased in it a to form it was at levels be We that form protein with for of monomers from and that there is of components We previously showed in immunofluorescence that sIgM and CD20 colocalize at the plasma membrane in resting cells and that sIgM its from CD20 to (13Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (70) Google Scholar). is an at shown of the after receptor ligation, These data are not in with one although it be observed by immunofluorescence in whole cells a at has to the of of light The the of from CD20 is was not by of or family or of protein C not with the data that and signaling is not a or of of the receptor in to of CD20, by cross-linking of the BCR engagement also the physical of the antigen-binding and signaling components of the BCR is to as a for receptor in B cells J.C. Full Text Full Text PDF PubMed Scopus Google Scholar, K.M. M. J.C. J. Immunol. 2002; PubMed Scopus Google Scholar), as as to of the receptor L. H. B. J. Immunol. PubMed Scopus Google Scholar, J.A. Int. Immunol. 2007; PubMed Scopus Google Scholar). the cell surface M. J. Immunol. PubMed Scopus Google Scholar, P. J.C. J. A. J.C. 2001; PubMed Scopus Google indicated that not in association with CD20. BCR from CD20 be necessary to CD20 to at the cell surface are or it the function of CD20. BCR ligation, phosphoproteins and calmodulin-binding proteins were detected in CD20 it is that some of these proteins were in the to receptor ligation, by or after However, these proteins are to be involved in of complexes, as signaling does not to be We that are involved in the calcium or signaling functions of CD20 activation and of the of these proteins be in the role and of the detected were and we have not yet been to We have here that CD20 is organized at the cell surface as a in association with a of it with the receptor that triggers its involvement in calcium entry. association was detected in B cells not as as in B cell and it be in the to whether CD20 forms complexes with other the many cell surface proteins expressed by B cells, in addition to CD20 and have been to with the BCR R.A. J. Immunol. PubMed Scopus Google Scholar, Bolen J.B. J. Immunol. 1997; Google Scholar). one of has been to CD20 (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). is a component of the receptor that signaling by the BCR by the of (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) that calcium influx by cross-linking was in CD20-deficient B CD20 and function together in calcium influx BCR activation, an that to be We for the

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