Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Jun 1;121(5):1121–1132. doi: 10.1083/jcb.121.5.1121

Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes

PMCID: PMC2119683  PMID: 7684739

Abstract

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20- associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alt F., Rosenberg N., Lewis S., Thomas E., Baltimore D. Organization and reorganization of immunoglobulin genes in A-MULV-transformed cells: rearrangement of heavy but not light chain genes. Cell. 1981 Dec;27(2 Pt 1):381–390. doi: 10.1016/0092-8674(81)90421-9. [DOI] [PubMed] [Google Scholar]
  2. Arispe N., Rojas E., Hartman J., Sorscher E. J., Pollard H. B. Intrinsic anion channel activity of the recombinant first nucleotide binding fold domain of the cystic fibrosis transmembrane regulator protein. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1539–1543. doi: 10.1073/pnas.89.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnstable C. J., Bodmer W. F., Brown G., Galfre G., Milstein C., Williams A. F., Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell. 1978 May;14(1):9–20. doi: 10.1016/0092-8674(78)90296-9. [DOI] [PubMed] [Google Scholar]
  4. Bosma M., Sidell N. Retinoic acid inhibits Ca2+ currents and cell proliferation in a B-lymphocyte cell line. J Cell Physiol. 1988 May;135(2):317–323. doi: 10.1002/jcp.1041350220. [DOI] [PubMed] [Google Scholar]
  5. Brent L. H., Butler J. L., Woods W. T., Jr, Bubien J. K. Transmembrane ion conductance in human B lymphocyte activation. J Immunol. 1990 Oct 15;145(8):2381–2389. [PubMed] [Google Scholar]
  6. Bubien J. K., Kirk K. L., Rado T. A., Frizzell R. A. Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes. Science. 1990 Jun 15;248(4961):1416–1419. doi: 10.1126/science.2162561. [DOI] [PubMed] [Google Scholar]
  7. Calvo J. C., Radicella J. P., Charreau E. H. Measurement of specific radioactivities in labelled hormones by self-displacement analysis. Biochem J. 1983 May 15;212(2):259–264. doi: 10.1042/bj2120259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  9. Clark E. A., Shu G., Ledbetter J. A. Role of the Bp35 cell surface polypeptide in human B-cell activation. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1766–1770. doi: 10.1073/pnas.82.6.1766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Corcia A., Pecht I., Hemmerich S., Ran S., Rivnay B. Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells. Biochemistry. 1988 Sep 20;27(19):7499–7506. doi: 10.1021/bi00419a048. [DOI] [PubMed] [Google Scholar]
  11. Dani J. A. Site-directed mutagenesis and single-channel currents define the ionic channel of the nicotinic acetylcholine receptor. Trends Neurosci. 1989 Apr;12(4):125–128. doi: 10.1016/0166-2236(89)90049-0. [DOI] [PubMed] [Google Scholar]
  12. Einfeld D. A., Brown J. P., Valentine M. A., Clark E. A., Ledbetter J. A. Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J. 1988 Mar;7(3):711–717. doi: 10.1002/j.1460-2075.1988.tb02867.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Flockerzi V., Oeken H. J., Hofmann F., Pelzer D., Cavalié A., Trautwein W. Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel. Nature. 1986 Sep 4;323(6083):66–68. doi: 10.1038/323066a0. [DOI] [PubMed] [Google Scholar]
  14. Fukushima Y., Hagiwara S. Voltage-gated Ca2+ channel in mouse myeloma cells. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2240–2242. doi: 10.1073/pnas.80.8.2240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Genot E., Valentine M. A., Degos L., Sigaux F., Kolb J. P. Hyperphosphorylation of CD20 in hairy cells. Alteration by low molecular weight B cell growth factor and IFN-alpha. J Immunol. 1991 Feb 1;146(3):870–878. [PubMed] [Google Scholar]
  16. Goding J. W. Structural studies of murine lymphocyte surface IgD. J Immunol. 1980 May;124(5):2082–2088. [PubMed] [Google Scholar]
  17. Golay J. T., Clark E. A., Beverley P. C. The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle. J Immunol. 1985 Dec;135(6):3795–3801. [PubMed] [Google Scholar]
  18. Golay J. T., Crawford D. H. Pathways of human B-lymphocyte activation blocked by B-cell specific monoclonal antibodies. Immunology. 1987 Oct;62(2):279–284. [PMC free article] [PubMed] [Google Scholar]
  19. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  20. Hagiwara S., Ohmori H. Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp. J Physiol. 1982 Oct;331:231–252. doi: 10.1113/jphysiol.1982.sp014371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoth M., Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. doi: 10.1038/355353a0. [DOI] [PubMed] [Google Scholar]
  22. Hupp K., Siwarski D., Mock B. A., Kinet J. P. Gene mapping of the three subunits of the high affinity FcR for IgE to mouse chromosomes 1 and 19. J Immunol. 1989 Dec 1;143(11):3787–3791. [PubMed] [Google Scholar]
  23. Kansas G. S., Tedder T. F. Transmembrane signals generated through MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent and independent adhesion in human B cells through a tyrosine kinase-dependent pathway. J Immunol. 1991 Dec 15;147(12):4094–4102. [PubMed] [Google Scholar]
  24. Klein E., Ben-Bassat H., Neumann H., Ralph P., Zeuthen J., Polliack A., Vánky F. Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia. Int J Cancer. 1976 Oct 15;18(4):421–431. doi: 10.1002/ijc.2910180405. [DOI] [PubMed] [Google Scholar]
  25. Kondorosi E., Kay J. E. The role of calcium in lymphocyte activation by the ionophore A23187 and phytohaemagglutinin [proceedings]. Biochem Soc Trans. 1977;5(4):967–970. doi: 10.1042/bst0050967. [DOI] [PubMed] [Google Scholar]
  26. Krafte D. S., Snutch T. P., Leonard J. P., Davidson N., Lester H. A. Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes. J Neurosci. 1988 Aug;8(8):2859–2868. doi: 10.1523/JNEUROSCI.08-08-02859.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Krueger B. K. Toward an understanding of structure and function of ion channels. FASEB J. 1989 Jun;3(8):1906–1914. doi: 10.1096/fasebj.3.8.2470631. [DOI] [PubMed] [Google Scholar]
  28. Lambert J. M., Senter P. D., Yau-Young A., Blättler W. A., Goldmacher V. S. Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins. J Biol Chem. 1985 Oct 5;260(22):12035–12041. [PubMed] [Google Scholar]
  29. Lindau M., Fernandez J. M. IgE-mediated degranulation of mast cells does not require opening of ion channels. Nature. 1986 Jan 9;319(6049):150–153. doi: 10.1038/319150a0. [DOI] [PubMed] [Google Scholar]
  30. Nadler L. M., Korsmeyer S. J., Anderson K. C., Boyd A. W., Slaughenhoupt B., Park E., Jensen J., Coral F., Mayer R. J., Sallan S. E. B cell origin of non-T cell acute lymphoblastic leukemia. A model for discrete stages of neoplastic and normal pre-B cell differentiation. J Clin Invest. 1984 Aug;74(2):332–340. doi: 10.1172/JCI111428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Noda M., Ikeda T., Suzuki H., Takeshima H., Takahashi T., Kuno M., Numa S. Expression of functional sodium channels from cloned cDNA. 1986 Aug 28-Sep 3Nature. 322(6082):826–828. doi: 10.1038/322826a0. [DOI] [PubMed] [Google Scholar]
  32. Oettgen H. C., Bayard P. J., Van Ewijk W., Nadler L. M., Terhorst C. P. Further biochemical studies of the human B-cell differentiation antigens B1 and B2. Hybridoma. 1983;2(1):17–28. doi: 10.1089/hyb.1983.2.17. [DOI] [PubMed] [Google Scholar]
  33. Olsen R. W., Tobin A. J. Molecular biology of GABAA receptors. FASEB J. 1990 Mar;4(5):1469–1480. doi: 10.1096/fasebj.4.5.2155149. [DOI] [PubMed] [Google Scholar]
  34. Ran S., Rivnay B. Activation of rat basophilic leukemia cells. Temporal identification of the signal calcium influx mediated by the receptor-operated channel pathway. Eur J Biochem. 1988 Feb 1;171(3):693–701. doi: 10.1111/j.1432-1033.1988.tb13841.x. [DOI] [PubMed] [Google Scholar]
  35. Rosenthal P., Rimm I. J., Umiel T., Griffin J. D., Osathanondh R., Schlossman S. F., Nadler L. M. Ontogeny of human hematopoietic cells: analysis utilizing monoclonal antibodies. J Immunol. 1983 Jul;131(1):232–237. [PubMed] [Google Scholar]
  36. Smeland E., Godal T., Ruud E., Beiske K., Funderud S., Clark E. A., Pfeifer-Ohlsson S., Ohlsson R. The specific induction of myc protooncogene expression in normal human B cells is not a sufficient event for acquisition of competence to proliferate. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6255–6259. doi: 10.1073/pnas.82.18.6255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sommer B., Seeburg P. H. Glutamate receptor channels: novel properties and new clones. Trends Pharmacol Sci. 1992 Jul;13(7):291–296. doi: 10.1016/0165-6147(92)90088-n. [DOI] [PubMed] [Google Scholar]
  38. Stamenkovic I., Seed B. Analysis of two cDNA clones encoding the B lymphocyte antigen CD20 (B1, Bp35), a type III integral membrane protein. J Exp Med. 1988 Jun 1;167(6):1975–1980. doi: 10.1084/jem.167.6.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stashenko P., Nadler L. M., Hardy R., Schlossman S. F. Characterization of a human B lymphocyte-specific antigen. J Immunol. 1980 Oct;125(4):1678–1685. [PubMed] [Google Scholar]
  40. Stashenko P., Nadler L. M., Hardy R., Schlossman S. F. Expression of cell surface markers after human B lymphocyte activation. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3848–3852. doi: 10.1073/pnas.78.6.3848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stevens C. F. Molecular neurobiology. Channel families in the brain. Nature. 1987 Jul 16;328(6127):198–199. doi: 10.1038/328198b0. [DOI] [PubMed] [Google Scholar]
  42. Tanabe T., Beam K. G., Powell J. A., Numa S. Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature. 1988 Nov 10;336(6195):134–139. doi: 10.1038/336134a0. [DOI] [PubMed] [Google Scholar]
  43. Tedder T. F., Boyd A. W., Freedman A. S., Nadler L. M., Schlossman S. F. The B cell surface molecule B1 is functionally linked with B cell activation and differentiation. J Immunol. 1985 Aug;135(2):973–979. [PubMed] [Google Scholar]
  44. Tedder T. F., Clement L. T., Cooper M. D. Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody. J Immunol. 1984 Aug;133(2):678–683. [PubMed] [Google Scholar]
  45. Tedder T. F., Disteche C. M., Louie E., Adler D. A., Croce C. M., Schlossman S. F., Saito H. The gene that encodes the human CD20 (B1) differentiation antigen is located on chromosome 11 near the t(11;14)(q13;q32) translocation site. J Immunol. 1989 Apr 1;142(7):2555–2559. [PubMed] [Google Scholar]
  46. Tedder T. F., Forsgren A., Boyd A. W., Nadler L. M., Schlossman S. F. Antibodies reactive with the B1 molecule inhibit cell cycle progression but not activation of human B lymphocytes. Eur J Immunol. 1986 Aug;16(8):881–887. doi: 10.1002/eji.1830160802. [DOI] [PubMed] [Google Scholar]
  47. Tedder T. F., Klejman G., Disteche C. M., Adler D. A., Schlossman S. F., Saito H. Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19. J Immunol. 1988 Dec 15;141(12):4388–4394. [PubMed] [Google Scholar]
  48. Tedder T. F., McIntyre G., Schlossman S. F. Heterogeneity in the B1 (CD20) cell surface molecule expressed by human B-lymphocytes. Mol Immunol. 1988 Dec;25(12):1321–1330. doi: 10.1016/0161-5890(88)90047-8. [DOI] [PubMed] [Google Scholar]
  49. Tedder T. F., Schlossman S. F. Phosphorylation of the B1 (CD20) molecule by normal and malignant human B lymphocytes. J Biol Chem. 1988 Jul 15;263(20):10009–10015. [PubMed] [Google Scholar]
  50. Tedder T. F., Streuli M., Schlossman S. F., Saito H. Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes. Proc Natl Acad Sci U S A. 1988 Jan;85(1):208–212. doi: 10.1073/pnas.85.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tsien R. Y., Pozzan T., Rink T. J. T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes. Nature. 1982 Jan 7;295(5844):68–71. doi: 10.1038/295068a0. [DOI] [PubMed] [Google Scholar]
  52. Valentine M. A., Cotner T., Gaur L., Torres R., Clark E. A. Expression of the human B-cell surface protein CD20: alteration by phorbol 12-myristate 13-acetate. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8085–8089. doi: 10.1073/pnas.84.22.8085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. White J. R., Ishizaka T., Ishizaka K., Sha'afi R. Direct demonstration of increased intracellular concentration of free calcium as measured by quin-2 in stimulated rat peritoneal mast cell. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3978–3982. doi: 10.1073/pnas.81.13.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zimmer D. B., Green C. R., Evans W. H., Gilula N. B. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J Biol Chem. 1987 Jun 5;262(16):7751–7763. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES