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. 1989 Dec 1;8(12):3641–3649. doi: 10.1002/j.1460-2075.1989.tb08538.x

Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells.

A Pelchen-Matthews 1, J E Armes 1, M Marsh 1
PMCID: PMC402046  PMID: 2583114

Abstract

The internalization of CD4, a T cell differentiation antigen and the receptor for the human immunodeficiency viruses (HIV-1 and -2), has been examined in HeLa and murine 3T3 cells transfected with CD4 cDNA. Fab' fragments of the anti-CD4 monoclonal antibody Leu3a were generated by pepsin digestion and used as a specific monovalent, non-crosslinking ligand for CD4. These Fab' fragments were shown to bind to CD4 on the transfected cells with an affinity similar to that of HIV gp120, and inhibited HIV infection of lymphocytic cells. The Fab' fragments were radioiodinated and used in an acid-stripping endocytosis assay to demonstrate that the CD4 expressed on transfected HeLa and NIH3T3 cells was internalized. Approximately 1.5-2% of the total cell-bound [125I]Fab' fragments were internalized per minute. Furthermore, the internalized [125I]Fab' fragments could be shown to recycle to the cell surface. After 30-60 min a steady state was reached between internalization and recycling, with approximately 30-40% of the total cellular CD4 pool residing inside the cell. Similar results were obtained in studies with the intact divalent radiolabelled Leu3a antibody. These data demonstrate that CD4 expressed on transfected non-lymphoid cells is constitutively endocytosed and recycled.

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Selected References

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

  1. Acres R. B., Conlon P. J., Mochizuki D. Y., Gallis B. Rapid phosphorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen. J Biol Chem. 1986 Dec 5;261(34):16210–16214. [PubMed] [Google Scholar]
  2. Anderson R. G., Brown M. S., Beisiegel U., Goldstein J. L. Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J Cell Biol. 1982 Jun;93(3):523–531. doi: 10.1083/jcb.93.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bank I., Chess L. Perturbation of the T4 molecule transmits a negative signal to T cells. J Exp Med. 1985 Oct 1;162(4):1294–1303. doi: 10.1084/jem.162.4.1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bedinger P., Moriarty A., von Borstel R. C., 2nd, Donovan N. J., Steimer K. S., Littman D. R. Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4. Nature. 1988 Jul 14;334(6178):162–165. doi: 10.1038/334162a0. [DOI] [PubMed] [Google Scholar]
  5. Bleil J. D., Bretscher M. S. Transferrin receptor and its recycling in HeLa cells. EMBO J. 1982;1(3):351–355. doi: 10.1002/j.1460-2075.1982.tb01173.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blue M. L., Hafler D. A., Craig K. A., Levine H., Schlossman S. F. Phosphorylation of CD4 and CD8 molecules following T cell triggering. J Immunol. 1987 Dec 15;139(12):3949–3954. [PubMed] [Google Scholar]
  7. Brown M. S., Goldstein J. L. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3330–3337. doi: 10.1073/pnas.76.7.3330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carrel S., Moretta A., Pantaleo G., Tambussi G., Isler P., Perussia B., Cerottini J. C. Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody. Eur J Immunol. 1988 Mar;18(3):333–339. doi: 10.1002/eji.1830180303. [DOI] [PubMed] [Google Scholar]
  9. Clapham P. R., Weiss R. A., Dalgleish A. G., Exley M., Whitby D., Hogg N. Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: receptor modulation and differentiation induced by phorbol ester. Virology. 1987 May;158(1):44–51. doi: 10.1016/0042-6822(87)90236-4. [DOI] [PubMed] [Google Scholar]
  10. Cruikshank W. W., Berman J. S., Theodore A. C., Bernardo J., Center D. M. Lymphokine activation of T4+ T lymphocytes and monocytes. J Immunol. 1987 Jun 1;138(11):3817–3823. [PubMed] [Google Scholar]
  11. Dalgleish A. G., Beverley P. C., Clapham P. R., Crawford D. H., Greaves M. F., Weiss R. A. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature. 1984 Dec 20;312(5996):763–767. doi: 10.1038/312763a0. [DOI] [PubMed] [Google Scholar]
  12. Doyle C., Strominger J. L. Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature. 1987 Nov 19;330(6145):256–259. doi: 10.1038/330256a0. [DOI] [PubMed] [Google Scholar]
  13. Ekblom P., Thesleff I., Lehto V. P., Virtanen I. Distribution of the transferrin receptor in normal human fibroblasts and fibrosarcoma cells. Int J Cancer. 1983 Jan 15;31(1):111–117. doi: 10.1002/ijc.2910310118. [DOI] [PubMed] [Google Scholar]
  14. Gay D., Maddon P., Sekaly R., Talle M. A., Godfrey M., Long E., Goldstein G., Chess L., Axel R., Kappler J. Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature. 1987 Aug 13;328(6131):626–629. doi: 10.1038/328626a0. [DOI] [PubMed] [Google Scholar]
  15. Goldmacher V. S., Tinnel N. L., Nelson B. C. Evidence that pinocytosis in lymphoid cells has a low capacity. J Cell Biol. 1986 Apr;102(4):1312–1319. doi: 10.1083/jcb.102.4.1312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoxie J. A., Matthews D. M., Callahan K. J., Cassel D. L., Cooper R. A. Transient modulation and internalization of T4 antigen induced by phorbol esters. J Immunol. 1986 Aug 15;137(4):1194–1201. [PubMed] [Google Scholar]
  17. Hoxie J. A., Rackowski J. L., Haggarty B. S., Gaulton G. N. T4 endocytosis and phosphorylation induced by phorbol esters but not by mitogen or HIV infection. J Immunol. 1988 Feb 1;140(3):786–795. [PubMed] [Google Scholar]
  18. Klatzmann D., Champagne E., Chamaret S., Gruest J., Guetard D., Hercend T., Gluckman J. C., Montagnier L. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature. 1984 Dec 20;312(5996):767–768. doi: 10.1038/312767a0. [DOI] [PubMed] [Google Scholar]
  19. Kornfeld H., Cruikshank W. W., Pyle S. W., Berman J. S., Center D. M. Lymphocyte activation by HIV-1 envelope glycoprotein. Nature. 1988 Sep 29;335(6189):445–448. doi: 10.1038/335445a0. [DOI] [PubMed] [Google Scholar]
  20. Lamoyi E., Nisonoff A. Preparation of F(ab')2 fragments from mouse IgG of various subclasses. J Immunol Methods. 1983 Jan 28;56(2):235–243. doi: 10.1016/0022-1759(83)90415-5. [DOI] [PubMed] [Google Scholar]
  21. Lanzavecchia A., Roosnek E., Gregory T., Berman P., Abrignani S. T cells can present antigens such as HIV gp120 targeted to their own surface molecules. Nature. 1988 Aug 11;334(6182):530–532. doi: 10.1038/334530a0. [DOI] [PubMed] [Google Scholar]
  22. Lasky L. A., Nakamura G., Smith D. H., Fennie C., Shimasaki C., Patzer E., Berman P., Gregory T., Capon D. J. Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor. Cell. 1987 Sep 11;50(6):975–985. doi: 10.1016/0092-8674(87)90524-1. [DOI] [PubMed] [Google Scholar]
  23. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  24. Maddon P. J., Littman D. R., Godfrey M., Maddon D. E., Chess L., Axel R. The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell. 1985 Aug;42(1):93–104. doi: 10.1016/s0092-8674(85)80105-7. [DOI] [PubMed] [Google Scholar]
  25. Maddon P. J., McDougal J. S., Clapham P. R., Dalgleish A. G., Jamal S., Weiss R. A., Axel R. HIV infection does not require endocytosis of its receptor, CD4. Cell. 1988 Sep 9;54(6):865–874. doi: 10.1016/s0092-8674(88)91241-x. [DOI] [PubMed] [Google Scholar]
  26. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [PubMed] [Google Scholar]
  27. McClure M. O., Marsh M., Weiss R. A. Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism. EMBO J. 1988 Feb;7(2):513–518. doi: 10.1002/j.1460-2075.1988.tb02839.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McDougal J. S., Nicholson J. K., Cross G. D., Cort S. P., Kennedy M. S., Mawle A. C. Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry. J Immunol. 1986 Nov 1;137(9):2937–2944. [PubMed] [Google Scholar]
  29. Parnes J. R. Molecular biology and function of CD4 and CD8. Adv Immunol. 1989;44:265–311. doi: 10.1016/s0065-2776(08)60644-6. [DOI] [PubMed] [Google Scholar]
  30. Pauza C. D., Price T. M. Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis. J Cell Biol. 1988 Sep;107(3):959–968. doi: 10.1083/jcb.107.3.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rivas A., Takada S., Koide J., Sonderstrup-McDevitt G., Engleman E. G. CD4 molecules are associated with the antigen receptor complex on activated but not resting T cells. J Immunol. 1988 May 1;140(9):2912–2918. [PubMed] [Google Scholar]
  32. Rosoff P. M., Burakoff S. J., Greenstein J. L. The role of the L3T4 molecule in mitogen and antigen-activated signal transduction. Cell. 1987 Jun 19;49(6):845–853. doi: 10.1016/0092-8674(87)90622-2. [DOI] [PubMed] [Google Scholar]
  33. Rudd C. E., Trevillyan J. M., Dasgupta J. D., Wong L. L., Schlossman S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5190–5194. doi: 10.1073/pnas.85.14.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Saizawa K., Rojo J., Janeway C. A., Jr Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor. Nature. 1987 Jul 16;328(6127):260–263. doi: 10.1038/328260a0. [DOI] [PubMed] [Google Scholar]
  35. Schwartz A. L., Fridovich S. E., Lodish H. F. Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J Biol Chem. 1982 Apr 25;257(8):4230–4237. [PubMed] [Google Scholar]
  36. Siliciano R. F., Knall C., Lawton T., Berman P., Gregory T., Reinherz E. L. Recognition of HIV glycoprotein gp120 by T cells. Role of monocyte CD4 in the presentation of gp120. J Immunol. 1989 Mar 1;142(5):1506–1511. [PubMed] [Google Scholar]
  37. Siliciano R. F., Lawton T., Knall C., Karr R. W., Berman P., Gregory T., Reinherz E. L. Analysis of host-virus interactions in AIDS with anti-gp120 T cell clones: effect of HIV sequence variation and a mechanism for CD4+ cell depletion. Cell. 1988 Aug 12;54(4):561–575. doi: 10.1016/0092-8674(88)90078-5. [DOI] [PubMed] [Google Scholar]
  38. Sleckman B. P., Bigby M., Greenstein J. L., Burakoff S. J., Sy M. S. Requirements for modulation of the CD4 molecule in response to phorbol myristate acetate. Role of the cytoplasmic domain. J Immunol. 1989 Mar 1;142(5):1457–1462. [PubMed] [Google Scholar]
  39. Sleckman B. P., Peterson A., Jones W. K., Foran J. A., Greenstein J. L., Seed B., Burakoff S. J. Expression and function of CD4 in a murine T-cell hybridoma. Nature. 1987 Jul 23;328(6128):351–353. doi: 10.1038/328351a0. [DOI] [PubMed] [Google Scholar]
  40. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  41. Stein B. S., Gowda S. D., Lifson J. D., Penhallow R. C., Bensch K. G., Engleman E. G. pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell. 1987 Jun 5;49(5):659–668. doi: 10.1016/0092-8674(87)90542-3. [DOI] [PubMed] [Google Scholar]
  42. Steinman R. M., Brodie S. E., Cohn Z. A. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976 Mar;68(3):665–687. doi: 10.1083/jcb.68.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tite J. P., Sloan A., Janeway C. A., Jr The role of L3T4 in T cell activation: L3T4 may be both an Ia-binding protein and a receptor that transduces a negative signal. J Mol Cell Immunol. 1986;2(4):179–190. [PubMed] [Google Scholar]
  44. Ukkonen P., Lewis V., Marsh M., Helenius A., Mellman I. Transport of macrophage Fc receptors and Fc receptor-bound ligands to lysosomes. J Exp Med. 1986 Apr 1;163(4):952–971. doi: 10.1084/jem.163.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  46. Veillette A., Bookman M. A., Horak E. M., Samelson L. E., Bolen J. B. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989 Mar 16;338(6212):257–259. doi: 10.1038/338257a0. [DOI] [PubMed] [Google Scholar]
  47. Wassmer P., Chan C., Lögdberg L., Shevach E. M. Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells. J Immunol. 1985 Oct;135(4):2237–2242. [PubMed] [Google Scholar]
  48. Weiss R. A., Clapham P. R., Weber J. N., Dalgleish A. G., Lasky L. A., Berman P. W. Variable and conserved neutralization antigens of human immunodeficiency virus. Nature. 1986 Dec 11;324(6097):572–575. doi: 10.1038/324572a0. [DOI] [PubMed] [Google Scholar]
  49. Weyand C. M., Goronzy J., Fathman C. G. Modulation of CD4 by antigenic activation. J Immunol. 1987 Mar 1;138(5):1351–1354. [PubMed] [Google Scholar]
  50. von Figura K., Gieselmann V., Hasilik A. Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts. EMBO J. 1984 Jun;3(6):1281–1286. doi: 10.1002/j.1460-2075.1984.tb01963.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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