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. 1985 Dec;62(3):579–585.

Binding of interleukin 2 to target cells: determination of high and low affinity binding sites on T-lymphoblasts by differential dissociation.

H J Horst, H D Flad
PMCID: PMC1577481  PMID: 3936657

Abstract

A new method for the determination of both low and high affinity binding sites on interleukin 2 (IL-2) target cells is described, which is based on differential dissociation of the ligand receptor complex. The technique requires highly purified but not radiolabelled IL-2. In the presence of activated charcoal the low affinity binding sites had a dissociation half time of less than 1 min, while that of the high affinity binding sites was 80 min. Human T-lymphocytes expressed both classes of binding sites within 48 h after PHA stimulation. During culture of PHA-stimulated T-lymphocytes, low affinity binding sites appeared on day 2 and remained high until day 8. High affinity binding sites appeared on day 1, were highest on day 2, and remained high until day 6. The changes in the concentration of the high affinity binding sites are considered as being the result of: receptor stimulation by PHA and/or IL-2 (days 1-2); receptor down regulation by extensive IL-2 production (day 3); increase of unoccupied IL-2 receptor after disappearance of IL-2 from the medium (days 4-6) and cessation of receptor synthesis and receptor breakdown (days 7-10).

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Selected References

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

  1. Bonnard G. D., Yasaka K., Jacobson D. Ligand-activated T cell growth factor-induced proliferation: absorption of T cell growth factor by activated T cells. J Immunol. 1979 Dec;123(6):2704–2708. [PubMed] [Google Scholar]
  2. Cantrell D. A., Smith K. A. Transient expression of interleukin 2 receptors. Consequences for T cell growth. J Exp Med. 1983 Dec 1;158(6):1895–1911. doi: 10.1084/jem.158.6.1895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dunn W. A., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Cell Biol. 1984 Jun;98(6):2148–2159. doi: 10.1083/jcb.98.6.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fagnani R., Cooper H. L., Mendelsohn J. Purification of human interleukin 2 to apparent homogeneity and partial characterization of its receptor. Anal Biochem. 1984 Nov 1;142(2):487–496. doi: 10.1016/0003-2697(84)90494-9. [DOI] [PubMed] [Google Scholar]
  5. Gillis S., Ferm M. M., Ou W., Smith K. A. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978 Jun;120(6):2027–2032. [PubMed] [Google Scholar]
  6. Gorden P., Carpentier J. L., Cohen S., Orci L. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5025–5029. doi: 10.1073/pnas.75.10.5025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gorden P., Carpentier J. L., Freychet P. O., Orci L. Internalization of polypeptide hormones: mechanism, intracellular localization and significance. Diabetologia. 1980 Apr;18(4):263–274. doi: 10.1007/BF00251003. [DOI] [PubMed] [Google Scholar]
  8. Henderson L. E., Hewetson J. F., Hopkins R. F., 3rd, Sowder R. C., Neubauer R. H., Rabin H. A rapid, large scale purification procedure for gibbon interleukin 2. J Immunol. 1983 Aug;131(2):810–815. [PubMed] [Google Scholar]
  9. Hunter T., Ling N., Cooper J. A. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 1984 Oct 4;311(5985):480–483. doi: 10.1038/311480a0. [DOI] [PubMed] [Google Scholar]
  10. Hunter T. The epidermal growth factor receptor gene and its product. Nature. 1984 Oct 4;311(5985):414–416. doi: 10.1038/311414a0. [DOI] [PubMed] [Google Scholar]
  11. Kindberg G. M., Ford T., Blomhoff R., Rickwood D., Berg T. Separation of endocytic vesicles in Nycodenz gradients. Anal Biochem. 1984 Nov 1;142(2):455–462. doi: 10.1016/0003-2697(84)90489-5. [DOI] [PubMed] [Google Scholar]
  12. Krupp M., Lane M. D. On the mechanism of ligand-induced down-regulation of insulin receptor level in the liver cell. J Biol Chem. 1981 Feb 25;256(4):1689–1694. [PubMed] [Google Scholar]
  13. Larsson E. L. Mechanism of T cell activation. II. Antigen- and lectin-dependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells. J Immunol. 1981 Apr;126(4):1323–1326. [PubMed] [Google Scholar]
  14. Leonard W. J., Depper J. M., Uchiyama T., Smith K. A., Waldmann T. A., Greene W. C. A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor; partial characterization of the receptor. Nature. 1982 Nov 18;300(5889):267–269. doi: 10.1038/300267a0. [DOI] [PubMed] [Google Scholar]
  15. Milgrom E., Baulieu E. E. A method for studying binding proteins, based upon differential dissociation of small ligand. Biochim Biophys Acta. 1969 Dec 23;194(2):602–605. doi: 10.1016/0005-2795(69)90124-x. [DOI] [PubMed] [Google Scholar]
  16. Poulson R. The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria. J Biol Chem. 1976 Jun 25;251(12):3730–3733. [PubMed] [Google Scholar]
  17. Reem G. H., Yeh N. H. Interleukin 2 regulates expression of its receptor and synthesis of gamma interferon by human T lymphocytes. Science. 1984 Jul 27;225(4660):429–430. doi: 10.1126/science.6429853. [DOI] [PubMed] [Google Scholar]
  18. Robb R. J., Greene W. C. Direct demonstration of the identity of T cell growth factor binding protein and the Tac antigen. J Exp Med. 1983 Oct 1;158(4):1332–1337. doi: 10.1084/jem.158.4.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robb R. J., Munck A., Smith K. A. T cell growth factor receptors. Quantitation, specificity, and biological relevance. J Exp Med. 1981 Nov 1;154(5):1455–1474. doi: 10.1084/jem.154.5.1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Taylor M. V., Metcalfe J. C., Hesketh T. R., Smith G. A., Moore J. P. Mitogens increase phosphorylation of phosphoinositides in thymocytes. 1984 Nov 29-Dec 5Nature. 312(5993):462–465. doi: 10.1038/312462a0. [DOI] [PubMed] [Google Scholar]
  21. Uchiyama T., Broder S., Waldmann T. A. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J Immunol. 1981 Apr;126(4):1393–1397. [PubMed] [Google Scholar]

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