Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Mar 15;90(6):2438–2442. doi: 10.1073/pnas.90.6.2438

Subcellular localization of T-cell receptor complexes containing tyrosine-phosphorylated zeta proteins in immature CD4+CD8+ thymocytes.

K P Kearse 1, D L Wiest 1, A Singer 1
PMCID: PMC46102  PMID: 7681596

Abstract

The T-cell antigen receptor (TCR) is a complex of at least six different proteins (alpha, beta, gamma, delta, epsilon, and zeta) that is assembled in the endoplasmic reticulum (ER) and transported to the cell surface. Unlike mature T cells, most immature CD4+CD8+ thymocytes retain within the ER and degrade greater than 90% of some of the TCR components they synthesize, resulting in low surface expression of TCR complexes. The few surface TCR complexes that most immature CD4+CD8+ thymocytes do express are only marginally capable of transducing signals mobilizing intracellular calcium. The inverse relationship with TCR expression and function suggested that phosphorylated zeta (P-zeta) molecules might function in CD4+CD8+ thymocytes either as an ER retention signal for newly synthesized TCR complexes or as a negative regulatory modification of TCR complexes present on the cell surface. The present study sought to evaluate these two possibilities by determining the subcellular location of TCR complexes containing P-zeta chains. We found that, unlike unmodified zeta chains, all P-zeta chains in CD4+CD8+ thymocytes existed in assembled TCR complexes and that all TCR complexes containing P-zeta molecules had undergone carbohydrate processing events indicative of transit through the Golgi apparatus. These results demonstrate that P-zeta chains are exclusively associated with mature TCR complexes, excluding the possibility that P-zeta serves as an ER retention signal in immature thymocytes. Although we could not directly determine the representation of P-zeta chains among surface TCR complexes, we found that 60-70% of surface TCR complexes on immature CD4+CD8+ thymocytes were associated with tyrosine-phosphorylated protein(s) and that this percentage was inversely correlated with their signaling competence. These results support the concept that tyrosine phosphorylation serves as a negative regulatory modification of certain TCR-associated proteins.

Full text

PDF
2438

Images in this article

2439Fig. 1
on p.2439
2440Fig. 2
on p.2440
2440Fig. 3
on p.2440
2440Fig. 4
on p.2440

Selected References

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

  1. Alarcon B., Berkhout B., Breitmeyer J., Terhorst C. Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains. J Biol Chem. 1988 Feb 25;263(6):2953–2961. [PubMed] [Google Scholar]
  2. Anderson P., Caligiuri M., Ritz J., Schlossman S. F. CD3-negative natural killer cells express zeta TCR as part of a novel molecular complex. Nature. 1989 Sep 14;341(6238):159–162. doi: 10.1038/341159a0. [DOI] [PubMed] [Google Scholar]
  3. Bonifacino J. S., Lippincott-Schwartz J., Chen C., Antusch D., Samelson L. E., Klausner R. D. Association and dissociation of the murine T cell receptor associated protein (TRAP). Early events in the biosynthesis of a multisubunit receptor. J Biol Chem. 1988 Jun 25;263(18):8965–8971. [PubMed] [Google Scholar]
  4. Bonifacino J. S., McCarthy S. A., Maguire J. E., Nakayama T., Singer D. S., Klausner R. D., Singer A. Novel post-translational regulation of TCR expression in CD4+CD8+ thymocytes influenced by CD4. Nature. 1990 Mar 15;344(6263):247–251. doi: 10.1038/344247a0. [DOI] [PubMed] [Google Scholar]
  5. Cenciarelli C., Hou D., Hsu K. C., Rellahan B. L., Wiest D. L., Smith H. T., Fried V. A., Weissman A. M. Activation-induced ubiquitination of the T cell antigen receptor. Science. 1992 Aug 7;257(5071):795–797. doi: 10.1126/science.1323144. [DOI] [PubMed] [Google Scholar]
  6. Chan A. C., Irving B. A., Fraser J. D., Weiss A. The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9166–9170. doi: 10.1073/pnas.88.20.9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunphy W. G., Brands R., Rothman J. E. Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell. 1985 Feb;40(2):463–472. doi: 10.1016/0092-8674(85)90161-8. [DOI] [PubMed] [Google Scholar]
  8. Finkel T. H., McDuffie M., Kappler J. W., Marrack P., Cambier J. C. Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking. Nature. 1987 Nov 12;330(6144):179–181. doi: 10.1038/330179a0. [DOI] [PubMed] [Google Scholar]
  9. Frank S. J., Niklinska B. B., Orloff D. G., Merćep M., Ashwell J. D., Klausner R. D. Structural mutations of the T cell receptor zeta chain and its role in T cell activation. Science. 1990 Jul 13;249(4965):174–177. doi: 10.1126/science.2371564. [DOI] [PubMed] [Google Scholar]
  10. Havran W. L., Poenie M., Kimura J., Tsien R., Weiss A., Allison J. P. Expression and function of the CD3-antigen receptor on murine CD4+8+ thymocytes. Nature. 1987 Nov 12;330(6144):170–173. doi: 10.1038/330170a0. [DOI] [PubMed] [Google Scholar]
  11. Irving B. A., Weiss A. The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. Cell. 1991 Mar 8;64(5):891–901. doi: 10.1016/0092-8674(91)90314-o. [DOI] [PubMed] [Google Scholar]
  12. Klausner R. D., Lippincott-Schwartz J., Bonifacino J. S. The T cell antigen receptor: insights into organelle biology. Annu Rev Cell Biol. 1990;6:403–431. doi: 10.1146/annurev.cb.06.110190.002155. [DOI] [PubMed] [Google Scholar]
  13. Klausner R. D., Samelson L. E. T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell. 1991 Mar 8;64(5):875–878. doi: 10.1016/0092-8674(91)90310-u. [DOI] [PubMed] [Google Scholar]
  14. Kubo R. T., Born W., Kappler J. W., Marrack P., Pigeon M. Characterization of a monoclonal antibody which detects all murine alpha beta T cell receptors. J Immunol. 1989 Apr 15;142(8):2736–2742. [PubMed] [Google Scholar]
  15. Lanier L. L., Yu G., Phillips J. H. Co-association of CD3 zeta with a receptor (CD16) for IgG Fc on human natural killer cells. Nature. 1989 Dec 14;342(6251):803–805. doi: 10.1038/342803a0. [DOI] [PubMed] [Google Scholar]
  16. Leo O., Foo M., Sachs D. H., Samelson L. E., Bluestone J. A. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1374–1378. doi: 10.1073/pnas.84.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Letourneur F., Klausner R. D. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8905–8909. doi: 10.1073/pnas.88.20.8905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lotan R., Beattie G., Hubbell W., Nicolson G. L. Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins. Biochemistry. 1977 May 3;16(9):1787–1794. doi: 10.1021/bi00628a004. [DOI] [PubMed] [Google Scholar]
  19. McCarthy S. A., Kruisbeek A. M., Uppenkamp I. K., Sharrow S. O., Singer A. Engagement of the CD4 molecule influences cell surface expression of the T-cell receptor on thymocytes. Nature. 1988 Nov 3;336(6194):76–79. doi: 10.1038/336076a0. [DOI] [PubMed] [Google Scholar]
  20. Mellman I., Simons K. The Golgi complex: in vitro veritas? Cell. 1992 Mar 6;68(5):829–840. doi: 10.1016/0092-8674(92)90027-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nakayama T., June C. H., Munitz T. I., Sheard M., McCarthy S. A., Sharrow S. O., Samelson L. E., Singer A. Inhibition of T cell receptor expression and function in immature CD4+CD8+ cells by CD4. Science. 1990 Sep 28;249(4976):1558–1561. doi: 10.1126/science.2120773. [DOI] [PubMed] [Google Scholar]
  22. Nakayama T., Samelson L. E., Nakayama Y., Munitz T. I., Sheard M., June C. H., Singer A. Ligand-stimulated signaling events in immature CD4+CD8+ thymocytes expressing competent T-cell receptor complexes. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9949–9953. doi: 10.1073/pnas.88.22.9949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nakayama T., Singer A., Hsi E. D., Samelson L. E. Intrathymic signalling in immature CD4+CD8+ thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain. Nature. 1989 Oct 19;341(6243):651–654. doi: 10.1038/341651a0. [DOI] [PubMed] [Google Scholar]
  24. Orloff D. G., Ra C. S., Frank S. J., Klausner R. D., Kinet J. P. Family of disulphide-linked dimers containing the zeta and eta chains of the T-cell receptor and the gamma chain of Fc receptors. Nature. 1990 Sep 13;347(6289):189–191. doi: 10.1038/347189a0. [DOI] [PubMed] [Google Scholar]
  25. Samelson L. E., Phillips A. F., Luong E. T., Klausner R. D. Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4358–4362. doi: 10.1073/pnas.87.11.4358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Samelson L. E., Weissman A. M., Robey F. A., Berkower I., Klausner R. D. Characterization of an anti-peptide antibody that recognizes the murine analogue of the human T cell antigen receptor-T3 delta-chain. J Immunol. 1986 Nov 15;137(10):3254–3258. [PubMed] [Google Scholar]
  27. Sussman J. J., Bonifacino J. S., Lippincott-Schwartz J., Weissman A. M., Saito T., Klausner R. D., Ashwell J. D. Failure to synthesize the T cell CD3-zeta chain: structure and function of a partial T cell receptor complex. Cell. 1988 Jan 15;52(1):85–95. doi: 10.1016/0092-8674(88)90533-8. [DOI] [PubMed] [Google Scholar]
  28. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  29. Wegener A. M., Letourneur F., Hoeveler A., Brocker T., Luton F., Malissen B. The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Cell. 1992 Jan 10;68(1):83–95. doi: 10.1016/0092-8674(92)90208-t. [DOI] [PubMed] [Google Scholar]
  30. Weissman A. M., Frank S. J., Orloff D. G., Merćep M., Ashwell J. D., Klausner R. D. Role of the zeta chain in the expression of the T cell antigen receptor: genetic reconstitution studies. EMBO J. 1989 Dec 1;8(12):3651–3656. doi: 10.1002/j.1460-2075.1989.tb08539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES