Skip to main content
PMC 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 Apr 15;90(8):3611–3615. doi: 10.1073/pnas.90.8.3611

Thy-1 glycoprotein and src-like protein-tyrosine kinase p53/p56lyn are associated in large detergent-resistant complexes in rat basophilic leukemia cells.

L Dráberová 1, P Dráber 1
PMCID: PMC46351  PMID: 7682713

Abstract

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cells (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with src-related protein-tyrosine kinase p53/p56lyn and other protein-tyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large ( > 10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.

Full text

PDF
3615

Images in this article

3612Fig. 1
on p.3612
3612Fig. 2
on p.3612
3612Fig. 3
on p.3612
3613Fig. 4
on p.3613
3613Fig. 5
on p.3613
3614Fig. 6
on p.3614
3614Fig. 7
on p.3614

Selected References

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

  1. Apgar J. R. Antigen-induced cross-linking of the IgE receptor leads to an association with the detergent-insoluble membrane skeleton of rat basophilic leukemia (RBL-2H3) cells. J Immunol. 1990 Dec 1;145(11):3814–3822. [PubMed] [Google Scholar]
  2. Benhamou M., Gutkind J. S., Robbins K. C., Siraganian R. P. Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5327–5330. doi: 10.1073/pnas.87.14.5327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  4. Cinek T., Horejsí V. The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol. 1992 Oct 1;149(7):2262–2270. [PubMed] [Google Scholar]
  5. Dráber P., Pokorná Z. Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies. Cell Differ. 1984 Dec;15(2-4):109–113. doi: 10.1016/0045-6039(84)90060-5. [DOI] [PubMed] [Google Scholar]
  6. Dráber P., Zikán J., Vogtísková M. Establishment and characterization of permanent murine hybridomas secreting monoclonal anti-thy-1 antibodies. J Immunogenet. 1980 Dec;7(6):455–474. doi: 10.1111/j.1744-313x.1980.tb00741.x. [DOI] [PubMed] [Google Scholar]
  7. Dráberová L., Dráber P. Functional expression of the endogenous Thy-1 gene and the transfected murine Thy-1.2 gene in rat basophilic leukemia cells. Eur J Immunol. 1991 Jul;21(7):1583–1590. doi: 10.1002/eji.1830210703. [DOI] [PubMed] [Google Scholar]
  8. Dráberová L. The involvement of Thy-1 antigen in the activation of rat mast cells. Eur J Immunol. 1989 Sep;19(9):1715–1720. doi: 10.1002/eji.1830190928. [DOI] [PubMed] [Google Scholar]
  9. Eiseman E., Bolen J. B. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature. 1992 Jan 2;355(6355):78–80. doi: 10.1038/355078a0. [DOI] [PubMed] [Google Scholar]
  10. Gunter K. C., Malek T. R., Shevach E. M. T cell-activating properties of an anti-Thy-1 monoclonal antibody. Possible analogy to OKT3/Leu-4. J Exp Med. 1984 Mar 1;159(3):716–730. doi: 10.1084/jem.159.3.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. He H. T., Naquet P., Caillol D., Pierres M. Thy-1 supports adhesion of mouse thymocytes to thymic epithelial cells through a Ca2(+)-independent mechanism. J Exp Med. 1991 Feb 1;173(2):515–518. doi: 10.1084/jem.173.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoessli D., Rungger-Brändle E. Association of specific cell-surface glycoproteins with a triton X-100-resistant complex of plasma membrane proteins isolated from T-lymphoma cells (P1798). Exp Cell Res. 1985 Jan;156(1):239–250. doi: 10.1016/0014-4827(85)90278-2. [DOI] [PubMed] [Google Scholar]
  13. Hooper N. M., Turner A. J. Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor. Biochem J. 1988 Mar 15;250(3):865–869. doi: 10.1042/bj2500865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kamps M. P., Sefton B. M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal Biochem. 1989 Jan;176(1):22–27. doi: 10.1016/0003-2697(89)90266-2. [DOI] [PubMed] [Google Scholar]
  15. Lake P., Clark E. A., Khorshidi M., Sunshine G. H. Production and characterization of cytotoxic Thy-1 antibody-secreting hybrid cell lines. Detection of T cell subsets. Eur J Immunol. 1979 Nov;9(11):875–886. doi: 10.1002/eji.1830091109. [DOI] [PubMed] [Google Scholar]
  16. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Low M. G. Anchoring of Thy-1 to the cell membrane. Immunol Ser. 1989;45:71–83. [PubMed] [Google Scholar]
  18. Mao S. Y., Alber G., Rivera J., Kochan J., Metzger H. Interaction of aggregated native and mutant IgE receptors with the cellular skeleton. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):222–226. doi: 10.1073/pnas.89.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mason D. W., Williams A. F. The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem J. 1980 Apr 1;187(1):1–20. doi: 10.1042/bj1870001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Metzger H. Molecular aspects of receptors and binding factors for IgE. Adv Immunol. 1988;43:277–312. doi: 10.1016/s0065-2776(08)60368-5. [DOI] [PubMed] [Google Scholar]
  21. Muñoz G., Marshall S. H. An alternative method for a fast separation of phosphotyrosine. Anal Biochem. 1990 Nov 1;190(2):233–237. doi: 10.1016/0003-2697(90)90185-c. [DOI] [PubMed] [Google Scholar]
  22. Park D. J., Min H. K., Rhee S. G. IgE-induced tyrosine phosphorylation of phospholipase C-gamma 1 in rat basophilic leukemia cells. J Biol Chem. 1991 Dec 25;266(36):24237–24240. [PubMed] [Google Scholar]
  23. Reif A. E. The discovery of Thy-1 and its development. Immunol Ser. 1989;45:3–45. [PubMed] [Google Scholar]
  24. Rudolph A. K., Burrows P. D., Wabl M. R. Thirteen hybridomas secreting hapten-specific immunoglobulin E from mice with Iga or Igb heavy chain haplotype. Eur J Immunol. 1981 Jun;11(6):527–529. doi: 10.1002/eji.1830110617. [DOI] [PubMed] [Google Scholar]
  25. Shenoy-Scaria A. M., Kwong J., Fujita T., Olszowy M. W., Shaw A. S., Lublin D. M. Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1. J Immunol. 1992 Dec 1;149(11):3535–3541. [PubMed] [Google Scholar]
  26. Stefanová I., Horejsí V., Ansotegui I. J., Knapp W., Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. doi: 10.1126/science.1719635. [DOI] [PubMed] [Google Scholar]
  27. Tamura G. S., Dailey M. O., Gallatin W. M., McGrath M. S., Weissman I. L., Pillemer E. A. Isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique. Anal Biochem. 1984 Feb;136(2):458–464. doi: 10.1016/0003-2697(84)90244-6. [DOI] [PubMed] [Google Scholar]
  28. Thomas P. M., Samelson L. E. The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. J Biol Chem. 1992 Jun 15;267(17):12317–12322. [PubMed] [Google Scholar]
  29. Yamanashi Y., Fukui Y., Wongsasant B., Kinoshita Y., Ichimori Y., Toyoshima K., Yamamoto T. Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1118–1122. doi: 10.1073/pnas.89.3.1118. [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