Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Aug 1;99(2):512–519. doi: 10.1083/jcb.99.2.512

A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton

PMCID: PMC2113253  PMID: 6378925

Abstract

The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein- denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Ash J. F., Louvard D., Singer S. J. Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5584–5588. doi: 10.1073/pnas.74.12.5584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  3. Bennett V., Stenbuck P. J. The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes. Nature. 1979 Aug 9;280(5722):468–473. doi: 10.1038/280468a0. [DOI] [PubMed] [Google Scholar]
  4. Bourguignon L. Y., Singer S. J. Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5031–5035. doi: 10.1073/pnas.74.11.5031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Branton D., Cohen C. M., Tyler J. Interaction of cytoskeletal proteins on the human erythrocyte membrane. Cell. 1981 Apr;24(1):24–32. doi: 10.1016/0092-8674(81)90497-9. [DOI] [PubMed] [Google Scholar]
  6. Bravo R., Small J. V., Fey S. J., Larsen P. M., Celis J. E. Architecture and polypeptide composition of HeLa cytoskeletons. Modification of cytoarchitectural polypeptides during mitosis. J Mol Biol. 1982 Jan 5;154(1):121–143. doi: 10.1016/0022-2836(82)90421-1. [DOI] [PubMed] [Google Scholar]
  7. Bretscher A., Weber K. Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J Cell Biol. 1980 Jul;86(1):335–340. doi: 10.1083/jcb.86.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bretscher A., Weber K. Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci U S A. 1979 May;76(5):2321–2325. doi: 10.1073/pnas.76.5.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  10. Carraway C. A., Jung G., Craik J. R., Rubin R. W., Carraway K. L. Identification of a cytoskeleton-associated glycoprotein from isolated microvilli of a mammary ascites tumor. Exp Cell Res. 1983 Feb;143(2):303–308. doi: 10.1016/0014-4827(83)90055-1. [DOI] [PubMed] [Google Scholar]
  11. Carter W. G., Hakomori S. A new cell surface, detergent-insoluble glycoprotein matrix of human and hamster fibroblasts. The role of disulfide bonds in stabilization of the matrix. J Biol Chem. 1981 Jul 10;256(13):6953–6960. [PubMed] [Google Scholar]
  12. Carter W. G. The cooperative role of the transformation-sensitive glycoproteins, GP140 and fibronectin, in cell attachment and spreading. J Biol Chem. 1982 Mar 25;257(6):3249–3257. [PubMed] [Google Scholar]
  13. Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Engvall E., Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer. 1977 Jul 15;20(1):1–5. doi: 10.1002/ijc.2910200102. [DOI] [PubMed] [Google Scholar]
  15. Ferracini R., Prat M., Comoglio P. M. Dissection of the antigenic determinants expressed on the cell surface of RSV-transformed fibroblasts by monoclonal antibodies. Int J Cancer. 1982 Apr 15;29(4):477–481. doi: 10.1002/ijc.2910290419. [DOI] [PubMed] [Google Scholar]
  16. Flanagan J., Koch G. L. Cross-linked surface Ig attaches to actin. Nature. 1978 May 25;273(5660):278–281. doi: 10.1038/273278a0. [DOI] [PubMed] [Google Scholar]
  17. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  18. Goldman R. D., Chojnacki B., Yerna M. J. Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid. J Cell Biol. 1979 Mar;80(3):759–766. doi: 10.1083/jcb.80.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hynes R. O. Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3170–3174. doi: 10.1073/pnas.70.11.3170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jung G., Helm R. M., Carraway C. A., Carraway K. L. Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells. J Cell Biol. 1984 Jan;98(1):179–187. doi: 10.1083/jcb.98.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koch G. L., Smith M. J. An association between actin and the major histocompatibility antigen H-2. Nature. 1978 May 25;273(5660):274–278. doi: 10.1038/273274a0. [DOI] [PubMed] [Google Scholar]
  22. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  25. Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
  26. Lehto V. P. 140 000 Dalton surface glycoprotein. A plasma membrane component of the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. Exp Cell Res. 1983 Feb;143(2):271–286. doi: 10.1016/0014-4827(83)90052-6. [DOI] [PubMed] [Google Scholar]
  27. Lehto V. P., Vartio T., Virtanen I. Enrichment of a 140 KD surface glycoprotein in adherent, detergent-resistant cytoskeletons of cultured human fibroblasts. Biochem Biophys Res Commun. 1980 Aug 14;95(3):909–916. doi: 10.1016/0006-291x(80)91559-4. [DOI] [PubMed] [Google Scholar]
  28. Lehto V. P., Vartio T., Virtanen I. Persistence of a 140 000 Mr surface glycoprotein in cell-free matrices of cultured human fibroblasts. FEBS Lett. 1981 Feb 23;124(2):289–292. doi: 10.1016/0014-5793(81)80158-5. [DOI] [PubMed] [Google Scholar]
  29. Lehto V. P., Virtanen I. Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230). J Cell Biol. 1983 Mar;96(3):703–716. doi: 10.1083/jcb.96.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lux S. E. Dissecting the red cell membrane skeleton. Nature. 1979 Oct 11;281(5731):426–429. doi: 10.1038/281426a0. [DOI] [PubMed] [Google Scholar]
  31. Marchesi V. T. The red cell membrane skeleton: recent progress. Blood. 1983 Jan;61(1):1–11. [PubMed] [Google Scholar]
  32. Mescher M. F., Jose M. J., Balk S. P. Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature. 1981 Jan 15;289(5794):139–144. doi: 10.1038/289139a0. [DOI] [PubMed] [Google Scholar]
  33. Moss D. J. Cytoskeleton-associated glycoproteins from chicken sympathetic neurons and chicken embryo brain. Eur J Biochem. 1983 Sep 15;135(2):291–297. doi: 10.1111/j.1432-1033.1983.tb07651.x. [DOI] [PubMed] [Google Scholar]
  34. Nelson W. J., Traub P. Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of Ehrlich ascites tumour cells with triton X-100. II. Ultrastructural analysis. J Cell Sci. 1982 Feb;53:77–95. doi: 10.1242/jcs.53.1.77. [DOI] [PubMed] [Google Scholar]
  35. Osborn M., Weber K. The detertent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exp Cell Res. 1977 May;106(2):339–349. doi: 10.1016/0014-4827(77)90179-3. [DOI] [PubMed] [Google Scholar]
  36. Painter R. G., Ginsberg M. Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton. J Cell Biol. 1982 Feb;92(2):565–573. doi: 10.1083/jcb.92.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Repasky E. A., Granger B. L., Lazarides E. Widespread occurrence of avian spectrin in nonerythroid cells. Cell. 1982 Jul;29(3):821–833. doi: 10.1016/0092-8674(82)90444-5. [DOI] [PubMed] [Google Scholar]
  38. Sheterline P., Hopkins C. R. Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes. J Cell Biol. 1981 Sep;90(3):743–754. doi: 10.1083/jcb.90.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  40. Streuli C. H., Patel B., Critchley D. R. The cholera toxin receptor ganglioside GM remains associated with triton X-100 cytoskeletons of BALB/c-3T3 cells. Exp Cell Res. 1981 Dec;136(2):247–254. doi: 10.1016/0014-4827(81)90002-1. [DOI] [PubMed] [Google Scholar]
  41. Tarone G., Ceschi P., Prat M., Comoglio P. M. Transformation-sensitive protein with molecular weight of 45,000 secreted by mouse fibroblasts. Cancer Res. 1981 Sep;41(9 Pt 1):3648–3652. [PubMed] [Google Scholar]
  42. Tarone G., Galetto G., Prat M., Comoglio P. M. Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins. J Cell Biol. 1982 Jul;94(1):179–186. doi: 10.1083/jcb.94.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Traub P., Nelson W. J. Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of Ehrlich ascites tumour cells with triton X-100. I. Biochemical analysis. J Cell Sci. 1982 Feb;53:49–76. doi: 10.1242/jcs.53.1.49. [DOI] [PubMed] [Google Scholar]
  44. Webster R. E., Henderson D., Osborn M., Weber K. Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5511–5515. doi: 10.1073/pnas.75.11.5511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yamada K. M. Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape. J Cell Biol. 1978 Aug;78(2):520–541. doi: 10.1083/jcb.78.2.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]

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

RESOURCES