Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Dec 1;103(6):2311–2322. doi: 10.1083/jcb.103.6.2311

Cell surface expression of membrane-anchored v-sis gene products: glycosylation is not required for cell surface transport

PMCID: PMC2114596  PMID: 3536965

Abstract

The v-sis gene is able to transform cells by production of a growth factor that is structurally related to platelet-derived growth factor. This growth factor has been detected in the conditioned media of v-sis transformed cells, and is able to stimulate the autophosphorylation of the platelet-derived growth factor receptor. We have used the v-sis gene product to analyze the role of protein-encoded signals in cell surface transport. We constructed several gene fusions that encode transmembrane forms of the v-sis gene product. These membrane-anchored forms of the v-sis gene product are properly folded into a native structure, as indicated by their dimerization, glycosylation, and NH2- terminal proteolytic processing. Indirect immunofluorescence demonstrated that several of these membrane-anchored gene products are transported to the cell surface. Removal of the N-linked glycosylation site from the v-sis gene product did not prevent cell surface transport. Several of these mutant genes are able to induce focus formation in NIH3T3 cells, providing further evidence that the membrane- anchored proteins are properly folded. These results demonstrate that N- linked glycosylation is not required for the cell surface transport of a protein that is in a native, biologically active conformation. These results provide a correlation between cell surface expression of the membrane-anchored v-sis gene products and transformation.

Full Text

The Full Text of this article is available as a PDF (3.4 MB).

Selected References

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

  1. Adams G. A., Rose J. K. Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein. Mol Cell Biol. 1985 Jun;5(6):1442–1448. doi: 10.1128/mcb.5.6.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Assoian R. K., Grotendorst G. R., Miller D. M., Sporn M. B. Cellular transformation by coordinated action of three peptide growth factors from human platelets. 1984 Jun 28-Jul 4Nature. 309(5971):804–806. doi: 10.1038/309804a0. [DOI] [PubMed] [Google Scholar]
  3. Bender W. Homeotic gene products as growth factors. Cell. 1985 Dec;43(3 Pt 2):559–560. doi: 10.1016/0092-8674(85)90224-7. [DOI] [PubMed] [Google Scholar]
  4. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deuel T. F., Huang J. S., Huang S. S., Stroobant P., Waterfield M. D. Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells. Science. 1983 Sep 30;221(4618):1348–1350. doi: 10.1126/science.6310754. [DOI] [PubMed] [Google Scholar]
  6. Devare S. G., Reddy E. P., Law J. D., Robbins K. C., Aaronson S. A. Nucleotide sequence of the simian sarcoma virus genome: demonstration that its acquired cellular sequences encode the transforming gene product p28sis. Proc Natl Acad Sci U S A. 1983 Feb;80(3):731–735. doi: 10.1073/pnas.80.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doolittle R. F., Hunkapiller M. W., Hood L. E., Devare S. G., Robbins K. C., Aaronson S. A., Antoniades H. N. Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science. 1983 Jul 15;221(4607):275–277. doi: 10.1126/science.6304883. [DOI] [PubMed] [Google Scholar]
  8. Friedlander M., Blobel G. Bovine opsin has more than one signal sequence. 1985 Nov 28-Dec 4Nature. 318(6044):338–343. doi: 10.1038/318338a0. [DOI] [PubMed] [Google Scholar]
  9. Gallione C. J., Rose J. K. A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. J Virol. 1985 May;54(2):374–382. doi: 10.1128/jvi.54.2.374-382.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garrett J. S., Coughlin S. R., Niman H. L., Tremble P. M., Giels G. M., Williams L. T. Blockade of autocrine stimulation in simian sarcoma virus-transformed cells reverses down-regulation of platelet-derived growth factor receptors. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7466–7470. doi: 10.1073/pnas.81.23.7466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  12. Graeve L., Garreis-Wabnitz C., Zauke M., Breindl M., Kruppa J. The soluble glycoprotein of vesicular stomatitis virus is formed during or shortly after the translation process. J Virol. 1986 Mar;57(3):968–975. doi: 10.1128/jvi.57.3.968-975.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greenwald I. lin-12, a nematode homeotic gene, is homologous to a set of mammalian proteins that includes epidermal growth factor. Cell. 1985 Dec;43(3 Pt 2):583–590. doi: 10.1016/0092-8674(85)90230-2. [DOI] [PubMed] [Google Scholar]
  14. Guan J. L., Machamer C. E., Rose J. K. Glycosylation allows cell-surface transport of an anchored secretory protein. Cell. 1985 Sep;42(2):489–496. doi: 10.1016/0092-8674(85)90106-0. [DOI] [PubMed] [Google Scholar]
  15. Guan J. L., Rose J. K. Conversion of a secretory protein into a transmembrane protein results in its transport to the Golgi complex but not to the cell surface. Cell. 1984 Jul;37(3):779–787. doi: 10.1016/0092-8674(84)90413-6. [DOI] [PubMed] [Google Scholar]
  16. Hannink M., Donoghue D. J. Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing. Mol Cell Biol. 1986 Apr;6(4):1343–1348. doi: 10.1128/mcb.6.4.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hannink M., Donoghue D. J. Requirement for a signal sequence in biological expression of the v-sis oncogene. Science. 1984 Dec 7;226(4679):1197–1199. doi: 10.1126/science.6095451. [DOI] [PubMed] [Google Scholar]
  18. Hannink M., Sauer M. K., Donoghue D. J. Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation. Mol Cell Biol. 1986 Apr;6(4):1304–1314. doi: 10.1128/mcb.6.4.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heldin C. H., Westermark B. Growth factors: mechanism of action and relation to oncogenes. Cell. 1984 May;37(1):9–20. doi: 10.1016/0092-8674(84)90296-4. [DOI] [PubMed] [Google Scholar]
  20. Hoffmann J. W., Steffen D., Gusella J., Tabin C., Bird S., Cowing D., Weinberg R. A. DNA methylation affecting the expression of murine leukemia proviruses. J Virol. 1982 Oct;44(1):144–157. doi: 10.1128/jvi.44.1.144-157.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang J. S., Huang S. S., Deuel T. F. Transforming protein of simian sarcoma virus stimulates autocrine growth of SSV-transformed cells through PDGF cell-surface receptors. Cell. 1984 Nov;39(1):79–87. doi: 10.1016/0092-8674(84)90193-4. [DOI] [PubMed] [Google Scholar]
  22. Johnsson A., Betsholtz C., Heldin C. H., Westermark B. Antibodies against platelet-derived growth factor inhibit acute transformation by simian sarcoma virus. Nature. 1985 Oct 3;317(6036):438–440. doi: 10.1038/317438a0. [DOI] [PubMed] [Google Scholar]
  23. Johnsson A., Heldin C. H., Wasteson A., Westermark B., Deuel T. F., Huang J. S., Seeburg P. H., Gray A., Ullrich A., Scrace G. The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor. EMBO J. 1984 May;3(5):921–928. doi: 10.1002/j.1460-2075.1984.tb01908.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Josephs S. F., Guo C., Ratner L., Wong-Staal F. Human-proto-oncogene nucleotide sequences corresponding to the transforming region of simian sarcoma virus. Science. 1984 Feb 3;223(4635):487–491. doi: 10.1126/science.6318322. [DOI] [PubMed] [Google Scholar]
  25. King C. R., Giese N. A., Robbins K. C., Aaronson S. A. In vitro mutagenesis of the v-sis transforming gene defines functional domains of its growth factor-related product. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5295–5299. doi: 10.1073/pnas.82.16.5295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nichols E. J., Manger R., Hakomori S., Herscovics A., Rohrschneider L. R. Transformation by the v-fms oncogene product: role of glycosylational processing and cell surface expression. Mol Cell Biol. 1985 Dec;5(12):3467–3475. doi: 10.1128/mcb.5.12.3467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Owen A. J., Pantazis P., Antoniades H. N. Simian sarcoma virus--transformed cells secrete a mitogen identical to platelet-derived growth factor. Science. 1984 Jul 6;225(4657):54–56. doi: 10.1126/science.6328659. [DOI] [PubMed] [Google Scholar]
  29. Pfeffer S., Ullrich A. Epidermal growth factor. Is the precursor a receptor? Nature. 1985 Jan 17;313(5999):184–184. doi: 10.1038/313184a0. [DOI] [PubMed] [Google Scholar]
  30. Robbins K. C., Antoniades H. N., Devare S. G., Hunkapiller M. W., Aaronson S. A. Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor. Nature. 1983 Oct 13;305(5935):605–608. doi: 10.1038/305605a0. [DOI] [PubMed] [Google Scholar]
  31. Robbins K. C., Leal F., Pierce J. H., Aaronson S. A. The v-sis/PDGF-2 transforming gene product localizes to cell membranes but is not a secretory protein. EMBO J. 1985 Jul;4(7):1783–1792. doi: 10.1002/j.1460-2075.1985.tb03851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rose J. K., Bergmann J. E. Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell. 1982 Oct;30(3):753–762. doi: 10.1016/0092-8674(82)90280-x. [DOI] [PubMed] [Google Scholar]
  33. Russell D. W., Schneider W. J., Yamamoto T., Luskey K. L., Brown M. S., Goldstein J. L. Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell. 1984 Jun;37(2):577–585. doi: 10.1016/0092-8674(84)90388-x. [DOI] [PubMed] [Google Scholar]
  34. Sprague J., Condra J. H., Arnheiter H., Lazzarini R. A. Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein. J Virol. 1983 Feb;45(2):773–781. doi: 10.1128/jvi.45.2.773-781.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Theilen G. H., Gould D., Fowler M., Dungworth D. L. C-type virus in tumor tissue of a woolly monkey (Lagothrix spp.) with fibrosarcoma. J Natl Cancer Inst. 1971 Oct;47(4):881–889. [PubMed] [Google Scholar]
  36. Warren T. G., Shields D. Expression of preprosomatostatin in heterologous cells: biosynthesis, posttranslational processing, and secretion of mature somatostatin. Cell. 1984 Dec;39(3 Pt 2):547–555. doi: 10.1016/0092-8674(84)90461-6. [DOI] [PubMed] [Google Scholar]
  37. Waterfield M. D., Scrace G. T., Whittle N., Stroobant P., Johnsson A., Wasteson A., Westermark B., Heldin C. H., Huang J. S., Deuel T. F. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature. 1983 Jul 7;304(5921):35–39. doi: 10.1038/304035a0. [DOI] [PubMed] [Google Scholar]
  38. Wharton K. A., Johansen K. M., Xu T., Artavanis-Tsakonas S. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell. 1985 Dec;43(3 Pt 2):567–581. doi: 10.1016/0092-8674(85)90229-6. [DOI] [PubMed] [Google Scholar]
  39. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES