Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Dec 1;109(6):3445–3453. doi: 10.1083/jcb.109.6.3445

Selective secretion of alternatively spliced fibronectin variants

PMCID: PMC2115891  PMID: 2600138

Abstract

We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0- V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein- protein interactions in FN dimer formation.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Bernard M. P., Kolbe M., Weil D., Chu M. L. Human cellular fibronectin: comparison of the carboxyl-terminal portion with rat identifies primary structural domains separated by hypervariable regions. Biochemistry. 1985 May 21;24(11):2698–2704. doi: 10.1021/bi00332a016. [DOI] [PubMed] [Google Scholar]
  2. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bulleid N. J., Freedman R. B. Defective co-translational formation of disulphide bonds in protein disulphide-isomerase-deficient microsomes. Nature. 1988 Oct 13;335(6191):649–651. doi: 10.1038/335649a0. [DOI] [PubMed] [Google Scholar]
  4. Choi M. G., Hynes R. O. Biosynthesis and processing of fibronectin in NIL.8 hamster cells. J Biol Chem. 1979 Dec 10;254(23):12050–12055. [PubMed] [Google Scholar]
  5. Copeland C. S., Doms R. W., Bolzau E. M., Webster R. G., Helenius A. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J Cell Biol. 1986 Oct;103(4):1179–1191. doi: 10.1083/jcb.103.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Copeland C. S., Zimmer K. P., Wagner K. R., Healey G. A., Mellman I., Helenius A. Folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin. Cell. 1988 Apr 22;53(2):197–209. doi: 10.1016/0092-8674(88)90381-9. [DOI] [PubMed] [Google Scholar]
  7. Creighton T. E., Hillson D. A., Freedman R. B. Catalysis by protein-disulphide isomerase of the unfolding and refolding of proteins with disulphide bonds. J Mol Biol. 1980 Sep 5;142(1):43–62. doi: 10.1016/0022-2836(80)90205-3. [DOI] [PubMed] [Google Scholar]
  8. Davies J., Jimenez A. A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg. 1980 Sep;29(5 Suppl):1089–1092. doi: 10.4269/ajtmh.1980.29.1089. [DOI] [PubMed] [Google Scholar]
  9. Ehrismann R., Roth D. E., Eppenberger H. M., Turner D. C. Arrangement of attachment-promoting, self-association, and heparin-binding sites in horse serum fibronectin. J Biol Chem. 1982 Jul 10;257(13):7381–7387. [PubMed] [Google Scholar]
  10. 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]
  11. Geetha-Habib M., Noiva R., Kaplan H. A., Lennarz W. J. Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER. Cell. 1988 Sep 23;54(7):1053–1060. doi: 10.1016/0092-8674(88)90120-1. [DOI] [PubMed] [Google Scholar]
  12. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  13. Hartman S. C., Mulligan R. C. Two dominant-acting selectable markers for gene transfer studies in mammalian cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8047–8051. doi: 10.1073/pnas.85.21.8047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Homandberg G. A., Amrani D. L., Evans D. B., Kane C. M., Ankel E., Mosesson M. W. Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers. Arch Biochem Biophys. 1985 May 1;238(2):652–663. doi: 10.1016/0003-9861(85)90211-5. [DOI] [PubMed] [Google Scholar]
  15. Homandberg G. A., Erickson J. W. Model of fibronectin tertiary structure based on studies of interactions between fragments. Biochemistry. 1986 Nov 4;25(22):6917–6925. doi: 10.1021/bi00370a027. [DOI] [PubMed] [Google Scholar]
  16. Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. doi: 10.1083/jcb.103.6.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Humphries M. J., Komoriya A., Akiyama S. K., Olden K., Yamada K. M. Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion. J Biol Chem. 1987 May 15;262(14):6886–6892. [PubMed] [Google Scholar]
  18. Hynes R. O. Fibronectins. Sci Am. 1986 Jun;254(6):42–51. doi: 10.1038/scientificamerican0686-42. [DOI] [PubMed] [Google Scholar]
  19. Kornblihtt A. R., Umezawa K., Vibe-Pedersen K., Baralle F. E. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 1985 Jul;4(7):1755–1759. doi: 10.1002/j.1460-2075.1985.tb03847.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kornblihtt A. R., Vibe-Pedersen K., Baralle F. E. Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain. EMBO J. 1984 Jan;3(1):221–226. doi: 10.1002/j.1460-2075.1984.tb01787.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Mautner V., Hynes R. O. Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells. J Cell Biol. 1977 Dec;75(3):743–768. doi: 10.1083/jcb.75.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCarthy J. B., Hagen S. T., Furcht L. T. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells. J Cell Biol. 1986 Jan;102(1):179–188. doi: 10.1083/jcb.102.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McDonald J. A. Extracellular matrix assembly. Annu Rev Cell Biol. 1988;4:183–207. doi: 10.1146/annurev.cb.04.110188.001151. [DOI] [PubMed] [Google Scholar]
  26. Merril C. R., Goldman D., Van Keuren M. L. Gel protein stains: silver stain. Methods Enzymol. 1984;104:441–447. doi: 10.1016/s0076-6879(84)04111-2. [DOI] [PubMed] [Google Scholar]
  27. Morrison S. L., Scharff M. D. Heavy chain-producing variants of a mouse myeloma cell line. J Immunol. 1975 Feb;114(2 Pt 1):655–659. [PubMed] [Google Scholar]
  28. Norton P. A., Hynes R. O. Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells. Mol Cell Biol. 1987 Dec;7(12):4297–4307. doi: 10.1128/mcb.7.12.4297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Patel R. S., Odermatt E., Schwarzbauer J. E., Hynes R. O. Organization of the fibronectin gene provides evidence for exon shuffling during evolution. EMBO J. 1987 Sep;6(9):2565–2572. doi: 10.1002/j.1460-2075.1987.tb02545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Paul J. I., Hynes R. O. Multiple fibronectin subunits and their post-translational modifications. J Biol Chem. 1984 Nov 10;259(21):13477–13487. [PubMed] [Google Scholar]
  31. Paul J. I., Schwarzbauer J. E., Tamkun J. W., Hynes R. O. Cell-type-specific fibronectin subunits generated by alternative splicing. J Biol Chem. 1986 Sep 15;261(26):12258–12265. [PubMed] [Google Scholar]
  32. Petersen T. E., Thøgersen H. C., Skorstengaard K., Vibe-Pedersen K., Sahl P., Sottrup-Jensen L., Magnusson S. Partial primary structure of bovine plasma fibronectin: three types of internal homology. Proc Natl Acad Sci U S A. 1983 Jan;80(1):137–141. doi: 10.1073/pnas.80.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Robinson R. M., Hermans J. Subunit interactions in human plasma fibronectin. Biochem Biophys Res Commun. 1984 Nov 14;124(3):718–725. doi: 10.1016/0006-291x(84)91017-9. [DOI] [PubMed] [Google Scholar]
  34. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  35. Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem. 1988;57:375–413. doi: 10.1146/annurev.bi.57.070188.002111. [DOI] [PubMed] [Google Scholar]
  36. Schwarzbauer J. E., Mulligan R. C., Hynes R. O. Efficient and stable expression of recombinant fibronectin polypeptides. Proc Natl Acad Sci U S A. 1987 Feb;84(3):754–758. doi: 10.1073/pnas.84.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schwarzbauer J. E., Patel R. S., Fonda D., Hynes R. O. Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J. 1987 Sep;6(9):2573–2580. doi: 10.1002/j.1460-2075.1987.tb02547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwarzbauer J. E., Paul J. I., Hynes R. O. On the origin of species of fibronectin. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1424–1428. doi: 10.1073/pnas.82.5.1424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schwarzbauer J. E., Tamkun J. W., Lemischka I. R., Hynes R. O. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 1983 Dec;35(2 Pt 1):421–431. doi: 10.1016/0092-8674(83)90175-7. [DOI] [PubMed] [Google Scholar]
  40. Sekiguchi K., Klos A. M., Kurachi K., Yoshitake S., Hakomori S. Human liver fibronectin complementary DNAs: identification of two different messenger RNAs possibly encoding the alpha and beta subunits of plasma fibronectin. Biochemistry. 1986 Aug 26;25(17):4936–4941. doi: 10.1021/bi00365a032. [DOI] [PubMed] [Google Scholar]
  41. Sekiguchi K., Titani K. Probing molecular polymorphism of fibronectins with antibodies directed to the alternatively spliced peptide segments. Biochemistry. 1989 Apr 18;28(8):3293–3298. doi: 10.1021/bi00434a026. [DOI] [PubMed] [Google Scholar]
  42. Skorstengaard K., Jensen M. S., Sahl P., Petersen T. E., Magnusson S. Complete primary structure of bovine plasma fibronectin. Eur J Biochem. 1986 Dec 1;161(2):441–453. doi: 10.1111/j.1432-1033.1986.tb10464.x. [DOI] [PubMed] [Google Scholar]
  43. Takei F., Oyama F., Kimura K., Hyodo A., Mizuno S., Shimura K. Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2). J Cell Biol. 1984 Dec;99(6):2005–2010. doi: 10.1083/jcb.99.6.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tamkun J. W., Hynes R. O. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem. 1983 Apr 10;258(7):4641–4647. [PubMed] [Google Scholar]
  45. Yamada K. M. Cell surface interactions with extracellular materials. Annu Rev Biochem. 1983;52:761–799. doi: 10.1146/annurev.bi.52.070183.003553. [DOI] [PubMed] [Google Scholar]

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

RESOURCES