Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Oct 2;127(2):557–565. doi: 10.1083/jcb.127.2.557

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility

PMCID: PMC2120206  PMID: 7523423

Abstract

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125- kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Albini A., Allavena G., Melchiori A., Giancotti F., Richter H., Comoglio P. M., Parodi S., Martin G. R., Tarone G. Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor. J Cell Biol. 1987 Oct;105(4):1867–1872. doi: 10.1083/jcb.105.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altruda F., Cervella P., Tarone G., Botta C., Balzac F., Stefanuto G., Silengo L. A human integrin beta 1 subunit with a unique cytoplasmic domain generated by alternative mRNA processing. Gene. 1990 Nov 15;95(2):261–266. doi: 10.1016/0378-1119(90)90369-3. [DOI] [PubMed] [Google Scholar]
  3. Balzac F., Belkin A. M., Koteliansky V. E., Balabanov Y. V., Altruda F., Silengo L., Tarone G. Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit. J Cell Biol. 1993 Apr;121(1):171–178. doi: 10.1083/jcb.121.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  5. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  7. Collo G., Starr L., Quaranta V. A new isoform of the laminin receptor integrin alpha 7 beta 1 is developmentally regulated in skeletal muscle. J Biol Chem. 1993 Sep 5;268(25):19019–19024. [PubMed] [Google Scholar]
  8. Defilippi P., Bozzo C., Volpe G., Romano G., Venturino M., Silengo L., Tarone G. Integrin-mediated signal transduction in human endothelial cells: analysis of tyrosine phosphorylation events. Cell Adhes Commun. 1994 Apr;2(1):75–86. doi: 10.3109/15419069409014203. [DOI] [PubMed] [Google Scholar]
  9. Defilippi P., Silengo L., Tarone G. Alpha 6.beta 1 integrin (laminin receptor) is down-regulated by tumor necrosis factor alpha and interleukin-1 beta in human endothelial cells. J Biol Chem. 1992 Sep 15;267(26):18303–18307. [PubMed] [Google Scholar]
  10. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Giancotti F. G., Ruoslahti E. Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells. Cell. 1990 Mar 9;60(5):849–859. doi: 10.1016/0092-8674(90)90098-y. [DOI] [PubMed] [Google Scholar]
  13. Guan J. L., Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature. 1992 Aug 20;358(6388):690–692. doi: 10.1038/358690a0. [DOI] [PubMed] [Google Scholar]
  14. Guan J. L., Trevithick J. E., Hynes R. O. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul. 1991 Nov;2(11):951–964. doi: 10.1091/mbc.2.11.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hogervorst F., Kuikman I., van Kessel A. G., Sonnenberg A. Molecular cloning of the human alpha 6 integrin subunit. Alternative splicing of alpha 6 mRNA and chromosomal localization of the alpha 6 and beta 4 genes. Eur J Biochem. 1991 Jul 15;199(2):425–433. doi: 10.1111/j.1432-1033.1991.tb16140.x. [DOI] [PubMed] [Google Scholar]
  16. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  17. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  18. Jaken S., Leach K., Klauck T. Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol. 1989 Aug;109(2):697–704. doi: 10.1083/jcb.109.2.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kintner C. Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain. Cell. 1992 Apr 17;69(2):225–236. doi: 10.1016/0092-8674(92)90404-z. [DOI] [PubMed] [Google Scholar]
  20. Kornberg L., Earp H. S., Parsons J. T., Schaller M., Juliano R. L. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem. 1992 Nov 25;267(33):23439–23442. [PubMed] [Google Scholar]
  21. Languino L. R., Ruoslahti E. An alternative form of the integrin beta 1 subunit with a variant cytoplasmic domain. J Biol Chem. 1992 Apr 5;267(10):7116–7120. [PubMed] [Google Scholar]
  22. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  23. Otey C. A., Pavalko F. M., Burridge K. An interaction between alpha-actinin and the beta 1 integrin subunit in vitro. J Cell Biol. 1990 Aug;111(2):721–729. doi: 10.1083/jcb.111.2.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reszka A. A., Hayashi Y., Horwitz A. F. Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association. J Cell Biol. 1992 Jun;117(6):1321–1330. doi: 10.1083/jcb.117.6.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rohrschneider L. R. Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3514–3518. doi: 10.1073/pnas.77.6.3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rossino P., Defilippi P., Silengo L., Tarone G. Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin. Cell Regul. 1991 Dec;2(12):1021–1033. doi: 10.1091/mbc.2.12.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sastry S. K., Horwitz A. F. Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr Opin Cell Biol. 1993 Oct;5(5):819–831. doi: 10.1016/0955-0674(93)90031-k. [DOI] [PubMed] [Google Scholar]
  28. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  30. Takada Y., Ylänne J., Mandelman D., Puzon W., Ginsberg M. H. A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques. J Cell Biol. 1992 Nov;119(4):913–921. doi: 10.1083/jcb.119.4.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tamura R. N., Cooper H. M., Collo G., Quaranta V. Cell type-specific integrin variants with alternative alpha chain cytoplasmic domains. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10183–10187. doi: 10.1073/pnas.88.22.10183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tamura R. N., Rozzo C., Starr L., Chambers J., Reichardt L. F., Cooper H. M., Quaranta V. Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4. J Cell Biol. 1990 Oct;111(4):1593–1604. doi: 10.1083/jcb.111.4.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Troyanovsky S. M., Eshkind L. G., Troyanovsky R. B., Leube R. E., Franke W. W. Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage. Cell. 1993 Feb 26;72(4):561–574. doi: 10.1016/0092-8674(93)90075-2. [DOI] [PubMed] [Google Scholar]
  34. Turner C. E., Burridge K. Transmembrane molecular assemblies in cell-extracellular matrix interactions. Curr Opin Cell Biol. 1991 Oct;3(5):849–853. doi: 10.1016/0955-0674(91)90059-8. [DOI] [PubMed] [Google Scholar]
  35. Wang N., Butler J. P., Ingber D. E. Mechanotransduction across the cell surface and through the cytoskeleton. Science. 1993 May 21;260(5111):1124–1127. doi: 10.1126/science.7684161. [DOI] [PubMed] [Google Scholar]
  36. Yatohgo T., Izumi M., Kashiwagi H., Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988 Aug;13(4):281–292. doi: 10.1247/csf.13.281. [DOI] [PubMed] [Google Scholar]
  37. van Kuppevelt T. H., Languino L. R., Gailit J. O., Suzuki S., Ruoslahti E. An alternative cytoplasmic domain of the integrin beta 3 subunit. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5415–5418. doi: 10.1073/pnas.86.14.5415. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES