Abstract
Many proteins involved in Cell-Cell and cell–matrix adhesion are regulated by signal transduction pathways and can activate signal transduction on ligation. Adhesion-related signal transduction is important throughout development, hemostasis, immunity, and in diseases such as cancer. Therefore, the identification of the various signaling pathways that are involved is crucial. Expression cloning is an unbiased way to isolate proteins with specific biological functions. This methodology has been adapted for the identification of proteins involved in cell signaling pathways that are mediated by Cell-Cell and cell–matrix interactions. We have successfully developed and used a novel expression cloning strategy to isolate the integrin-regulated apoptosis signaling protein BIT-1. This screen was based on previous observations that integrin-mediated adhesion upregulates the anti-apoptotic protein bcl-2. Our strategy described in this chapter uses flow cytometry and a reporter construct in which the bcl-2 promoter is linked to enhanced green fluorescence protein. The advantage of using flow cytometry in expression cloning is that it increases the sensitivity of the screen by enabling us to examine function quantitatively at the level of a single cell millions of times in one experiment. The following protocol provides a detailed method for the isolation of proteins that are regulated by cell adhesion.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hynes, R. O. (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687.
Juliano, R. L. (2002) Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulinsuperfamily members. Annu. Rev. Pharmacol. Toxicol. 42, 283–323.
Wheelock, M. J. and Johnson, K. R. (2003) Cadherin-mediated cellular signaling. Curr. Opin. Cell Biol. 15, 509–514.
Seed, B. (1995) Developments in expression cloning. Curr. Opin. Biotechnol. 6, 567–573.
Tanaka, T., Camerini, D., Seed, B., et al. (1992) Cloning and functional expression of the T cell activation antigen CD26. J. Immunol. 149, 481–486.
Adams, J. C., Seed, B., and Lawler, J. (1998) Muskelin, a novel intracellular mediator of cell adhesive and cytoskeletal responses to thrombospondin-1. EMBO J. 17, 4964–4974.
Fenczik, C. A., Sethi, T., Ramos, J. W., Hughes, P. E., and Ginsberg M. H. (1997) Complementation of dominant suppression implicates CD98 in integrin activation. Nature 390, 81–85.
Ramos, J. W., Kojima, T. K., Hughes, P. E., Fenczik, C. A., and Ginsberg, M. H. (1998) The death effector domain of PEA-15 is involved in its regulation of integrin activation. J. Biol. Chem. 273, 3897–3900.
Matter, M. L., Ginsberg, M. H., and Ramos, J. W. (2001) Identification of cell signaling molecules by expression cloning. Sci. STKE 9, L9.
Jan, Y., Matter, M., Pai, J. T., et al. (2004) A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell 116, 751–762.
Frisch, S. M. and Screaton, R. A. (2001) Anoikis mechanisms. Curr. Opin. Cell Biol. 13, 555–562.
Frisch, S. M. and Ruoslahti, E. (1997) Integrins and anoikis. Curr. Opin. Cell Biol. 9, 701–706.
Matter, M. L. and Ruoslahti, E. (2001) A signaling pathway from the alpha5beta1 and alpha(v)beta3 integrins that elevates bcl-2 transcription. J. Biol. Chem. 276, 27,757–27,763.
Ong, E., Yeh, J. C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids. J. Biol. Chem. 273, 5190–5195.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Matter, M.L., Ramos, J.W. (2006). Expression Cloning of Signaling Proteins Regulated by Cell Adhesion. In: Colgan, S.P. (eds) Cell-Cell Interactions. Methods in Molecular Biology™, vol 341. Humana Press. https://doi.org/10.1385/1-59745-113-4:155
Download citation
DOI: https://doi.org/10.1385/1-59745-113-4:155
Publisher Name: Humana Press
Print ISBN: 978-1-58829-523-1
Online ISBN: 978-1-59745-113-0
eBook Packages: Springer Protocols