Abstract
Receptor tyrosine kinases (RTKs) bind to their ligands with high affinity and specificity. Soluble receptor approaches exploit these biological properties to make affinity probes that can be used to detect or to purify the cognate ligands (1,2). In many respects, these soluble receptor reagents resemble antibodies, and they can be used in almost all the same types of procedure. They can also have important advantages over antibodies. They can be used to identify and clone previously unknown ligands of orphan receptors (1–9). They can be produced much more quickly than antibodies. Also, because they exploit natural receptor-ligand interactions, they can give information not available with antibodies, for example permitting quantitative characterization of ligandreceptor binding interactions (1,2,10), or allowing the simultaneous detection of multiple cross-reacting ligands in an embryo (5,11,12).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Flanagan J. G. and Leder P. (1990) The kit ligand: a cell surface molecule altered in Steel mutant fibroblasts. Cell 63, 185–194.
Aruffo A., Stamenkovic I., Melnick M., Underhill C. B., and Seed B. (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61,1303–1313.
Armitage R. J., et al. (1992) Molecular and biological characterization of a murine ligand for CD40. Nature 357, 80–82.
Lyman S. D., et al. (1993) Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 75, 1157–1167.
Cheng H.-J. and Flanagan J. G. (1994) Identification and cloning of ELF-1, a developmentally expressed ligand for the Mek4 and Sek receptor tyrosine kinases. Cell 79, 157–168.
Bartley T. D., et al. (1994) B61 is a ligand for the ECK receptor protein-tyrosine kinase. Nature 368, 558–560.
Davis S., Gale N. W., Aldrich T. H., Maisonpierre P. C., Lhotak V., Pawson T., et al. (1994) Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity. Science 266, 816–819.
Winslow J. W., Moran P., Valverde J., Shih A., Yuan J. Q., Wong S. C., et al. (1995) Cloning of AL-1, a ligand for an Eph-related tyrosine kinase receptor involved in axon bundle formation. Neuron 14, 973–981.
Davis S., Aldrich T. H., Jones P. F., Acheson A., Compton D. L., Jain V., et al. (1996) Isolation of angiopoietin-1, a ligand for the tie-2 receptor, by secretion-trap expression cloning. Cell 87, 1161–1169.
Wang Z. E., Myles G. M., Brandt C. S., Lioubin M. N., and Rohrschneider L. (1993) Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain. Mol. Cell. Biol. 13, 5348–5359.
Cheng H.-J., Nakamoto M., Bergemann A. D., and Flanagan J. G. (1995) Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82, 371–381.
Gale N. W., Holland S. J., Valenzuela D. M., Flenniken A., Pan L., Ryan T. E., et al. (1996) Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 17,9–19.
Berger J., Howard A. D., Brink L., Gerber L., Hauber J., Cullen B. R., and Udenfriend S. (1988) COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein. J. Biol. Chem. 263, 10,016–10,021.
Flanagan J. G., Chan D. C., and Leder P. (1991) Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant. Cell 64,1025–1035.
Chiang M.-K. and Flanagan J. G. (1995) Interactions between the Flk-1 receptor, vascular endothelial growth factor, and cell surface proteoglycan identified with a soluble receptor reagent. Growth Factors 12,1–10.
Bergemann A. D., Cheng H.-J., Brambilla R., Klein R., and Flanagan J. G. (1995) ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites. Mol. Cell. Biol. 15,4921–4929.
He Z. G. and Tessier-Lavigne M. (1997) Neuropilin is a receptor for the axonal chemorepellent semaphorin III. Cell 90,739–751.
Kolodkin A. L., Levengood D. V., Rowe E. G., Tai Y. T., Giger R. J., and Ginty D. D. (1997) Neuropilin is a semaphorin III receptor. Cell 90, 753–762.
Koppel A. M., Feiner L., Kobayashi H., and Raper J. A. (1997) A 70 amino acid region within the semaphorin domain activates specific cellular response of semaphorin family members. Neuron 19,531–537.
Aruffo A. and Seed B. (1987) Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc. Natl. Acad. Sci. USA 84,8573–8577.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Cheng, HJ., Flanagan, J.G. (2000). Cloning and Characterization of RTK Ligands Using Receptor-Alkaline Phosphatase Fusion Proteins. In: Reith, A.D. (eds) Protein Kinase Protocols. Methods in Molecular Biology™, vol 124. Humana Press. https://doi.org/10.1385/1-59259-059-4:313
Download citation
DOI: https://doi.org/10.1385/1-59259-059-4:313
Publisher Name: Humana Press
Print ISBN: 978-0-89603-700-7
Online ISBN: 978-1-59259-059-9
eBook Packages: Springer Protocols