Generation of a Minimal α5β1 Integrin-Fc Fragment

• Published in: The Journal of biological chemistry 2001-09, Vol.276 (38), p.35854-35866
• Main Authors: Coe, Alexander P.F., Askari, Janet A., Kline, Adam D., Robinson, Martyn K., Kirby, Hishani, Stephens, Paul E., Humphries, Martin J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M103639200

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains andN-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377–390), a series of recombinant soluble human α5β1 integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional α5-subunit truncation contained the N-terminal 613 residues, whereas the shortest β1-subunit was a fragment containing residues 121–455. Each of these minimally truncated integrins displayed the antibody binding characteristics of α5β1 purified from human placenta and bound ligand with the same apparent affinity as the native receptor.