Integrin alpha 3 beta 1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

• Type of Material: Article
• Authors: Ashley C. Brown, North Carolina State University; Marilyn M. Dysart, Georgia Institute of Technology; Kimberly C. Clarke, Georgia Institute of Technology; Sarah E. Stabenfeldt, Arizona State University; Thomas Barker, Emory University
• Language: English
• Date: 2015-10-16
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M115.656702
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 290
• Issue: 42
• Start Page: 25534
• End Page: 25547
• Grant/Funding Information: All authors reviewed the results and approved the final version of the manuscript.
• Keywords: DOMAINS; Biochemistry & Molecular Biology; Science & Technology; CELL-ADHESION; MESENCHYMAL TRANSITION; SPECIFICITY; BIOLOGICAL-ACTIVITY; RECEPTOR; Life Sciences & Biomedicine; SYNERGY SITE; BETA-CATENIN; BETA-1-INTEGRIN; ADSORPTION
• Research Categories: Chemistry, Biochemistry; Engineering, Biomedical

Abstract

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9R →A 10), (iii) a two-Gly (FnIII92G 10) insertion, and (iv) a four-Gly (FNIII94G 10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9'10 and FnIII92G 10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.