title: Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat.
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 (FnIII9(R)→(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)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 FnIII9(2G)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.
authors: From the Department of Biomedical Engineering, North Carolina State University and the University of North Carolina, Raleigh, North Carolina 27606.
authors: Ashley C
authors: AC
authors: Brown
authors: the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta Georgia 30332.
authors: Marilyn M
authors: MM
authors: Dysart
authors: the School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332.
authors: Kimberly C
authors: KC
authors: Clarke
authors: the School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona 85287, and.
authors: Sarah E
authors: SE
authors: Stabenfeldt
authors: the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta Georgia 30332, the Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332 thomas.barker@bme.gatech.edu.
authors: Thomas H
authors: TH
authors: Barker
mesh_terms: D000595:Amino Acid Sequence; D002448:Cell Adhesion; D002460:Cell Line; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D039201:Integrin alpha3beta1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding
chemical_list: D005353:Fibronectins; D039201:Integrin alpha3beta1
keywords: cell adhesion; epithelial cell; extracellular matrix; fibronectin; integrin; laminin; surface plasmon resonance (SPR)