Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

Abstract

We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp 41 and Gly 42 residues within the C-C loop of EIIIA are necessary for integrin ␣9␤1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C loop encode sufficient information to completely inhibit ␣9␤1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in ␣9␤1-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability, and peptide sequences for probing EIIIA functions in vitro and in vivo.Guidance of cell function during adult tissue repair, the tumor microenvironment, and embryogenesis is highly orchestrated and involves the concerted action of extracellular matrix (ECM), 3 growth factors, and mechanical forces. Although it is now clear that the ECM serves both structural and instructional roles, the mechanisms that regulate the presentation of this "instruction set" to cells remain unclear. Potential mechanisms include alternative splicing to enhance the sequence complexity for an ECM gene product at a needed site. In addition, once expressed, ECM proteins can harbor cryptic sites that are exposed by specific proteolysis or by conformation changes induced by mechanical forces (3-6). The fibronectins (FNs) comprise a family of ECM proteins in which all three potential mechanisms have been implicated in its function.FNs are high molecular weight, multifunctional adhesive glycoproteins present in the ECM, connective tissues, basement membrane, and various body fluids. They provide excellent substrates for cell adhesion and spreading, thereby promoting cell migration during embryonic development, wound healing, and tumor progression and interact with other ECM proteins and cellular ligands, such as glycosaminoglycans, collagen, fibrin, and integrins (7). FNs are disulfide-bonded dimers of two closely related subunits, each consisting of three types of homologous repeating modules termed types I, II, and III (8). FN molecules have multiple isoforms generated from a single gene by alternative splicing of combinations of three exons: extra domain A (EDA/EIIIA), extra domain B (EDB/EIIIB), and connecting segment III (V). Both EIIIA and EIIIB exons are type III repeating units (7). Plasma fibronectin (pFN), produced by hepatocytes and abundant in plasma, lacks both the EIIIA and EIIIB domains. However, cellular FNs, produced by fibroblasts and epithelial and other cell types, are insoluble, and incorporated into the pericellular matrix, they contain the EIIIA and EIIIB segments in various combinations (9).

Integrin α9β1 mediates enhanced cell migration through nitric oxide synthase activity regulated by Src tyrosine kinase

“…Although much is known about the various ligands that can bind to integrin α9β1 to induce cell migration (Yokosaki et al, 1998; Yokasaki and Sheppard, 2000;Vlahakis et al, 2005;Vlahakis et al, 2007;Shinde et al, 2008;Staniszewska et al, 2008), the extent and nature of signaling pathways distal to the activated integrin remain unclear. Fig.…”

Eukaryotic Translation Elongation Factor 1A Induces Anoikis by Triggering Cell Detachment

“…Fibronectin, a key protein component of the ECM, plays a major role in survival and proliferation of many cell types through signal transduction via the fibronectin receptors (integrin ␣5␤1 and ␣4␤1) (9,10). Several cryptic functional sites have also been found in fibronectin, particularly in the fibronectin type III repeat (FNIII) (11)(12)(13)(14). We found previously that the amino acid sequence YTIYVIAL, buried within the structure of the 14th FNIII repeat, opposes cell adhesion to the extracellular matrix (15,16).…”

Integrin Regulation of Epidermal Functions in Wounds

“…8,92,93 Distinct binding motifs for a 9 b 1 have been identified, including the AEIDGIEL in tenascin-C, the PEDGIHE motif that occurs within an exposed loop of the fibronectin EIIIA domain, and distinct motifs within ADAM family members. 93,94 While a 9 b 1 has been studied less extensively than other integrins, its roles are likely to be complex given the wide range of potential ligands for this integrin that are present in wounds. Genetic studies have revealed an important role for a 9 b 1 in wound re-epithelialization.…”