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).

We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for ␣4 integrins. Here we show that ␣4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of ␣4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the ␣4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when ␣4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the ␣4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the ␣4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for ␣4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury. Key words: integrin; peripheral nerve regeneration; fibronectin; alternative splicing; paxillin; ␣4; PC12 cell; dorsal root ganglia; chimera; LDVDamage to the peripheral nervous system (PNS) is followed by Wallerian degeneration of axons distal to the lesion site associated with increased expression of extracellular matrix (ECM) molecules including fibronectin (FN) (Lefcort et al., 1992;Martini, 1994;Scherer and Salzer, 1996). Antibody-blocking experiments suggest that these increased levels of FN contribute to the subsequent repair (Toyota et al., 1990;Wang et al., 1992;Bailey et al., 1993;Agius and Cochard, 1998). FN is expressed as different isoforms generated by alternative splicing of the primary gene transcript. Two type III repeats EIIIA and EIIIB are either included or excluded, whereas the V (IIICS) region can be partly or completely excluded in patterns that differ between species (Schwarzbauer et al., 1983(Schwarzbauer et al., , 1987Tamkun et al., 1984;Kornblihtt et al., 1985;Gutman and Kornblihtt, 1987;Zardi et al., 1987). In the rat, three different forms (V0, V95, and V120) can be generated (Fig. 1). The latter two differ by the inclusion or exclusion of the first 25 amino acids, a segment called V25. This segment contains a cell-binding sequence Leu-Asp-Val (LDV) that is recognized by the integrins ␣4␤1 and ␣4␤7 (Wayner et al., 1989;Guan and Hynes, 1990). This cell-binding site is distinct from the Arg-Gly-Asp (RGD) sequence within the 10 th type III repeat (Fig. 1), recognized by other integrins including ␣5␤1 (Ruoslahti, 1996). The expression of the alternatively spliced isoforms of FN in vivo is developmentally regulated. Most FN mRNA early in development is EIIIAϩ, EIIIBϩ, and...

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that α4, α5, and β1 are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage α4β1-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, α4β1-integrin–dependent phase.