Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell ␣-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor--mediated smooth muscle cell ␣-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His 44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2 ) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile 43 and His 44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.The fibronectins (FNs) 1 comprise a group of extracellular matrix proteins that mediate cell adhesion, migration, proliferation, and differentiation (1). FNs play significant roles in embryonic development and are prominent components of the provisional matrix following tissue injury in adults (2, 3). The fundamental importance of the FNs is substantiated by the observation that homozygous mutations in either the FN gene or in the ␣ 5 integrin, a FN-specific receptor, are lethal (4, 5). The FNs are disulfide-linked, dimeric glycoproteins with structural domains that bind cells, collagen, proteoglycans, and fibrin. Each FN consists of homologous repeats, either type I, II, or III. Individual type III repeats within FN exhibit high sequence similarity between species (greater than 90% identity (6)). Despite variations in protein sequence identity between different type III repeats within FN (20 -40% (6)), these repeats show a high degree of structural homology (7-11). X-ray crystallographic studies demonstrate that each type III repeat consists of two  sheets, made up of four strands (G, F, C, CЈ) and three strands (A, B, E) respectively, folded into a  sandwich (7). This structural arrangement is also conserved in other proteins, including growth hormone (12), tenascin (13), neuroglian (14), tissue factor (15), and chitinases (16).Diversity in the FNs occurs by alternative splicing in two type III repeats termed EIIIA (or ED-A) and EIIIB (or ED-B) and one non-homologous repeat called V (or IIICS) (1). The EIIIA and EIIIB segments are either entirely included or excluded, whereas the V region may be included, excluded, or partial...
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