Biol. Pharm. Bull. 30(5) 891—897 (2007)

tant determinants of cellular morphology and function. The ECM proteins, such as fibronectin (FN), collagen and laminin, support cell adhesion and thereby influences various cellular processes, such as proliferation, differentiation, survival and migration. Cell adhesion should be controlled reversibly in vivo, and it has therefore been presumed that cell adhesion-inhibiting factors as well as cell adhesion-stimulating factors are required for normal development. From this view point, endogenous factors modulating negatively cell adhesion to the ECM, so-called “antiadhesive factors,” have been explored, and some protein factors such as thrombospondine, tenascin, SPARC and osteopontin have been identified. FN which is one of the essential cell adhesive ECM proteins, harbors several cell adhesive sites, such as RGD central cell adhesive site recognized by integrin a5b1 and CS-1 and -5 in alternative splicing domain recognized by integrin a4b1. We previously found that the FN has not only these cell adhesive sites but also an additional functional site opposing cell adhesion to the ECM, so-called “antiadhesive site.” A synthetic FN peptide derived from the 14th FN type III-like (FN-III) repeat, termed peptide FNIII14 (Fig. 1), strongly inhibits b1 integrin-mediated cell adhesion to the FN without binding to integrins, whose activity depends on its C-terminal amino acid sequence YTIYVIAL. The antiadhesive sequence YTIYVIAL is cryptic at least in the plasma FN molecule, but it is exposed by either FN processing with MMP-2 or FN interaction with heparin. As a negative modulator of cell-ECM interaction, peptide FNIII14 influences physiological cellular processes in vitro, such as survival, differentiation and gene expression, as well as pathological events including tumor metastasis. Therefore, it might be possible that this cryptic antiadhesive sequence YTIYVIAL, once exposed, plays important roles in cell regulation in vivo. Although an intracellular signaling pathway by which peptide FNIII14 expresses the antiadhesive activity remains unclear, our following previous findings have indicated that antiadhesive activity of peptide FNIII14 is mediated by a putative membrane receptor: (i) peptide FNIII14 conjugated with polyethylenglycol (Mr. 10000), which is membrane impermeable hydrophilic polymer, is able to inhibit cell adhesion to FN (Miura, S. and Fukai, F., unpublished data), (ii) peptide FNIII14 has the antiadhesive effect even when coated on culture plates, (iii) peptide FNIII14 specifically binds to a membrane protein with molecular mass of 50-kDa (p50), not to b1 integrins. There are several different sequences analogous to the antiadhesive sequence YTIYVIAL in another FN-III repeat of the FN molecule and also in the FN-III repeats of other ECM proteins. They include the YTITVYAV of the 10th FN-III repeat of the FN, the YQVTVIAL in the 1st FN-III repeat of type VII collagen and the YTITVSSL and YKITVIAV in the 13th and 16th FN-III repeats, respectively, of type XII collagen (Fig. 1). These analogous sequences may be important for these ECM protein molecules in serving as scaffoldings for cell adhesion. In this study, we first investigate whether the presence of p50, a specific binding protein of peptide FNIII14, on cell surface is necessary for peptide FNIII14 in expressing its antiadhesive activity. The results show that cellular susceptibility to the antiadhesive effect of peptide FNIII14 is in parallel with the presence of p50 on cell surfaces, suggesting that this p50 serves as a receptor for peptide FNIII14. Next, we examine whether peptides derived May 2007 891