The alpha 3(IV)185-206 peptide from noncollagenous domain 1 of type IV collagen interacts with a novel binding site on the beta(3) subunit of integrin alpha(v)beta(3) and stimulates focal adhesion kinase and phosphatidylinositol 3-kinase phosphorylation

We have recently identified integrin alpha (v)beta (3), and the associated CD47/integrin-associated protein (IAP) together with three other proteins a s the potential tumor cell receptors for the ly, chain of basement membrane type IV collagen (Shahan, T.A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G ., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590) . Using different cell lines expressing alpha (v)beta (3), alpha (IIb)beta (3), and/or CD47 and a liquid phase receptor capture assay, we now provide direct evidence that the synthetic and biologically active alpha3(IV)185-20 6 peptide, derived from the alpha3(IV) chain, interacts with the beta (3) s ubunit of integrin alpha (v)beta (3), independently of CD47. Increased alph a3(IV) peptide binding was observed on transforming growth factor-beta (1)- stimulated HT-144 cells shown to up-regulate alpha (v)beta (3) independentl y of CD47. Also, incubation of HT-144 melanoma cells in suspension induced de novo exposure of ligand-induced binding site epitopes on the beta (3) su bunit similar to those observed following Arg-Gly-Asp-Ser (RGDS) stimulatio n. However, RGDS did not prevent HT-144 cell attachment and spreading on th e alpha3(TV) peptide, suggesting that the alpha3(TV) binding domain on the beta (3) subunit is distinct from the RGD recognition site. alpha3(IV) pept ide binding to HT-144 cells in suspension stimulated time-dependent tyrosin e phosphorylation, while the RGDS peptide did not. Two major phosphotyrosin e proteins of 120-130 and 85 kDa were immunologically identified as focal a dhesion kinase and phosphatidylinositol 3-kinase (PI3-kinase). A direct inv olvement of PI3-kinase in alpha3(IV)-dependent beta (3) integrin signaling could be documented, since pretreatment of HT-144 cells with wortmannin, a PI3-kinase inhibitor, reverted the known inhibitory effect of alpha3(IV) on HT-144 cell proliferation as well as membrane type 1-matrix metalloprotein ase gene expression. These results provide evidence that the alpha3(TV)185- 206 peptide, by directly interacting with the beta (3) subunit of alpha (v) beta (3), activates a signaling cascade involving focal adhesion kinase and PI3-kinase.