We have recently identified integrin alpha(v)beta(3) and the associated CD47/integrin-associated protein (IAP) together with three other proteins as the potential tumor cell receptors for the alpha(3) 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)185-206 peptide, derived from the alpha3(IV) chain, interacts with the beta(3) subunit of integrin alpha(v)beta(3), independently of CD47. Increased alpha3(IV) peptide binding was observed on transforming growth factor-beta(1)-stimulated HT-144 cells shown to up-regulate alpha(v)beta(3) independently 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) subunit similar to those observed following Arg-Gly-Asp-Ser (RGDS) stimulation. However, RGDS did not prevent HT-144 cell attachment and spreading on the alpha3(IV) peptide, suggesting that the alpha3(IV) binding domain on the beta(3) subunit is distinct from the RGD recognition site. alpha3(IV) peptide binding to HT-144 cells in suspension stimulated time-dependent tyrosine phosphorylation, while the RGDS peptide did not. Two major phosphotyrosine proteins of 120-130 and 85 kDa were immunologically identified as focal adhesion kinase and phosphatidylinositol 3-kinase (PI3-kinase). A direct involvement 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 metalloproteinase gene expression. These results provide evidence that the alpha3(IV)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.