The α3(IV)185–206 Peptide from Noncollagenous Domain 1 of Type IV Collagen Interacts with a Novel Binding Site on the β3Subunit of Integrin αvβ3and Stimulates Focal Adhesion Kinase and Phosphatidylinositol 3-Kinase Phosphorylation

Sylvie Pasco, Jean-Claude Monboisse, Nelly Kieffer
2000 Journal of Biological Chemistry  
We have recently identified integrin ␣ v ␤ 3 and the associated CD47/integrin-associated protein (IAP) together with three other proteins as the potential tumor cell receptors for the ␣ 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 ␣ v ␤ 3 , ␣ IIb ␤ 3 , and/or CD47 and a liquid phase receptor capture assay, we now provide
more » ... , we now provide direct evidence that the synthetic and biologically active ␣3(IV)185-206 peptide, derived from the ␣3(IV) chain, interacts with the ␤ 3 subunit of integrin ␣ v ␤ 3 , independently of CD47. Increased ␣3(IV) peptide binding was observed on transforming growth factor-␤ 1 -stimulated HT-144 cells shown to up-regulate ␣ v ␤ 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 ␤ 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 ␣3(IV) peptide, suggesting that the ␣3(IV) binding domain on the ␤ 3 subunit is distinct from the RGD recognition site. ␣3(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 ␣3(IV)-dependent ␤ 3 integrin signaling could be documented, since pretreatment of HT-144 cells with wortmannin, a PI3-kinase inhibitor, reverted the known inhibitory effect of ␣3(IV) on HT-144 cell proliferation as well as membrane type 1-matrix metalloproteinase gene expression. These results provide evidence that the ␣3(IV)185-206 peptide, by directly interacting with the ␤ 3 subunit of ␣ v ␤ 3 , activates a signaling cascade involving focal adhesion kinase and PI3-kinase.
doi:10.1074/jbc.m005235200 pmid:10934203 fatcat:pt3troasnfcobemfc257dq357i