Abstract
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.
Highlights
We have previously shown that the anterior lens capsule (ALC) type IV collagen, which contains an ␣3(IV) chain, as well as the NC1 domain derived from the ␣3(IV) chain inhibited HT-144 melanoma cell proliferation and migration, whereas Engelbreth-Holm-Swarm collagen, which does not contain the ␣3(IV) chain, did not [17, 18]
We demonstrate that the ␣3(IV) peptide identifies a novel type IV collagen binding site on the 3 subunit of integrin ␣v3, distinct from the RGD recognition site, and initiates an ␣v3-dependent intracellular signaling process leading to focal adhesion kinase (FAK) and PI3-kinase phosphorylation
As shown by flow cytometry analysis (Fig. 1A), erythrocytes expressed CD47 in the absence of ␣v3 and HT-144 melanoma cells were positive for both human ␣v3 and CD47, whereas Chinese hamster ovary (CHO) transfectants expressing the recom
Summary
Horseradish peroxidase-conjugated sheep anti-mouse IgG was purchased from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). Fluorescein-conjugated goat anti-mouse IgG and fluoresceinconjugated streptavidin were from Jackson Immunoresearch Laboratories Inc. Anti-CD47 monoclonal antibody (mAb) (B6H12) was purchased from Pharmingen (San Diego, CA); polyclonal anti-FAK (C903) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); polyclonal anti-PI3-kinase p85 was from Upstate Biotechnologies, Inc. PCR primers were obtained from Eurogentec (Seraing, Belgium). The NC1 domain from ALC type IV collagen was prepared as described previously [21]. The biotinylated peptide corresponding to residues 185–206 in the NC1 domain of the ␣3 chain of type IV collagen, 185CNYYSNSYSFWLASLNPERMFR206-KKK(Biotin)NH2, as well as the corresponding scrambled peptide, MPSWRFASLEYCSRNFNYNYSL-KKK(Biotin)-NH2, were purchased from Neosystem (Strasbourg, France). The following monoclonal antibodies were generous gifts: anti-␣IIb (S1.3) and anti-3 (4D10G3)
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