Identification of CD47/integrin-associated protein and alpha(v)beta3 as two receptors for the alpha3(IV) chain of type IV collagen on tumor cells.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.