Demonstration of receptors for insulin-like growth factor binding protein- 3 on Hs578T human breast cancer cells

Hs578T human breast cancer cells are from an estrogen receptor-negative breast cell line derived from a highly aggressive mammary tumor. Our previous insulin-like growth factor binding protein-3 (IGFBP-3) binding studies (Oh, Y., Muller, H. L., Lamson, G., and Rosenfeld, R. G. (1993) J. Biol. Chem. 268, 14964-14971) have demonstrated specific binding of IGFBP-3 on the Hs578T cell surface and a significant inhibitory effect of IGFBP-3, itself, on monolayer growth. In this study, we have demonstrated cell surface association proteins that are specific for IGFBP-3 by showing: 1) detection of 20-, 26-, and 50-kDa proteins by affinity cross-linking with ¹²⁵I- IGFBP-3(E. coli) and immunoprecipitation of cell monolayers and cell lysates with anti-IGFBP-3 antibodies; 2) dose-dependent competition of ¹²⁵I- IGFBP-3(E. coli) by unlabeled IGFBP-3(E. coli). 3) inhibition of IGFBP-3 binding to these cell surface proteins by EDTA and by coincubation with native insulin-like growth factor II (IGF-II), but not by coincubation with [Gln⁶, Ala⁷, Tyr¹⁸, Leu¹⁹, Leu²⁷]IGF-II, an IGF-II analog with decreased affinity for IGFBP-3; and 4) partial purification of 20- and 26- kDa species by IGFBP-3 anti-IGFBP-3 antibody immunoaffinity membranes. Characteristics of these specific IGFBP-3 cell surface association proteins are identical to those observed in our previous monolayer binding assay and monolayer growth assay experiments. The specificity of binding and the inhibitory effect of IGFBP-3 binding on Hs578T cell growth suggest that these cell surface proteins are IGFBP-3-specific receptors or receptor subunits mediating the direct inhibitory effect of IGFBP-3 on monolayer growth of Hs578T cells.