Collagen-binding proteins of mammary epithelial cells are related to Ca2(+)- and phospholipid-binding annexins

Three major proteins of 34, 36, and 38 kDa were isolated from membrane preparations of chemically induced mammary tumors of the rat by collagen type I affinity chromatography and therefore were termed collagen-binding proteins (CBP). Three proteins in the same molecular weight range isolated from cell extracts by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on hydroxylapatite were demonstrated to be immunologically related to CBP. As shown by immunoblot analysis, an antiserum directed against the cluster of the 34-38 kDa proteins reacted strongly with porcine intestinal protein I, weakly with porcine lipocortin I, and very weakly with porcine intestinal protein II. Antiserum against the 34 kDa protein reacted weakly with protein I but strongly with protein II. All three CBP reacted with protein I/calpactin I-specific antiserum of immunoblots and in immunoprecipitation experiments. However, antisera directed against CBP failed to show cross-reaction with collagen-binding protein anchorin II from chicken chondrocytes. Conversely, antisera against anchorin II did not react with CBP. Antiserum AS/87 immunoprecipitated CBP of 38 kDa that was labeled in a lactoperoxydase-catalyzed iodination, suggesting that this polypeptide is associated with the cell surface. Further, all three CBP were found to be phosphorylated by incubating mammary cells with 32P-orthophosphate. CBP bound to epithelial cell membranes in a Ca2+ dependent manner (= Triton X 100 insoluble form). Fractionated extraction and immunofluorescence microscopy also show that another form of CBP (= Triton X 100 soluble form) exists in these cells and is associated with a granular fraction. We therefore conclude that mammary collagen-binding proteins represent members of a family of Ca2(+)-binding membrane proteins. The 38 kDa CBP seems closely related to the pp60src kinase substrate protein I/calpactin I monomer, the 34 kDa CBP seems to be related or equivalent to protein II, while the relationship of the 36 kDa CBP to other defined proteins is still unclear.