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The Journal of biological chemistry

Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties.


PMID 2808373

Abstract

We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. These observations demonstrate an interactive relationship between np69 and soluble fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.