Ligand binding characteristics of a glycosylphosphatidyl inositol membrane-anchored HeLa cell folate receptor epitope-related to human milk folate binding protein

The folate receptor (FR) in HeLa cells was characterized as to ligand bindi ng mechanism, antigenic properties and membrane anchor in order to obtain i nformation to be used for the design of biological agents targeting FR in m alignant tumors. The receptor displayed the following binding characteristi cs in equilibrium dialysis experiments (37 degrees C, pH 7.4) with [H-3] fo late: a high-affinity type of binding that exhibited positive cooperativity with a Hill coefficient > 1.0 and an upward convex Scatchard plot, a slow radioligand dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of other folates. The molecular size of the receptor was 10 0 kDa on gel filtration with Triton X-100, or similar to that of high molec ular weight human milk folate binding protein (FBP). The latter protein rep resents a 25 kDa molecule which equipped with a hydrophobic glycosylphospha tidyl inositol (GPI) membrane anchor susceptible to cleavage by phosphatidy linositol specific phospholipase C (PI-PLC) forms micelles of 100 kDa size with Triton X-100. The HeLa cell FR immunoreacted with antibodies against p urified human milk FBP in ELISA, and in a fluorescence activated cell sorti ng system, where HeLa cells exposed to increasing concentrations of antibod y showed a dose-dependent response. Exposure to PI-PLC decreased the fracti on of immunolabeled cells indicating a linkage of FR to cell membranes by a GPI anchor. HeLa cells incubated with radiofolate showed a continuous upta ke with time, however, with a complete suppression of uptake in the presenc e of an excess of cold folate. Prewash of cells at acidic pH to remove endo genous folate increased the uptake. Binding and uptake of [H-3] folate was increased in cells grown in a folate-deprived medium. The HeLa FR seems to be epitope related to human milk FBP.