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 binding mechanism, antigenic properties and membrane anchor in order to obtain information to be used for the design of biological agents targeting FR in malignant tumors. The receptor displayed the following binding characteristics in equilibrium dialysis experiments (37 degrees C, pH 7.4) with [3H] folate: 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 100 kDa on gel filtration with Triton X-100, or similar to that of high molecular weight human milk folate binding protein (FBP). The latter protein represents a 25 kDa molecule which equipped with a hydrophobic glycosylphosphatidyl inositol (GPI) membrane anchor susceptible to cleavage by phosphatidylinositol specific phospholipase C (PI-PLC) forms micelles of 1kDa size with Triton X-100. The HeLa cell FR immunoreacted with antibodies against purified human milk FBP in ELISA, and in a fluorescence activated cell sorting system, where HeLa cells exposed to increasing concentrations of antibody showed a dose-dependent response. Exposure to PI-PLC decreased the fraction of immunolabeled cells indicating a linkage of FR to cell membranes by a GPI anchor. HeLa cells incubated with radiofolate showed a continuous uptake with time, however, with a complete suppression of uptake in the presence of an excess of cold folate. Prewash of cells at acidic pH to remove endogenous folate increased the uptake. Binding and uptake of [3H] folate was increased in cells grown in a folate-deprived medium. The HeLa FR seems to be epitope related to human milk FBP.
Holm
J
J
Department of Clinical Chemistry, Herning Hospital, Denmark.
Hansen
S I
SI
Høier-Madsen
M
M
Korsbaek
L
L
Beckmann
H
H
Josefsen
K
K
D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D058973:Folate Receptors, GPI-Anchored; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008895:Milk, Human / Q000378:metabolism*; D010716:Phosphatidylinositols; D011956:Receptors, Cell Surface / Q000378:metabolism
D002352:Carrier Proteins; D000939:Epitopes; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D010716:Phosphatidylinositols; D011956:Receptors, Cell Surface