The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [3H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [3H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.
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Research Article|
April 01 2000
Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein
Jan Holm;
Jan Holm
1Department of Clinical Chemistry, Herning Hospital, Herning, DK-7400, Denmark
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Steen Ingemann Hansen;
Steen Ingemann Hansen
2Department of Clinical Chemistry, Central Hospital Hilleød, DK-3400, Denmark
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Mimi Høier-Madsen;
Mimi Høier-Madsen
3Laboratory for Autoimmune Serology, State Serum Institute, Copenhagen, DK-2300 S, Denmark
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Lars Korsbaek;
Lars Korsbaek
4Bartholin Institute, Municipal Hospital, Copenhagen, DK-1399, Denmark
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Heidi Beckmann;
Heidi Beckmann
4Bartholin Institute, Municipal Hospital, Copenhagen, DK-1399, Denmark
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Knud Josefsen
Knud Josefsen
4Bartholin Institute, Municipal Hospital, Copenhagen, DK-1399, Denmark
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Publisher: Portland Press Ltd
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© 2000 Plenum Publishing Corporation
2000
Biosci Rep (2000) 20 (2): 109–118.
Citation
Jan Holm, Steen Ingemann Hansen, Mimi Høier-Madsen, Lars Korsbaek, Heidi Beckmann, Knud Josefsen; Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein. Biosci Rep 1 April 2000; 20 (2): 109–118. doi: https://doi.org/10.1023/A:1005567417123
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