Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.
KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/micrograms protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/10(6) cells, and this is approximately 50% of the surface binding capacity of KG cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of [3H] folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions.
Luhrs
C A
CA
Department of Medicine, Brooklyn Veterans Affairs Hospital, New York 11209.
Raskin
C A
CA
Durbin
R
R
Wu
B
B
Sadasivan
E
E
McAllister
W
W
Rothenberg
S P
SP
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002470:Cell Survival; D004247:DNA / Q000235:genetics*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism / Q000494:pharmacology*; D006017:Glycolipids / Q000502:physiology*; D017261:Glycosylphosphatidylinositols; D006801:Humans; D007624:KB Cells; D051379:Mice; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000502:physiology*; D011956:Receptors, Cell Surface; D014162:Transfection
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D011956:Receptors, Cell Surface; D004247:DNA; D005492:Folic Acid