Clustered folate receptors deliver 5-methyltetrahydrofolate to cytoplasm of MA104 cells

Previously, a high affinity, glycosylphosphatidylinositol-anchored receptor for folate and a caveolae internalization cycle have been found necessary for potocytosis of 5-methyltetrahydrofolate in MA104, We now show by cell fractionation that folate receptors also must be clustered in caveolae for potocytosis. An enriched fraction of caveolae from control cells retained 65-70% of the [H-3]folic acid bound to cells in culture. Exposure of cells to the cholesterol-binding drug, filipin, which is known to uncluster receptors, shifted similar to 50% of the bound [H-3]folic acid from the caveolae fraction to the noncaveolae membrane fraction and markedly inhibited internalization of [H-3]folic acid, An mAb directed against the folate receptor also shifted similar to 50% of the caveolae-associated [H-3]folic acid to noncaveolae membrane, indicating the antibody perturbs the normal receptor distribution. Concordantly, the mAb inhibited the delivery of 5-methyl[H-3]tetrahydrofolate to the cytoplasm. Receptor bound 5-methyl[H-3]tetrahydrofolate moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor-mediated endocytosis. These results suggest cell fractionation can be used to study the uptake of molecules by caveolae.