CLUSTERED FOLATE RECEPTORS DELIVER 5-METHYLTETRAHYDROFOLATE TO CYTOPLASM OF MA104 CELLS

Previously, a high affinity, glycosylphosphatidylinositol-anchored rec eptor 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 cluster ed 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 b ound [H-3]folic acid from the caveolae fraction to the noncaveolae mem brane fraction and markedly inhibited internalization of [H-3]folic ac id, An mAb directed against the folate receptor also shifted similar t o 50% of the caveolae-associated [H-3]folic acid to noncaveolae membra ne, indicating the antibody perturbs the normal receptor distribution. Concordantly, the mAb inhibited the delivery of 5-methyl[H-3]tetrahyd rofolate to the cytoplasm. Receptor bound 5-methyl[H-3]tetrahydrofolat e moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor-mediated endocytosis. Th ese results suggest cell fractionation can be used to study the uptake of molecules by caveolae.