Abstract. The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surfaee of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.VER 25 different membrane receptors have been identiffed that participate in receptor-mediated endocytosis (4, 24). Characteristically, these receptors are transmembrane glycoproteins that bind protein ligands and carry them into an endocytic system. In many cases, the receptor is recycled back to the cell surface where it participates in subsequent rounds of internalization while at the same time the ligand is transported to other intracellular compartments such as the lysosome (11). The clathrin-coated pit is the staging area for this process since it controls both receptor clustering and the formation of the endosome (3, 25).The folate receptor, also known as the membrane folate binding protein, is a membrane protein that has some features of a receptor that is involved in receptor-mediated endocytosis. The molecule is maximally expressed on the surface of folate-depleted tissue culture cells and is responsible for the high affinity accumulation of 5-methyltetrahydrofolic acid (5-CH3FI-L) t in the cytoplasm of these cells (8,(26)(27)(28)35). In MA104 cells, the surface membrane has two equal sets of receptors: one set is exposed to the extracellular environment, and the other is not accessible to this space. Several lines of evidence suggest that ordinarily the two sets of receptors completely exchange with each other once every hour (27,28). Kinetic studies have shown that this exchange reaction results in the delivery of bound 5-methyltetrahydrofolic acid to the cytoplasm (28) and that delivery is blocked by 1. Abbreviations used in this paper: 5-CH3FI-I4, 5-methyltetrahydrofolic acid; DNP, dinitrophenol; GPI, glycophospholipid; LDL, low density lipoprotein; TNP, 2,4,6 trinitrophenol.ionophores that disrupt proton gradients (27). Therefore, the folate receptor internalizes 5-CH3FH4 and recycles.Other information about the folate receptor is not consistent with the receptor-mediated endocytosis paradigm. Recently, we showed (30) that this receptor is anchored in the membrane by a glycosyl-phosphatidylinositol (GPI) linkage. Although a varied of membrane proteins are membrane anchored in this manner (19,33), none of these p...

A B S T R A C T In man, use of the general anesthetic nitrous oxide, N20, is associated with hematologic and neurologic abnormalities that mimic those seen in cobalamin (Cbl, vitamin

The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58±17 (mean±SD) and 5±2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182±34 and 26±6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing < 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 ;&M 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.

A B S T R A C T Analogues of cobalamin (

The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl)