Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.
Potocytosis is an endocytic process that is specialized for the internalization of small molecules. Recent studies on the uptake of 5-methyltetrahydrofolate by the folate receptor have suggested that the glycosyl-phosphatidylinositol anchor on this protein causes it to cluster and be internalized by caveolae instead of coated pits. To test this hypothesis directly, we have constructed a chimeric folate receptor that has the glycosyl-phosphatidylinositol anchor replaced with the transmembrane domain and cytoplasmic tail of the low density lipoprotein receptor. The cells with wild-type receptors delivered 5-methyltetrahydrofolate to the cytoplasm more rapidly than did cells expressing the chimeric receptor. This suggests that efficient delivery to the cytoplasm depends on caveolae. In sharp contrast to cells with wild-type folate receptors, cells internalizing folate by clathrin-coated pits were unable to decrease vitamin uptake when they were either folate replete or confluent.Cells use specific membrane receptors to concentrate various types of molecules before they are sequestered and delivered to the interior of the cell. There are at least two different sets of receptors: those that govern the uptake of macromolecules such as low density lipoprotein (LDL) and those that handle small molecules such as 5-methyltetrahydrofolate (5-MeTHF). Cells take up macromolecules by receptor-mediated endocytosis (1), using receptors that are internalized by clathrincoated pits. Many of these receptors use a tight ,B-turn motif (2, 3) in their cytoplasmic domain to cluster in coated pits (4, 5) prior to internalization. Small molecules, by contrast, appear to enter cells through caveolae by a process called potocytosis (6, 7). The receptors in this latter group are linked to the membrane by glycosyl-phosphatidylinositol (GPI) and it is the lipid anchor that mediates receptor clustering in association with caveolae (8-13).The molecules that are internalized by these two pathways have quite different fates. Clathrin-coated pits bud from the membrane and form vesicles that merge by a series of regulated fusion reactions with endosomes, lysosomes, and portions of the trans-Golgi network (14,15). The principal function of this pathway is to deliver macromolecules by lysosomes for hydrolytic processing or to transport them across polarized cells by transcytosis (16). Caveolae, on the other hand, seal off from the plasma membrane but appear to remain separate from other endocytic compartments. The small molecules that are concentrated within each closed caveola reach the cytoplasm by diffusing across the membrane through water-filled channels.The existence of two separate endocytic pathways, operating side-by-side in the same cell, suggests that caveolae are better able to deliver small molecules to the cytoplasm than areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fac...
The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor ineases the icy of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.Potocytosis is a high-affinity uptake process that cells use to obtain essential, low molecular weight molecules (1) from their environment. Membrane-bound proteins at the cell surface concentrate the molecules within closed caveolae. Once the molecules are released into the caveolar space, they diffuse into the cytoplasm through carrier proteins in the membrane.Caveolae are the membrane-bound transport vehicles for potocytosis (1). This membrane specialization is present on the surface of a wide variety of cells (2, 3) but has been studied most thoroughly in endothelial cells (4-6). In these cells, caveolae form vesicles or open channels that appear to transport molecules from the blood to the tissue spaces. The invaginated morphology of caveolae makes them easy to recognize in thin-section electron micrographs (7). Rapidfreeze, deep-etch images have shown that the cytoplasmic surface of invaginated and flat caveolae membranes is decorated with a characteristic striated coat. This coat is resistant to removal with high salt (7). A protein component ofthe coat has recently been identified and named caveolin (7). The membrane coat may play a role in controlling the closing or pinching off of caveolae to form the transport compartment for potocytosis.Potocytosis was discovered by studying the receptormediated uptake of 5-methyltetrahydrofolate in folatedepleted MA104 cells (8)(9)(10)(11)(12). The folate receptor is a glycosyl-phosphatidylinositol-anchored membrane protein that cycles in and out of the cell by caveolae. Each cycle delivers a quantity of the vitamin to the interior of closed caveolae, where it dissociates from the receptor and diffuses through anion carriers in the membrane into the cytoplasm. When cells are grown in physiological concentrations of 5-methyltetrahydrofolate, the receptor increases the efficiency of vitamin uptake by 30-fold (9). It is not known whether this increased efficiency confers any survival advantage on cells growing under physiological conditions. If it does, then cells cultured in the presence of 1-10 nM 5-methyltetrahydrofolate should grow more rapidly if they express the folate receptor. The availability ofthe cDNA for the folate receptor (13) Transfection. PAM 212 cells (16) were plated onto 100-mm dishes (1.5 x 105 cells per dish) and cultured in 10 ml of cDMEM containing Polybrene at 3 mg/ml (15). After 18 hr, the medium was replaced with 3 ml of cDMEM containing Polybrene at 3 mg/ml....
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