Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake.

Abstract

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...

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

“…Several studies of the endocytic pathway of folate receptors in which kidney cells, MA104, as well as other cell lines were used have indicated that caveolae are involved in folate receptor endocytosis (1,8,42,43). Other studies have shown that folate receptors are localized to clathrin-coated-pits and vesicles (2,3,22).…”

Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase

“…This involves nonclathrin-coated pit transport of certain ions, low-molecular-weight molecules or macromolecules from the cell surface to the cytoplasm, to internal organelles such as the ER (4), and from one plasma membrane to another plasma membrane site (e.g., apical to basolateral). Folate transport, which involves a glycerolphosphoinositol (GPI)-anchored receptor, is a well-known example of potocytosis (160,205), but Fe 2ϩ , Ca 2ϩ , alkaline phosphatase, and insulin are also transported in this manner (4,85,168). Caveolae-based endocytosis can be employed opportunistically by toxins, viruses, bacteria (227), and trypanosomes (63,232) to gain access to the cell's interior.…”

The Caveolae Membrane System

“…Proteins modified with either GPI or fatty acids are found to be enriched in caveolae fractions obtained by most methods of purification (7,8). Mutations that abolish either the GPI-anchor addition (51,52) or fatty acylation (53,54) shift the protein to other fractions, which suggests that the lipid moiety is required for targeting to caveolae. These two different covalent modifications, therefore, are responsible for targeting proteins with a wide range of biochemical activities to opposing surfaces of the same membrane domain.…”

“…A minor population of folate receptors appear in the coated vesicle pathway of cells expressing high numbers of receptors (139). But when chimeric receptors are targeted specifically to coated pits, folate delivery is inefficient and unregulated (52). The process was named potocytosis (5) to emphasize the special ability of caveolae to concentrate and move molecules or ions into the cell.…”