The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

Abstract

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...

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).…”

Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

“…Conceptually, the folate-FBP complex might be internalized in caveolae by a process termed potocytosis, following which folate is dissociated by low pH and crosses into the cytoplasm by an anion carrier and is polyglutamylated while the FBP recycles back to the cell surface [29]. Although this concept was developed from studies with isolated monkey kidney MA104 cells [30], it is also applicable to the proposed mechanism for folate uptake by Caco-2 cells [8] and the rat kidney proximal tubule [31] in which uptake was prevented by the cleavage of membrane-bound [$H]folic acid from transfected cells and isolated rat kidney brush border membranes by phosphatidylinositol phospholipase C [8,31]. The sequence analysis of pig liver FBP might be consistent with the glycosylphosphatidylinositol anchoring system, because 9 of the 14 C-terminal residues were identical with those described in isolated human Caco-2 and KB cells ( Figure 2) [2,7,8].…”

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging

“…Apparently, sufficient recycling machinery is available in early endosomes to allow some degree of membrane recycling. We also speculate that some type of tether may exist between these peripherally stalled vesicles and the plasma membrane that enables the return of the FR-containing endosome back to the cell surface (23,24).…”