Annexin VI stimulates endocytosis and is involved in the trafficking of low density lipoprotein to the prelysosomal compartment

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.     

Demonstration of two distinct transferrin receptor recycling pathways and transferrin-independent receptor internalization in K562 cells

The endocytosis and recycling of the human transferrin receptor were evaluated by several experimental modalities in K562 cells perturbed with 10(-5) M monensin. The work presented is an extension of a previous study demonstrating both complete inhibition of release of internalized human transferrin and a 50% reduction in the number of cell surface transferrin binding sites in K562 cells treated with monensin (Stein, B. S., Bensch, K. G., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772). The data directly reveal the existence of two distinct transferrin receptor recycling pathways. One pathway is monensin-sensitive and is felt to represent recycling of transferrin receptors through the Golgi apparatus, and the other pathway is monensin-resistant and most likely represents non-Golgi-mediated transferrin receptor recycling. A transferrin-free K562 cell culture system was developed and used to demonstrate that cell surface transferrin receptors can be endocytosed without antecedent ligand binding, indicating that there are factors other than transferrin binding which regulate receptor internalization. Evidence is presented suggesting that two transferrin receptor recycling pathways are also operant in K562 cells under ligand-free conditions, signifying that trafficking of receptor into either recycling pathway is not highly ligand-dependent.     

Internalization and recycling pathways of the thyrotropin receptor.

Scant information is available to date on the intracellular trafficking of the TSH receptor. In the present study we have used stably transfected L cells that express the TSH receptor, 225I-labeled TSH, and antireceptor antibodies as well as gold-conjugated antireceptor monoclonal antibodies and hormone. The latter allowed us to study, by electron microscopy, the cellular distribution and endocytosis of TSH receptor. The receptor was initially localized on the plasmalemma proper and in clathrin-coated pits but was excluded from smooth vesicles open to the cell surface. It was internalized through clathrin-coated vesicles. Constitutive endocytosis represented 10% of cell surface receptor molecules. Endocytosis was increased 3-fold by incubation with hormone. The majority of internalized receptor molecules (90%) was recycled to the cell surface, whereas the hormone was degraded in lysosomes. This recycling of receptor was inhibited by administration of monensin. Electron microscopic and confocal microscopic studies were repeated in primary cultures of human thyroid cells and showed a distribution, and endocytosis pathways, very similar to those observed in transfected L cells. A previous study has shown the LH receptor to be endocytosed in high proportion and to be degraded in lysosomes. Confocal microscopy and colocalization studies with transferrin receptor confirmed that the highly homologous LH and TSH receptors exhibit, when expressed in the same cells, very different cellular trafficking properties. The use of LH/TSH receptor chimeras showed that transmembrane-intracellular domains contain information orienting the protein toward recycling or degradative pathways. The extracellular domain seems to play a role in the extent of intemalization. These observations should now allow the identification of the molecular signals involved.     

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.     

Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells

Three cell-permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin-mediated endocytosis in mammalian cells. These compounds had variable effects on receptor-mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jasplakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos-7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor-mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.     

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell''s waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell''s waltzer fibroblasts.     

Formation of coated vesicles from coated pits in broken A431 cells

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti- transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.     

Sorting and recycling of cell surface receptors and endocytosed ligands: the asialoglycoprotein and transferrin receptors

With few exceptions, receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. During this process, ligand-receptor dissociation occurs, most probably in a low pH prelysosomal compartment. In most cases the ligand is ultimately directed to the lysosomes, wherein it is degraded, while the receptor recycles to the cell surface. We have studied the kinetics of internalization and recycling of both the asialoglycoprotein receptor and the transferrin receptor in a human hepatoma cell line. By employing both biochemical and morphological/immunocytochemical approaches, we have gained some insight into the complex mechanisms which govern receptor recycling as well as ligand sorting and targeting. We can, in particular, explain why transferrin is exocytosed intact from the cells, while asialoglycoproteins are degraded in lysosomes. We have also localized the intracellular site at which endocytosed receptor and ligand dissociate.     

Functional and genetic analysis of annexin VI

This study is concerned with the determination of the function of the 68kDa calcium-binding protein, annexin VI. Studies on the structure and regulation of the gene include a detailed analysis of annexin VI expressed heterologously in human A431 carcinoma cells. We have recently discovered that annexin VI is subject to a novel growth dependent post-translational modification. Interestingly, the protein exerts a negative effect on A431 cells. This effect was manifested as a partial reversal of the transformed phenotype. We are currently exploring the hypothesis that the post-translational modification of annexin VI is required for sub-cellular targeting, and that correct localisation within the cell is essential for function.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.     

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.     

Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.     

Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements.

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.     

Human transferrin receptor contains O-linked oligosaccharides

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.     

Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid

The human transferrin receptor is post-translationally modified by the covalent attachment of palmitic acid to Cys62 and Cys67 via a thio-ester bond. To investigate the role of the acylation of the transferrin receptor, Cys62 and Cys67 were substituted with serine and alanine residues. The properties of the mutant receptors were compared with wild-type receptors after expression in Chinese hamster ovary cells that lack endogenous transferrin receptors. Rapid incorporation of [3H]palmitate into the wild-type transferrin receptor was observed, but the mutant receptors were found to be palmitoylation-defective. The kinetics of endocytosis and recycling of the wild-type and mutant receptors were compared. It was observed that the rate of endocytosis of the palmitoylation-defective transferrin receptors was significantly greater than the rate measured for the wild-type transferrin receptor. In contrast, the mutation of Cys62 and Cys67 was found to have no significant effect on the rate of transferrin receptor recycling. Consistent with these observations, it was found that cells expressing palmitoylation-defective transferrin receptors exhibited an increased rate of accumulation of [59Fe]diferric transferrin. Together, these data indicate that the palmitoylation of the transferrin receptor is associated with an inhibition of the rate of transferrin receptor endocytosis. Addition of insulin to cultured cells causes an increase in the palmitoylation of cell surface transferrin receptors and a decrease in the rate of transferrin receptor internalization. It was observed that the effect of insulin to inhibit the endocytosis of the acylation-defective [Ala62 Ala67]transferrin receptor was attenuated in comparison with the wild-type receptor. The decreased effectiveness of insulin to inhibit the internalization of the acylation-defective transferrin receptor is consistent with the hypothesis that palmitoylation represents a potential mechanism for the regulation of transferrin receptor endocytosis.     

地衣霉素对细胞膜表面运铁蛋白受体功能的影响

应用抑制糖蛋白Ｎ－糖链合成的地衣霉素处理ＳＭＭＣ－７７２１人肝癌细胞,３Ｈ甘露糖掺入实验显示细胞膜表面糖蛋白Ｎ－糖链的合成受到显著抑制,但细胞膜表面运铁蛋白受体内吞再循环的过程无显明变化,进一步的研究表明受体与运铁蛋白的亲和力亦无改变,但细胞膜表面运铁蛋白受体数减少。结果提示用地衣霉素处理细胞后,在内质网合成的无Ｎ－糖链的运铁蛋白受体影响其运输到细胞膜表面表达。     

A role for the cytoplasmic domain in transferrin receptor sorting and coated pit formation during endocytosis

The cytoplasmic domain of transferrin receptor (TR) is essential for endocytosis of this transmembrane protein. We have investigated by electron microscopy the association of wild-type and cytoplasmic deletion mutant human TR with coated pits at the surface of transfected L cell lines. Approximately 15% of wild-type TR was concentrated in coated pits, regardless of the level of TR expression. In contrast, only 2% of deletion mutant TR was present in these structures. We also correlated the frequency of coated pits with the level of TR expression in different transfected L cell lines. Expression of more than 3 x 10(6) wild-type TR per cell was accompanied by up to a 4-fold increase in coated pits compared with nontransfected Ltk- cells. No such increase was observed in a cell line expressing a similarly high level of cytoplasmic deletion mutant TR. These results indicate that the cytoplasmic domain plays an active role in sorting and endocytosis of TR by providing an assembly site for coated pit formation.