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At least 19 recordsLinked to original sources
PubMed1991

Spontaneous endocytosis in human neonatal and adult red blood cells: comparison to drug-induced endocytosis and to receptor-mediated endocytosis.

Neonatal RBC contain many more spontaneous endocytic vacuoles than do adult RBC. It is not known if this difference is a result of an increase in production of vacuoles in the neonatal RBC (as is the case in drug-induced endocytosis), or is the result of a less effective neonatal macrophagic "pitting" process. Using an in vitro model of spontaneous endocytosis, we compared the rate and quantity of vacuoles and the shape of cord and adult RBC containing pits, visible by interference contrast microscopy (Nomarski method). The mechanism of the spontaneous endocytosis was explored using different inhibitors: sodium vanadate an inhibitor of ATPases, sodium fluoride which inhibits the generation of ATP and sodium cyanide a potent inhibitor of oxidative phosphorylation. We then compared spontaneous endocytosis with two other forms of RBC endocytosis: drug-induced endocytosis and receptor-mediated endocytosis. Spontaneous endocytosis is in fact increased in neonatal RBC initially but the increase in number of RBC containing pits after 144 hr of incubation is almost the same in adult RBC and neonatal RBC. Comparing spontaneous endocytosis with drug-induced endocytosis, it appears that their mechanisms are different in that spontaneous endocytosis is not preceded by stomatocytic shape change and is not inhibited by sodium vanadate or sodium fluoride as is the case for drug-induced endocytosis. Spontaneous endocytosis is different than transferrin receptor-mediated endocytosis because it occurs in many RBC, not only in the motile R1 reticulocytes and is not inhibited by sodium cyanide as is receptor-mediated endocytosis. Thus spontaneous endocytosis appears to be different than drug-induced endocytosis and transferrin receptor-mediated endocytosis. The increase in spontaneous endocytosis in cord RBC seen in vivo is probably a consequence of an immaturity of the neonatal macrophage pitting process.

Adult↗
PubMed2002

Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells.

Transient stimulation of secretion in calf chromaffin cells is invariably followed by rapid endocytosis (RE), a clathrin- and K(+)-independent process with a half time of several seconds. Here we show that when exocytosis is triggered in a more sustained manner, a much slower form of endocytosis (SE) replaces RE. SE is complete within 10 min and is abolished when anticlathrin antibodies are introduced into the cell or when intracellular K(+) is removed. RE, but not SE, is blocked by intracellular administration of antidynamin-1 antibodies; the inverse specificity was found for antidynamin-2 antibodies. Replacement of extracellular Ca(2+) by Ba(2+) or Sr(2+) completely blocked RE but had little effect on SE. Thus chromaffin cells exhibit two kinetically and mechanistically distinct forms of endocytosis that are coupled to different extents of exocytosis and are mediated by different isoforms of dynamin. We surmise that RE is associated with the transient fusion ("kiss-and-run") mechanism of transmitter release and is the prevalent means of vesicle recapture and recycling under normal physiological conditions, whereas the clathrin-based SE mechanism comes into play only at higher levels of stimulation and may be associated with complete fusion of vesicles with the plasma membrane.

Animals↗
PubMed1988

Comparison of transferrin receptor-mediated endocytosis and drug-induced endocytosis in human neonatal and adult RBCs.

Neonatal RBCs can undergo receptor-mediated endocytosis; normal adult RBCs cannot. Previously, we showed that drug-induced endocytosis, which can occur in adult RBCs exposed to amphipathic cations like primaquine, is greatly enhanced in all density-defined fractions of neonatal RBCs. To investigate the similarities and differences between receptor-mediated endocytosis and drug-induced endocytosis, we characterized transferrin receptor-mediated endocytosis in neonatal RBCs and compared it with drug-induced endocytosis. Primaquine drug-induced endocytosis is dependent on RBC ATP levels, is invariably preceded by stomatocytosis, and is inhibited by vanadate. In contrast, receptor-mediated endocytosis of transferrin is not preceded by stomatocytosis, is not nearly so dependent on ATP levels as is drug-induced endocytosis, and is not inhibited by vanadate. Furthermore, receptor-mediated endocytosis is quantitatively blocked by preincubation of neonatal RBCs with sodium cyanide, whereas cyanide does not inhibit drug-induced endocytosis in either adult or neonatal RBCs. Morphologic observation of the neonatal RBCs established the fact that only puckered RBCs that exhibited brilliant cresyl blue staining reticulum were capable of undergoing receptor-mediated endocytosis of transferrin. These characteristics identify them as motile R-1 reticulocytes. Reticulocytes in normal adult RBCs were incapable of exhibiting this phenomenon. Thus, receptor-mediated endocytosis, a property of motile reticulocytes in neonatal RBCs, differs from drug-induced endocytosis in its energy requirements, response to inhibitors, and morphologic concomitants.

Adenosine Triphosphate↗
PubMed1992

M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages.

Although coated vesicles can mediate both solute and receptor-mediated endocytosis, there are other kinds of endocytic vesicles that contribute to these processes. The relative contributions of these other organelles, particularly regarding solute influx, remains unsettled. Here we describe a physiological uncoupling of solute and receptor-mediated endocytosis that occurs during growth factor-stimulated macropinocytosis. We examined how recombinant human macrophage colony-stimulating factor (rM-CSF), which rapidly stimulates solute endocytosis in murine bone marrow-derived macrophages, affected ligand internalization via receptor-mediated endocytosis. Although rM-CSF stimulated internalization and accumulation of Lucifer Yellow (LY), a probe for solute endocytosis, it had no effect on accumulation of fluorescent acetylated low-density lipoprotein (acLDL), a ligand for the macrophage scavenger receptor, or on the endocytosis of 125I-labelled diferric transferrin. Video microscopy revealed that rM-CSF immediately induced active cell ruffling and the formation of phase-bright macropinosomes. Nocodazole pretreatment of macrophages inhibited both ruffling and macropinocytosis. Macropinosomes were fluorescently labelled by incubating macrophages briefly with probes for both solute endocytosis (fluorescent dextrans) and ligand endocytosis (fluorescein-labelled transferrin or diI-labelled acLDL). Macrophages incubated for one or two minutes formed macropinosomes that were labelled predominantly with the fluorescent solute probes but with little or none of the ligand probes; the latter were localized within smaller pinosomes. When cells pulsed with the fluorescent probes were washed and chased for an additional two minutes, solute and ligand probes occasionally co-localized in macropinosomes. Nocodazole inhibited macropinocytosis with little apparent effect on endocytosis via smaller vesicles. These experiments show that macropinosome formation is dependent on microtubules and also that the macropinosomes induced by rM-CSF are solute-rich and receptor-poor. Macropinosomes differ from coated vesicles in these respects, and therefore provide a physiologically regulated mechanism for uncoupling solute and receptor-mediated endocytosis.

Animals↗
PubMed1999

Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes.

Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [(125)I]tyramine cellobiose ([(125)I]TC). [(125)I]TC-labelled bovine serum albumin ([(125)I]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of [(125)I]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min. The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [(125)I]TC-BSA and [(125)I]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([(125)I]TC-AOM and [(125)I]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [(125)I]TC-AOM and [(125)I]TC-BSA; the uptake of [(125)I]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes.

Animals↗
PubMed2006

Aquaporin 2 (AQP2) and vasopressin type 2 receptor (V2R) endocytosis in kidney epithelial cells: AQP2 is located in 'endocytosis-resistant' membrane domains after vasopressin treatment.

BACKGROUND INFORMATION: Aquaporin 2 (AQP2) plays an important, VP (vasopressin)-regulated role in water reabsorption by the kidney. The amount of AQP2 expressed at the surface of principal cells results from an equilibrium between the AQP2 in intracellular vesicles and the AQP2 on the plasma membrane. VP shifts the equilibrium in favour of the plasma membrane and this allows osmotic equilibration to occur between the collecting duct lumen and the interstitial space. Membrane accumulation of AQP2 could result from a VP-induced increase in exocytosis, a decrease in endocytosis, or both. In the present study, we further investigated AQP2 accumulation at the cell surface, and compared it with V2R (VP type 2 receptor) trafficking using cells that express epitope-tagged AQP2 and V2R. RESULTS: Endocytosis of V2R and of AQP2 are independent events that can be separated temporally and spatially. The burst of endocytosis seen after VP addition to target cells, when AQP2 accumulates at the cell surface, is primarily due to internalization of the V2R. Increased endocytosis is not induced by forskolin, which also induces membrane accumulation of AQP2 by direct stimulation of adenylate cyclase. This indicates that cAMP elevation is not the primary cause of the initial, VP-induced endocytic process. After VP exposure, AQP2 is not located in endosomes with internalized V2R. Instead, it remains at the cell surface in 'endocytosis-resistant' membrane domains, visualized by confocal imaging. After VP washout, AQP2 is progressively internalized with the fluid-phase marker FITC-dextran, indicating that VP washout releases an endocytotic block that maintains AQP2 at the cell surface. Finally, polarized application of VP to filter-grown cells shows that apical VP can induce basolateral endocytosis and V2R down-regulation, and vice versa. CONCLUSIONS: After VP stimulation of renal epithelial cells, AQP2 accumulates at the cell surface, while the V2R is actively internalized. This endocytotic block may involve a reduced capacity of phosphorylated AQP2 to interact with components of the endocytotic machinery. In addition, a complex cross-talk exists between the apical and basolateral plasma-membrane domains with respect to endocytosis and V2R down-regulation. This may be of physiological significance in down-regulating the VP response in the kidney in vivo.

Animals↗
PubMed1986

Endocytosis of aggregated immunoglobulin G by rat basophilic leukemia cells; rate, extent, and effects on the endocytosis of immunoglobulin E.

Rat basophilic leukemia (RBL) cells have distinct receptors for IgE and IgG. We assessed the endocytosis of chemically and immunochemically cross-linked mouse-IgG and its influence on the simultaneous endocytosis of IgE. We found that at 37 degrees C, aggregates of IgG and IgE were endocytosed at about the same rate with one-half of the maximal endocytosis occurring in 5 to 13 min, and the efficiency of endocytosis for both ligands ranging from 40 to 70%. We also found that endocytosis of cross-linked IgE and IgG occurred simultaneously and neither ligand significantly affected the rate or extent of endocytosis of the other. The cells accumulated the cross-linked IgG, and then released it to the extracellular environment, at a rate (less than 3%/hr) slower than the released endocytosed IgE (greater than 10%/hr). Using an assay that discriminates between unbound and receptor-bound oligomeric IgG, we found that oligomeric IgG is endocytosed with its receptor, and that the bulk of the ligand remains bound to its receptor for greater than 120 min after endocytosis. The differences in the rate of release of endocytosed IgG vs IgE suggests that the intracellular fate or pathway of these two oligomeric ligands may differ.

Animals↗
PubMed1985

Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis.

Yeast cells have been shown to internalize lucifer yellow CH by endocytosis. Internalization of the fluorescent dye is time-, temperature-, and energy-dependent, it is not saturable, and the dye is accumulated in the vacuole. Some of the yeast secretory mutants that accumulate endoplasmic reticulum or Golgi bodies are defective for endocytosis at restrictive temperature, while others are not. All of the mutants that accumulate secretory vesicles are defective for endocytosis. These results suggest that efficient transport of proteins from the endoplasmic reticulum to the Golgi apparatus and from the Golgi to secretory vesicles is not necessary for endocytosis. In contrast, endocytosis may be obligatorily coupled with the latest steps of secretion.

Cytoplasmic Granules↗
PubMed1999

Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation.

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56(lck), undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56(lck), we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis.

Receptor-mediated endocytosis of decorin depends on its core-protein-mediated interaction with a 51 kDa membrane protein, which, in addition to its core-protein-binding site, carries a binding site for glycosaminoglycan chains. Membrane-associated heparan sulphate as well as heparin are known to have an inhibitory effect on decorin endocytosis by cultured skin fibroblasts. In this study, structural features of both glycosaminoglycans required for binding to the 51 kDa protein and for inhibiting decorin endocytosis, were investigated. Upon digestion of [(3)H]glucosamine-labelled heparan sulphate with heparinase III, dodeca- and higher saccharides were able to interact with the receptor protein. In comparison with unbound fragments of the same size, bound fragments were enriched in N-sulphated disaccharides carrying one or two sulphate ester groups. Using heparinase III-generated fragments from [(35)S]sulphate-labelled heparan sulphate chains, binding of fragments as small as octasaccharides could be detected. Competition experiments between dermatan sulphate and chemically modified heparin revealed that N- and 6-O-sulphation of glucosamine residues are important structural elements for binding to the receptor, whereas iduronate-2-O-sulphate groups contribute to binding only to a limited extent. However, with respect to the inhibition of decorin endocytosis, 2-O-desulphation had a quantitatively similar effect to 6-O-desulphation. Furthermore, for maximal inhibition of decorin endocytosis, longer fragments were required than for binding to the receptor. Thus, it appears that heparin/heparan sulphate has to interact with additional component(s) for effective inhibition of decorin uptake.

Cell Line↗
PubMed2001

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. Distribution of clathrin after stimulation of EGR endocytosis in various cell lines during inhibition of Src-kinase activity].

A distribution of EGF receptor and clathrin during EGF endocytosis in A431, HER14, WT and PURO cell lines was studied by indirect immunofluorescence. Though the initial distribution of EGF-receptors on A431 and HER14 cells was somewhat different, the late stages of endocytosis proceeded equally and were marked by formation of bright spots in the juxtanuclear region characteristic of the late endosomes. The Src-family kinase inhibitor CGP77675 had no influence on the dynamics of receptor endocytosis at the immunofluorescent level in both cell lines. Stimulation of EGF-receptor endocytosis in A431 cells did not also result in any redistribution of clathrin in the areas where the majority of EGF-receptors are localized, i.e. in the lateral plasma membrane both in the control cells and under CGP77675 treatment. Clathrin in A431, WT and PURO cells demonstrated even a punctuated pattern throughout the cytoplasm with some accumulation in the juxtanuclear region. This distribution depended neither on the absence or presence of Src activity nor on EGF addition. The data obtained indicate that 1) EGF-receptors do not serve as the initiation sites during clathrin coated pit assembly; 2) Src-kinase activation does not result in significant clathrin redistribution in the plasma membrane, and its influence on EGF endocytosis can be considered as a secondary effect.

Animals↗
PubMed1997

Differences between fluid-phase endocytosis (pinocytosis) and receptor-mediated endocytosis in isolated rat hepatocytes.

To characterize possible differences between the fluid-phase endocytosis (pinocytosis) of bovine serum albumin and the receptor-mediated endocytosis of asialo-orosomucoid (AOM) in isolated rat hepatocytes, both probes were conjugated to radioiodinated tyramine-cellobiose, [125I]TC. The use of these conjugates made it possible to measure the uptake and intracellular distribution of the intact proteins as well as of their acid-soluble, membrane-impermeant degradation products. [125I]TC-albumin was taken up at a very low rate (0.5%/h) compared to [125I]TC-AOM (45%/h), suggesting that neither membrane adsorption nor membrane permeation compromised its suitability as a fluid-phase marker. Sucrose gradient analysis indicated that both probes sequentially entered light endosomes (1.11 g/ml), dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml), but [125I]TC-albumin traversed the endocytic compartments more rapidly than [125I]TC-AOM, and was partially degraded intralysosomally already after 15 min. The microtubule inhibitor, vinblastine, had a stronger inhibitory effect on the uptake and degradation of [125I]TC-AOM (80% and 95%, respectively) than on the uptake and degradation of [125I]TC-albumin (50% and 70%, respectively). In the presence of vinblastine, [125I]TC-AOM was retained both in light and dense endosomes, whereas [125I]TC-albumin was retained in dense endosomes only, suggesting that the early steps of fluid-phase endocytosis were less critically dependent on microtubular function than the early steps of receptor-mediated endocytosis. A perturbant of vacuolar pH, propylamine, inhibited the degradation of both probes strongly (75-100%), as would be expected from its lysosomotropic effect. Propylamine also inhibited endocytic uptake, with a stronger effect on [125I]TC-AOM uptake (95% inhibition) than on [125I]TC-albumin uptake (60% inhibition), probably reflecting a reduction in endosomal acidity, reduced receptor-ligand dissociation and diminished recycling of free asialoglycoprotein receptors to the cell surface in addition to a general trapping of membrane in swollen vacuoles. A protein phosphatase inhibitor, okadaic acid, strongly (80-100%) inhibited the uptake and degradation of both [125I]TC-albumin and [125I]TC-AOM. An inhibitor of lysosomal proteinases, leupeptin, strongly suppressed the degradation of both probes and moderately reduced the uptake of [125I]TC-AOM, whereas the uptake of [125I]TC-albumin was unaffected. In contrast, an inhibitor of autophagic sequestration, 3-methyladenine, reduced both the uptake and degradation of [125I]TC-albumin markedly (55% and 75%, respectively), with considerably less effect on [125I]TC-AOM (25% and 35%, respectively). As autophagy-inhibitory amino acid mixture did not share these effects, suggesting that 3-methyladenine may suppress endocytic fluid-phase uptake by an autophagy-independent mechanism. Fluid-phase and receptor-mediated endocytosis in hepatocytes thus appear to differ with respect to uptake mechanisms as well as in the kinetics by which endocytosed material traverses the endocytic-lysosomal pathway.

Animals↗
PubMed1996

CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) protein has been reported to be a cAMP-regulator of plasma membrane recycling in epithelial cells overexpressing CFTR. To assess its role in the different endocytic processes in human respiratory epithelial cells, the rates of internalization of membrane, fluid-phase and receptor-mediator tracers were compared, under control conditions and after treatment with the cAMP agonist forskolin in normal and cystic fibrosis (CF) cells. In both control and treated-cells, CFTR was only present in the plasma membrane of normal but not in CF cells. Similarly, activation of Cl- efflux only occurred in normal and not in CF-treated cells. The rate of membrane endocytosis was significantly decreased by 35% in normal treated-cells, whereas it was not significantly decreased (12%) in CF-treated cells. Upon forskolin treatment, the decrease of the rate of both fluid-phase and receptor-mediated endocytosis was not significantly different between normal and CF cells. These results demonstrate that CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cell Line↗
PubMed1978

Studies on secretion and endocytosis of macromolecules by cultivated skin fibroblasts. Effects of anti-microtubular agents on secretion and endocytosis of lysosomal hydrolases and of sulphated glycosaminoglycans.

Fibroblasts were incubated in the presence of the anti-microtubular drugs colchicine, vinblastine and vincristine. In concentrations between 10nm and 1 mM these drugs stimulated the secretion of beta-N-acetylglucosaminidase, alpha-N-acetylglucosaminidase and beta-glucuronidase, but not of beta-galactosidase. The endocytosis of beta-N-acetylhexosaminidase and alpha-N-acetylglucosaminidase, but not of beta-glucuronidase, was inhibited at drug concentrations higher than 0.1 micrometer. Formation, secretion and association with the cell membrane of sulphated proteoglycans were not affected by anti-microtubular drugs. Endocytosis of sulphated proteoglycans and their subsequent degradation was inhibited by drug concentrations above 0.1 micrometer. The inhibition of intracellular glycosaminoglycan degradation led to a moderate storage of these compounds. These results suggest that microtubules participate in the control of secretion and endocytosis of lysosomal enzymes, and in the endocytosis and degradation of lysosomal substrates such as sulphated proteoglycans.

Acetylglucosaminidase↗
PubMed2005

Pseudorabies virus glycoprotein gD contains a functional endocytosis motif that acts in concert with an endocytosis motif in gB to drive internalization of antibody-antigen complexes from the surface of infected monocytes.

Viral glycoproteins gB and gD of the swine alphaherpesvirus pseudorabies virus (PRV), which is closely related to human herpes simplex virus and varicella-zoster virus, are able to drive internalization of antibody-antigen complexes that may form at the cell surface of infected monocytes, thereby protecting these cells from efficient antibody-mediated lysis. We found earlier that gB relies on an endocytosis motif in its cytoplasmic domain for its function during this internalization process. Here, we report that the PRV gD protein also contains a functional endocytosis motif (YRLL) in its cytoplasmic domain that drives spontaneous endocytosis of gD from the cell surface early in infection and that acts in concert with the endocytosis motif in gB to contribute to efficient internalization of antibody-antigen complexes in PRV-infected monocytes.

Amino Acid Motifs↗
PubMed1994

Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited.

The cytokine interleukin 2 (IL2) is produced by activated helper T lymphocytes and modulates the growth and activity of cells expressing high-affinity surface IL2 receptors that transduce its signaling. After ligand binding to receptors on the plasma membrane, receptor-ligand complexes are rapidly endocytosed and IL2 is degraded in acidic compartments. The best known receptor-mediated endocytosis pathway involves clathrin-coated pits. Receptors that carry an internalization signal recognized by adaptors on the cytosolic side of the plasma membrane are clustered into the coated pits and enter cells very efficiently. Many receptors use this pathway, but other endocytic pathways have also been reported, for ricin, EGF and insulin, for instance, which seem to be less efficient than the coated one. We compared the endocytosis of IL2 and its receptors to that of transferrin, a marker of the coated pit pathway. Under normal conditions, the kinetics of entry of IL2 was two times slower than that of transferrin. When internalization via coated pits was inhibited by two different methods, potassium depletion and cytosol acidification, endocytosis of IL2 and its receptors was only partly inhibited, while transferrin entry was strongly affected. Treatment with the cationic amphiphilic drug chlorpromazine, which induces a redistribution of a clathrin-coated pit component, AP-2, to endosomes, reduced transferrin, but not IL2 internalization. Thus, unexpectedly, this cytokine and its receptors can still be rapidly endocytosed in the absence of functional clathrin-coated structures. We propose a model for receptor-mediated endocytosis that may account for these results and published data on other receptors.

Acids↗
PubMed2006

Kinins promote B2 receptor endocytosis and delay constitutive B1 receptor endocytosis.

Upon sustained insult, kinins are released and many kinin responses, such as inflammatory pain, adapt from a B2 receptor (B2R) type in the acute phase to a B1 receptor (B1R) type in the chronic phase. In this study, we show that kinins modulate receptor endocytosis to rapidly decrease B2R and increase B1R on the cell surface. B2Rs, which require agonist for activity, are stable plasma membrane components without agonist but recruit beta-arrestin 2, internalize in a clathrin-dependent manner, and recycle rapidly upon agonist treatment. In contrast, B1Rs, which are inducible and constitutively active, constitutively internalize without agonist via a clathrin-dependent pathway, do not recruit beta-arrestin 2, bind G protein-coupled receptor sorting protein, and target lysosomes for degradation. Agonist delays B1R endocytosis, thus transiently stabilizing the receptor. Most of the receptor trafficking phenotypes are transplantable from one receptor to the other through exchange of the C-terminal receptor tails, indicating that the tails contain epitopes that are important for the binding of protein partners that participate in the endocytic and postendocytic receptor choices. It is noteworthy that the agonist delay of B1R endocytosis is not transplanted to the B2R via the B1R tail, suggesting that this property of the B1R requires another domain. These events provide a rapid kinin-dependent mechanism for 1) regulating the constitutive B1R activity and 2) shifting the balance of accessible receptors in favor of B1R.

Arrestins↗
PubMed1999

Compensated endocytosis of LDL by hamster cells co-expressing the two distinct mutant LDL receptors defective in endocytosis and ligand binding.

The low density lipoprotein receptor (LDLR) regulates the plasma cholesterol level by mediating endocytosis of LDL. We established stable hamster cell lines expressing two LDLRs with distinct functional defects, i.e., endocytosis and ligand binding. In the cell line expressing only I189D h/r (human-rat chimeric) LDLR, defective in LDL binding, very little amount of LDL was internalized, although the receptor was endocytosed efficiently. In the cell line expressing Y807C LDLR solely, very few receptors were located in coated pits or endocytosed, while LDL binding to the receptor was not disrupted. In striking contrast, in the cells co-expressing both receptors, a much larger number of Y807C LDLR were internalized and co-located with I189D h/r LDLR in the perinuclear region. In these cells, LDL was bound exclusively to Y807C LDLR and its uptake was enhanced by 80% as compared to the cell expressing Y807C LDLR solely, whereas LDL binding affinity was not changed. These results suggest that a defect of the essential motif for endocytosis, cysteine 807, could be compensated by co-expression of I189D h/r LDLR, but the LDL binding was not affected.

Animals↗