Stochastic entry of enveloped viruses: fusion versus endocytosis

Infection by membrane-enveloped viruses requires the binding of receptors on the target cell membrane to glycoproteins, or "spikes," on the viral membrane. The initial entry mechanism is usually classified as fusogenic or endocytotic. However, binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. Both fusion and internalization have been observed to be viable pathways for many viruses. We develop a stochastic model for viral entry that incorporates a competition between receptor-mediated fusion and endocytosis. The relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. We find different parameter regimes where the entry pathway probabilities can be analytically expressed. Experimental tests of our mechanistic hypotheses are proposed and discussed.     

Theoretical study on temperature dependence of cellular uptake of QDs nanoparticles

Cellular uptake kinetics of nanoparticles is one of the key issues determining the design and application of the particles. Models describing nanoparticles intrusion into the cell mostly take the endocytosis process into consideration, and the influences of electrical charges, sizes, concentrations of the particles have been investigated. In this paper, the temperature effect on the cellular uptake of Quantum Dots (QDs) is studied experimentally. QDs are incubated with the SPCA-1 human lung tumor cells, and the nanoparticles on the cell membrane and inside the cell are quantified according to the fluorescence intensities recorded. It is found that the amounts of nanoparticles attached onto the cell membrane and inside the cell both increase with temperature. Based on the experimental results, a model is proposed to describe the cellular uptake dynamic process of nanoparticles. The process consists of two steps: nanoparticles adsorption onto the cell membrane and the internalization. The dynamic parameters are obtained through curve fitting. The simulated results show that the internalization process can be categorized into different phases. The temperature dependent internalization rate constant is very small when below 14?°C. It increases distinctly when temperature rises from 14?°C to 22?°C, but there is no evident increase as temperature further increases above 22?°C. Results show that by incorporating a temperature-independent internalization factor, the model predictions well fit the experimental results.     

Receptor-mediated endocytosis of asialoglycoproteins and diferric transferrin is independent of second messengers.

The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.     

Visualization of cell surface vasopressin V1a receptors in rat hepatocytes with a fluorescent linear antagonist.

To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis, we used a high-affinity fluorescent linear antagonist, Rhm8-PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm8-PVA alone did not stimulate Ca2+ signals and competitively blocked Ca2+ signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm8-PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm8-PVA binding was confirmed by its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin at 4C. Prior vasopressin-mediated endocytosis of V1a receptors at 37C abolished binding of the labeled antagonist, whereas in non-preincubated cells, Rhm8-PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm8-PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm8-PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm8-PVA. We conclude that Rhm8-PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.     

近场光学显微镜结合量子点标记人胃腺癌SGC-7901细胞靶点的观察

扫描近场光学显微镜突破衍射极限,具有纳米量级的空间分辨率,量子点(QD s)标记有荧光强度高且抗光漂白能力强等优点。结合上述两种技术,对人胃腺癌SGC-7901细胞膜表面特异性结合的叶酸受体(FR)进行成像探测,获得了叶酸受体在SGC-7901细胞膜表面上的分布,以及细胞内化外源性叶酸过程中叶酸受体在细胞膜表面的分布变化,成像的光学分辨率达到120 nm。实验结果表明:特异性结合的叶酸受体在SGC-7901细胞膜表面的分布,绝大部分是以聚集体的形式存在。随着SGC-7901细胞内化叶酸量的增加,叶酸受体在细胞膜表面的分布密度逐渐降低,并在经过120 m in左右趋于稳定。上述方法和手段为实现单细胞水平上靶点分布和变化的长期监测,肿瘤细胞内化受体的机制研究提供了新的技术途径。     

Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4 degrees C and the temperature increased to 37 degrees C and where initial incubation was at 37 degrees C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Acute upregulation of interleukin-1 receptor by ligand.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.     

Targeting and sensing cancer cells with ZnO nanoprobes in vitro

A new class of zinc oxide quantum dots (ZnO QDs) was investigated as nanoprobes for targeting cancer cells in vitro. ZnO nanoparticles were synthesized using conventional sol–gel method and encapsulated using trimethoxy aminopropyl silane. Transferrin, the ligand targeting the cancer cells, was conjugated to the ZnO QDs. In vitro imaging studies using MDA-MB-231 showed the biocompatible ZnO nanoprobe selectively binding to the cell surface receptor and internalizing through receptor-mediated endocytosis. Time-lapsed photobleaching studies indicate the ZnO QDs to be resistant to photobleaching, making them suitable for long term imaging purpose. Investigation of the ZnO nanoprobe as a platform for sensitive bioassays indicates that it can be used as an alternative fluoroprobe for cancer cell targeting and sensing applications.     

An enzymatic method to determine receptor-mediated endocytosis

Many studies have measured receptor-mediated endocytosis using radiolabeled ligands or antibodies. Upon ligation and cross-linking, the labeled ligand or antibody is endocytosed and the internalization of the radioisotope is assayed after stripping the uninternalized ligand from the cell membrane. This study reports on an enzymatic assay to measure receptor-mediated endocytosis and compares it with the radioactive method. The results show that receptor-mediated endocytosis measured using the peroxidase conjugated antibody is two fold higher than that measured with a radiolabeled antibody. Thus, approximately 38% endocytosis of CD3 is measured using an ¹²⁵I-labeled antibody, whereas approximately 79% endocytosis is detected by peroxidase conjugated antibody method. Similar increases are also found with CD2 receptor-mediated endocytosis. Our study has demonstrated that the enzymatic method could be employed in determining receptor-mediated endocytosis. In addition to increased sensitivity, the enzymatic assay eliminates the use of radioactive materials.     

Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells

We have studied the phosphorylation state of the insulin receptor during receptor-mediated endocytosis in the well-differentiated rat hepatoma cell line Fao. Insulin induced the rapid internalization of surface-iodinated insulin receptors into a trypsin-resistant compartment, with a 3-fold increase in the internalization rate over that seen in the absence of insulin. Within 20 min of insulin stimulation, 30-35% of surface receptors were located inside the cell. This redistribution was half-maximal by 10.5 min. Similar results were obtained when the loss of surface receptors was measured by 125I-insulin binding. Tyrosyl phosphorylation of internalized insulin receptors was measured by immunoprecipitation with antiphosphotyrosine antibody. Immediately after insulin stimulation, 70-80% of internalized receptors were tyrosine phosphorylated. Internalized receptors persisted in a phosphorylated state after the dissociation of insulin but were dephosphorylated prior to their return to the plasma membrane. After 45-60 min of insulin stimulation, the tyrosine phosphorylation of the internal receptor pool decreased by 45%, whereas the phosphorylation of surface receptors was unchanged. These data suggest that insulin induces the internalization of phosphorylated insulin receptors into the cell and that the phosphorylation state of the internal receptor pool may be regulated by insulin.     

Chronic ethanol administration impairs the binding and endocytosis of asialo-orosomucoid in isolated hepatocytes

We have examined the effect of ethanol administration on receptor-mediated endocytosis of asialo-orosomucoid by isolated hepatocytes. Significantly less ligand was bound, internalized, and degraded by hepatocytes isolated from rats fed an ethanol diet for 5-7 weeks than by cells isolated from chow-fed or pair-fed controls. Reduced binding was shown to be primarily due to a decreased number of cell surface receptors rather than to a lowered affinity of the receptor for its ligand. This reduction in cell surface receptors resulted in a marked inhibition of internalization and degradation of ligand by hepatocytes from the ethanol-fed rats. In addition, a defect in the initial stages of receptor-ligand internalization was also indicated, since less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared to controls. Rates of internalization and degradation of internalized ligand were, however, similar for all three groups, suggesting that neither degradation per se nor rate of delivery of internalized ligand to the lysosomes was affected by ethanol feeding. Receptor recycling was impaired in ethanol-fed rats, as indicated by a decrease in the binding site number after stimulation of endocytosis for 120 min when compared to initial binding capacity. Receptor recycling was not impaired in hepatocytes from control animals. These results indicate that chronic ethanol feeding impairs the process of receptor-mediated endocytosis by the liver; the major cause of this impairment appears to be due to a decreased number of cell surface asialoglycoprotein receptors in the ethanol-fed animals, along with a decreased ability of these cells to internalize all of the surface-bound ligand.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors

Animal cells internalize specific extracellular macromolecules (ligands) by using specialized cell surface receptors that operate through a complex and highly regulated process known as receptor-mediated endocytosis, which involves the binding, internalization, and transfer of ligands through a series of distinct intracellular compartments. For the uptake of a variety of carbohydrate-containing macromolecules, such as glycoproteins, animal cells use specialized membrane-bound lectins as endocytic receptors that recognize different sugar residues or carbohydrate structures present on various ligands. The hepatic asialoglycoprotein receptor, which recognizes glycoconjugates containing terminal galactose or N-acetylgalactosamine residues, was the first membrane lectin discovered and has been a classical system for studying receptor-mediated endocytosis. Studies of how the asialoglycoprotein receptor functions have led to the discovery of two functionally distinct, parallel pathways of clathrin-mediated endocytosis (called the State 1 and State 2 pathways), which may also be utilized by all the other endocytic recycling receptor systems. Another endocytic membrane lectin, the hyaluronan/chondroitin sulfate receptor, which has recently been purified and cloned, is responsible for the turnover in mammals of these glycosaminoglycans, which are important components of extracellular matrices. We discuss the characteristics and physiological importance of these two proteins as examples of how lectins can function as endocytic receptors.     

Internalization and cycling of nerve growth factor in PC12 cells: interconversion of type II (fast) and type I (slow) nerve growth factor receptors

The effects of agents that inhibit receptor-mediated endocytosis on type I (slow or high-affinity) and type II (fast or low-affinity) NGF binding have been examined in rat PC12 cells. Compounds interfering with endocytosis eliminate type I NGF binding; those interfering with acidification of endosomal vesicles cause increased type I binding at the expense of type II binding. Measurement of NGF binding during and after treatment with inhibitors indicates that NGF receptors rapidly cycle from the cell surface into an undefined endocytotic compartment and back to the surface with little degradation of receptor or NGF, consistent with a model in which NGF receptors are rapidly and reversibly endocytosed or sequestered; those receptors free on the surface represent type II NGF receptors, while those in the process of endocytosis represent type I NGF receptors. The type I and type II NGF receptor species can be interconverted by agents that can manipulate the position of the receptor in the internalization cycle.     

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.     

Fluorescence studies of macrophage recognition and endocytosis of native and acetylated low-density lipoprotein

Macrophage recognition and endocytosis of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labeled low-density lipoprotein (LDL) and acetyl LDL (Ac-LDL) was studied using fluorescence flow cytometry and fluorescence video intensification microscopy. RAW264 macrophages and U937 monocytes were grown in the tissue culture media in the presence and absence of LDL and Ac-LDL. Several lines of evidence indicate that receptor-mediated endocytosis of diI-labeled LDL or Ac-LDL was taking place. Binding can be distinguished from binding plus endocytosis by incubation at 4 and 37 degrees C, respectively. Binding was saturable at 4 degrees C and uptake at 37 degrees C was time- and ligand dose-dependent. Also, unlabeled LDL and Ac-LDL compete for their receptors. Macrophages grown in the presence or absence of LDL demonstrated distinct labeling patterns. LDL receptors were significantly increased by culture in defined medium without serum lipoproteins, while Ac-LDL receptors remained unaffected. Flow cytometry can provide an important tool to examine receptor levels, modulation of these levels and receptor-mediated endocytosis. Video intensification microscopy of similarly labeled cells has been performed. Receptors appear as punctate fluorescence, usually distributed randomly across the cell surface.     

Loss of alpha 2-macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides

We have found that certain naphthalenesulfonamides [e.g., N-6(-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell-surface receptors for alpha 2-macroglobulin, and epidermal growth factor (EGF) in fibroblasts. The loss of alpha 2-macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for alpha 2-macroglobulin, and the EGF receptors do not reappear at the surface when W-7 is removed. Receptor loss was measured as a change in the capacity for binding iodinated ligands; no change in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W-7 did not induce a loss of cell surface beta 2-microglobulin, a membrane protein which is excluded from coated pits and which is not internalized, indicating that the effect of W-7 was specific for membrane receptors and not a result of bulk depletion of plasma membrane. The loss of alpha 2-macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W-7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsible for the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: 1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; 2) the microinjection of calmodulin into cells did not reverse the effects of W-7; using pressure microinjection, we introduced up to a 100-fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rhodamine-labeled alpha 2-macroglobulin, we saw that the W-7 induced inhibition of receptor-mediated endocytosis was the same in injected and uninjected cells; 3) we injected calcineurin, a calmodulin-binding protein, into cells (1-3 pg/cell) and observed no effect on the receptor-mediated endocytosis of rhodamine-labeled alpha 2-macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.     

Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides

GRP94(gp96) elicits CD8(+) T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in alpha(2)-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated alpha(2)-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.     

Human low density lipoprotein receptor expressed in Xenopus oocytes. Conserved signals for O-linked glycosylation and receptor-mediated endocytosis

The human low density lipoprotein (LDL) receptor is shown to carry out efficient receptor-mediated endocytosis in Xenopus laevis oocytes. Microinjection of mRNAs encoding the human receptor led to synthesis of a 120-kDa precursor possessing high mannose N-linked sugars and core O-linked sugars. During its transport to the cell surface, the protein increased in apparent size to 160 kDa, which is similar to the change that occurs in human cells. This increase was not seen when the receptor lacked the serine/threonine-rich region that undergoes O-linked glycosylation. The surface receptors bound 125I-LDL at 0 degrees C and internalized it with a half-time of 2 min when the cells were warmed to 19 degrees C. The rate of internalization was slowed by 7-fold when a single residue in the cytoplasmic domain (Tyr807) was changed to a cysteine, an alteration that slows incorporation into coated pits in mammalian cells. Deletion of the cytoplasmic domain abolished rapid internalization. We conclude that the signals for O-linked glycosylation and receptor-mediated endocytosis of the LDL receptor have been conserved throughout vertebrate evolution.