Endocytosis of antigen, anti-idiotype, and anti-immunoglobulin antibodies and receptor re-expression by murine B cells

The endocytosis of Ag mediated by membrane-associated Ig (mIg) molecules has been spectrophotometrically monitored using a cell line (2C3) specific for the hapten phthalate (Xmp) and employing conjugates of Xmp and horseradish peroxidase (HRP) as the labeled ligand. Approximately 50% of both Xmp-HRP, or the larger ligand, Xmp-keyhole limpet hemocyanin-HRP, are internalized rapidly, reaching an initial plateau by 30 min. The rate of endocytosis of anti-idiotype-HRP is similar to the rates that were observed for the hapten-bearing ligands, while a slower rate of endocytosis of anti-Ig-HRP was observed. The percent of ligand bound that is internalized and the rate of endocytosis appear to be largely independent of the size and amount of ligand bound per cell. However, mIg-mediated endocytosis is markedly reduced when mIg-ligand complexes are more extensively cross-linked by the binding of a second antibody. In addition to the initial rapid phase of endocytosis, there is a prolonged phase during which more of the bound ligand is internalized, and up to 90% of the internalized ligand is degraded. Re-expression of Ag-binding receptors by the 2C3 cells is independent of new protein synthesis and is accomplished in part by the translocation of a presynthesized pool of mIg molecules from the cytoplasm to the plasma membrane of the cell. The kinetics of endocytosis of HRP-labeled anti-Ig antibodies by BALB/c splenic B-lymphocytes and other B-lymphocyte cell lines is very similar to the endocytosis of Ag and anti-idiotype observed with the 2C3 cell line. Light and electron microscopy are also performed to visually confirm that the HRP-labeled ligands are being internalized and to determine the percentage of cells involved in this process. Finally it was determined that the transmembrane and cytoplasmic domains of the mIg molecules are required for endocytosis since the secreted form of the molecule (which lacks these domains) fails to mediate the internalization of bound ligand.     

The effects of chronic ethanol administration on the rates of internalization of various ligands during hepatic endocytosis.

The purpose of the present study was to further characterize the ethanol-induced impairments in hepatic endocytosis. Specifically, we examined the effects of ethanol treatment on receptor-ligand internalization via the coated and noncoated pit pathways. Insulin, epidermal growth factor (EGF) and asialoorosomucoid (ASOR) were used as model ligands to study internalization by isolated hepatocytes. ASOR and EGF are thought to be internalized strictly in coated pit regions of the cell membrane, while insulin may be internalized in both coated and uncoated membrane regions. Ethanol administration for 5-7 weeks decreased internalization of ASOR and EGF while internalization of insulin was unchanged during a single round of endocytosis of surface-bound ligand. Similarly, a more quantitative measure of endocytosis, the endocytic rate constant, was decreased for EGF and ASOR but not for insulin in livers of experimental rats. When endocytosis of Lucifer yellow, a fluorescent dye known to be internalized in the cell by fluid-phase endocytosis was examined, the initial rates of dye uptake were not significantly altered by alcohol administration. These results indicate that ethanol may selectively impair internalization occurring by coated pits while it has a minimal effect on initial uptake of molecules which are internalized by noncoated membrane regions.     

Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.     

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.     

Endocytosis and recycling of subunit H1 of the asialoglycoprotein receptor is independent of oligomerization with H2.

The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expressing the two subunits in transfected fibroblast cell lines, it has been shown previously that the formation of a hetero-oligomeric complex is necessary for the transport of H2 to the plasma membrane and for high-affinity ligand binding. Here we show that subunit H1, when expressed in the absence of H2, is capable of internalization through coated pits and recycling. The kinetics of these processes are very similar to those of the H1-H2 complex. To study endocytosis in the absence of ligand binding, the cell surface was labeled at 4 degrees C with the 125I-iodinated impermeant reagent sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, the cells were incubated at 37 degrees C for different times and the amount of internalized receptor was determined by protease digestion of surface proteins and immunoprecipitation. Similarly, recycling of surface-labeled and then internalized receptor protein was studied by monitoring its reappearance on the surface in the presence of exogenous protease. Our results show that subunit H1 contains all the signals necessary for receptor endocytosis and recycling independent of ligand binding.     

Either part of a Drosophila epsin protein,divided after the ENTH domain,functions in endocytosis of delta in the developing eye

Epsin is part of a protein complex that performs endocytosis in eukaryotes. Drosophila epsin, Liquid facets (Lqf), was identified because it is essential for patterning the eye and other imaginal disc derivatives [2]. Previous work has provided only indirect evidence that Lqf is required for endocytosis in Drosophila [2, 3]. Epsins are modular and have an N-terminal ENTH (epsin N-terminal homology) domain that binds PIP(2) at the cell membrane and four different classes of protein-protein interaction motifs. The current model for epsin function in higher eukaryotes is that epsin bridges the cell membrane, a transmembrane protein to be internalized, and the core endocytic complex. Here, we show directly that Drosophila epsin (Lqf) is required for endocytosis. Specifically, we find that Lqf is essential for internalization of the Delta (Dl) transmembrane ligand in the developing eye. Using this endocytic defect in lqf mutants, we develop a transgene rescue assay and perform a structure/function analysis of Lqf. We find that when we divide Lqf into two pieces, an ENTH domain and an ENTH-less protein, each part retains significant ability to function in Dl internalization and eye patterning. These results challenge the model for epsin function that requires an intact protein.     

Rate transition and regulatory coupling in endocytosis of interferon-alpha and tumor necrosis factor-alpha in human epithelial tumor cells.

The time-dependent concentrations of interferon-alpha and tumor necrosis factor-alpha associated with the membrane and internalized by cells contain information on the kinetics of endocytosis and their cellular processing. This information can be reduced quantitatively by application of the respective compartmental models. In our studies of human epithelial tumor cells interacting with human interferon-alpha and human tumor necrosis factor-alpha, we accounted only for actual endocytosis and elimination of the tracer from cells by a novel method sensitive to changes in the rate of endocytosis, to the delay in tracer elimination, and to the nonlinear regulatory coupling between endocytosis and the internalized ligand. Data reduced by this method resulted in best-fit parameter values statistically superior to values obtained by previous methods (Bajzer et al., 1989). The results indicate a change with time in the rate of endocytosis of tumor necrosis factor-alpha and the inhibition of endocytosis by the endocytosed ligand-receptor complex. We conclude that sorting and processing of interferon-alpha and tumor necrosis factor-alpha are restricted by the type of both the receptor and the cell.     

Binding, internalization, and intracellular processing of proteins interacting with recycling receptors. A kinetic analysis

We measured time-dependent concentration changes of human interferon-alpha 2a (IFN) and human tumor necrosis factor-alpha (TNF) bound at the plasma membrane and internalized by human lung alveolar carcinoma A549 cells in the presence of excess free ligand. Concentration changes for these two ligands were substantially different. We modified our compartmental kinetic model encompassing receptor synthesis and receptor loss (Myers, A. C., Kovach, J. S., and Vuk-Pavlovi?, S. (1987) J. Biol. Chem. 262, 6494-6499) to include receptor recycling. We solved analytically the equations of three variants of the model of receptor recycling. All parameters (rate constants) were identifiable when the data sets consisted of time-resolved concentrations of IFN and TNF at the cell surface and internalized by cells. By least squares fitting we derived the best fit values for the first order rate constants for internalization of the ligand-receptor complex, receptor recycling, turnover of free receptors, elimination of the ligand from cells, and the rate of insertion of free receptors into the membrane. The best fit to data for interactions of cells with IFN was obtained without inclusion of the term for recycling of receptors to the membrane. The simplest model including receptor recycling was necessary and sufficient for the fit to the respective data for TNF. These results demonstrate that the contribution of receptor recycling to the metabolism of the ligand and the receptor can be quantitated by compartmental modeling. Receptor recycling does not contribute to the kinetics of Type I IFN receptor in A549 cells. In contrast, recycling contributes significantly to endocytosis mediated by the TNF receptor.     

Perturbational effects of inorganic cations on human erythrocyte membranes.

The perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy in incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni++, Co++ and Mn++ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.     

Divalent cation-induced surface tension increase in acidic phospholipid membranes: Ion binding and membrane fusion

Chelation binding of divalent cations to phospholipid membranes may cause deformation in the headgroup regions of these lipid molecules. This deformation may be responsible for the observed large increase in surface tension of acidic phospholipid membranes induced by divalent cations. On the other hand, simple binding of monovalent cations without being followed by such a deformation of membrane molecules, does not result in a large surface tension increase in the membrane. A theoretical explanation for the above situation is given and the divalent cation-induced acidic phospholipid membrane fusion as well as other lipid membrane fusions are discussed in terms of the increased surface energy of membranes.     

Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells.

The internalization of CD4, a T cell differentiation antigen and the receptor for the human immunodeficiency viruses (HIV-1 and -2), has been examined in HeLa and murine 3T3 cells transfected with CD4 cDNA. Fab' fragments of the anti-CD4 monoclonal antibody Leu3a were generated by pepsin digestion and used as a specific monovalent, non-crosslinking ligand for CD4. These Fab' fragments were shown to bind to CD4 on the transfected cells with an affinity similar to that of HIV gp120, and inhibited HIV infection of lymphocytic cells. The Fab' fragments were radioiodinated and used in an acid-stripping endocytosis assay to demonstrate that the CD4 expressed on transfected HeLa and NIH3T3 cells was internalized. Approximately 1.5-2% of the total cell-bound [125I]Fab' fragments were internalized per minute. Furthermore, the internalized [125I]Fab' fragments could be shown to recycle to the cell surface. After 30-60 min a steady state was reached between internalization and recycling, with approximately 30-40% of the total cellular CD4 pool residing inside the cell. Similar results were obtained in studies with the intact divalent radiolabelled Leu3a antibody. These data demonstrate that CD4 expressed on transfected non-lymphoid cells is constitutively endocytosed and recycled.     

Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.     

Perturbational effects of inorganic cations on human erythrocyte membranes

Summary The perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy of incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni⁺⁺, Co⁺⁺ and Mn⁺⁺ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.     

Flow and shuttle of plasma membrane during endocytosis

A striking feature of endocytosis is the large amount of surface membrane that is brought into the cells through the formation of endocytic vesicles. Little is known about the fate of this membrane material. It is implausible that it would be destroyed in lysosomes, as the rate of turnover of the constituents of plasma membrane is much too low with respect to the rate of endocytosis in all cells studied so far. Conversely, plasma membrane fragments, internalized by endocytosis cannot merely be incorporated in lysosomes, as these organelles have been shown to maintain their size, despite continuous and active endocytosis. We present evidence that plasma membrane antigens, detected by means of specific antibodies, are internalized during endocytosis and reach lysosomes. They are thereafter returned back to cell surface. These results indicate the existence of a shuttle of membrane elements between the cell surface and lysosomes.     

The crossroads of receptor-mediated signaling and endocytosis in plants

Plants deploy numerous plasma membrane receptors to sense and rapidly react to environmental changes. Correct localization and adequate protein levels of the cell-surface receptors are critical for signaling activation and modulation of plant development and defense against pathogens. After ligand binding, receptors are internalized for degradation and signaling attenuation. However, one emerging notion is that the ligand-induced endocytosis of receptor complexes is important for the signal duration, ampli tude, and specificity. Recently, mutants of major endocytosis players, including clathrin and dynamin have been shown to display defects in activation of a subset of signal transduction pathways, implying that signaling in plants might not be solely restricted to the plasma membrane. Here, we summarize the up-to-date knowledge of receptor complex endocytosis and its effect on the signaling outcome, in the context of plant development and immunity.     

A Highlights from MBoC Selection: Neuralized Promotes Basal to Apical Transcytosis of Delta in Epithelial Cells

Notch receptors mediate short-range signaling controlling many developmental decisions in metazoans. Activation of Notch requires the ubiquitin-dependent endocytosis of its ligand Delta. How ligand endocytosis in signal-sending cells regulates receptor activation in juxtaposed signal-receiving cells remains largely unknown. We show here that a pool of Delta localizes at the basolateral membrane of signal-sending sensory organ precursor cells in the dorsal thorax neuroepithelium of Drosophila and that Delta is endocytosed in a Neuralized-dependent manner from this basolateral membrane. This basolateral pool of Delta is segregated from Notch that accumulates apically. Using a compartimentalized antibody uptake assay, we show that murine Delta-like 1 is similarly internalized by mNeuralized2 from the basolateral membrane of polarized Madin-Darby canine kidney cells and that internalized ligands are transcytosed to the apical plasma membrane where mNotch1 accumulates. Thus, endocytosis of Delta by Neuralized relocalizes Delta from the basolateral to the apical membrane domain. We speculate that this Neuralized-dependent transcytosis regulates the signaling activity of Delta by relocalizing Delta from a membrane domain where it cannot interact with Notch to another membrane domain where it can bind and activate Notch.     

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLa^K44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

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.     

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.     

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.