Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.     

Reversible pinocytosis of horseradish peroxidase in lymphoid cells

A detailed study of fluid phase endocytosis of horseradish peroxidase (HRP) in rat lymph node cells (LNC) is presented in this paper. Preliminary experiments have shown that HRP was internalized by non-receptor-mediated endocytosis and interacted minimally or not at all with plasma membrane of LNC, and can then be considered as a true fluid phase marker for these cells. Kinetics of uptake of HRP was found not to be linear with incubation time at 37 degrees C and deviation from linearity can be attributed to constant exocytosis of HRP. The kinetics of exocytosis cannot be described by a single exponential process. Rather, a minimum of two exponentials is required to account for exocytosis. This suggests that at least two intracellular compartments are involved in this process. The first turns over very rapidly with a t 1/2 release of about 3 min and is saturated after 10 min of exposure with HRP. The second, which turns over very slowly, is characterized by a t 1/2 release of about 500 min and accounts for the intracellular accumulation of HRP. Similar biphasic kinetics of exocytosis were observed with unfractionated LNC, with T lymphocyte-enriched LNC and with lymphocytes purified according to their density. This suggests that most, if not all, LNC are able to release HRP and that each cell type is endowed with the two intracellular compartments. Kinetics of uptake of HRP in these two compartments indicated that they are probably filled by two endocytic pathways, at least partially independent. Taken together, these results seem to indicate that a rapid membrane recycling occurs in lymphocytes. Furthermore, the weak base ammonium chloride and the carboxylic ionophore monensin were shown in our study to inhibit fluid phase endocytosis of HRP. The inhibition was time-dependent and required a preincubation of the cells with the drugs to be observed. Our results suggest that a perturbation of the vesicular traffic or a sequestration of membranes involved in HRP uptake is induced by these drugs. Under these conditions the release of cell-associated HRP was also reduced and to the same extent as the inhibition of uptake. Distribution of HRP between the two compartments and the t 1/2 release of HRP from either compartment were not perturbed. Taken together these results seem to indicate that exocytosis is not specifically affected by these drugs. Inhibition of uptake in drug-treated cells could result from a general decrease of membrane recycling or to the formation of smaller pinocytic vesicles with a different surface to volume ratio.     

Surface immunoglobulin on rabbit lymphoid cells. II. Ultrastructural distribution of b4 allotypic determinants and morphology of spleen lymphocytes

Surface immunoglobulin allotypic determinants on rabbit spleen lymphoid cells are ultrastructurally localized by labeling with antiallotype antisera and soluble complexes of ferritin and rabbit antiferritin of a given allotype. At 0 °C surface Ig is visualized in patches on the membrane of 54% of the spleen lymphocytes examined. Four morphologically distinct categories of spleen lymphocytes display different amounts of labeled surface Ig. Type I cells are essentially identical to peripheral blood lymphocytes and demonstrate rapid endocytosis of surface Ig at 37 °C. Type II cells greater amounts of surface Ig, demonstrate little endocytosis, and are consistent with lymphoblast cells. Type III cells have the greatest amount of surface Ig, reveal some endocytosis, and are morphologically consistent with proplasmacytes and plasmablast cells. Type IV cells are immature plasma cells and have very little detectable surface Ig. The percentage of each cell type making up the labeled population is Type I, 28%; Type II, 21%; Type III, 50%, and Type IV, 1%. Immunoferritin labeled Ig determinants may be modulated from the surface of these cells at 37 °C by endocytosis and/or by shedding after reaction with anti-Ig antisera.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.     

Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.     

Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

The cellular uptake of horseradish peroxidase and its poly(lysine) conjugate by cultured fibroblasts is qualitatively similar despite a 900-fold difference in rate

When horseradish peroxidase (HRP) is conjugated to poly(L-lysine) of molecular weight (MW) 13,000, its transport into cultured L929 fibroblasts in 1 hour at 37 degrees C is increased 918-fold. The kinetics of uptake are linear with time and concentration, reflecting a process of nonreceptor-mediated adsorptive endocytosis. Neither HRP-poly(Lys) conjugate nor free poly(Lys) of any size cause any increase in fluid phase endocytosis. All evidence indicates that the covalently bound poly(Lys) increases the binding of HRP to the cell surface and that this adequately accounts for an increased internalization occurring at a steady rate. In the absence of Ca++, both surface binding and uptake of the conjugate, but not of free HRP, are decreased. Cytochalasin B does not significantly inhibit the transport of either form of HRP. The half-lives of HRP and HRP-poly(Lys) are 7.6 and 5.6 hours, respectively, when measured over a period of 12 hours. Electron microscopic analysis of cells that have ingested comparable amounts of HRP shows that the intracellular localization of free and conjugated HRP are comparable and that both are found inside the same array of structures. Thus HRP, which binds very poorly to the cell surface and is considered a fluid-phase marker for the "contents" of pinocytotic vesicles, and HRP-poly(Lys), which binds very strongly to the cell surface and is considered a membrane marker for adsorptive endocytosis, are taken up along the same endocytotic pathway. Moreover, despite the fact that neither markers are transported by receptor-mediated endocytosis, both are seen in structures that were described as receptosomes. It appears, therefore, that the pathways utilized in receptor-mediated transport are available to all other forms of pinocytosis.     

Internalization of lectins in neuronal GERL

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin- labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.     

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Potential mechanism of the stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipose cell. Apparent redistribution of receptors cycling between a large intracellular pool and the plasma membrane

Previous studies have proposed that insulin increases the binding of insulin-like growth factor II (IGF-II) in isolated rat adipose cells at 24 degrees C by increasing receptor affinity (Ka). This study re-examines these observations under conditions in which receptor-ligand internalization is blocked by 1 mM KCN. In the absence of KCN, adipose cells bind 0.71 amol of IGF-II/cell with low apparent affinity (0.030 nM-1), of which greater than 75% is not accessible to trypsin. In contrast, in the presence of KCN, IGF-II binding is decreased by 95% and its apparent affinity increased to 0.21 nM-1. Moreover, greater than 60% of the bound IGF-II now is sensitive to trypsin. In either the absence or presence of KCN, approximately 20% of the cell's total IGF-II receptors are present in the plasma membranes and approximately 80% in the low density microsomes. Insulin induces a 5-fold increase in cell surface IGF-II receptors without a change in affinity when IGF-II binding is measured in the presence of KCN. Similarly, insulin increases IGF-II receptor concentration in the plasma membranes and concomitantly decreases that in the low density microsomes. Receptor affinity in these two subcellular membrane fractions is not affected by incubation of intact cells with either insulin or KCN and is similar to that observed in intact cells in the presence of KCN. Addition of KCN prior to insulin abolishes all of these effects of insulin. These data suggest that (a) the effects of KCN reflect a selective blockade of endocytosis; (b) in the absence of KCN, IGF-II binds to receptors of constant affinity that cycle between the plasma membrane and an intracellular pool resulting in an accumulation of intracellular IGF-II; (c) insulin induces an increase in IGF-II binding by causing a steady state redistribution of receptors from this intracellular pool to the plasma membrane; and (d) this redistribution in the intact cell can only be detected using Scatchard analysis when recycling of the receptors is prevented by KCN.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.     

Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5’- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.     

Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling

Receptor-mediated endocytosis of rat preputial beta-glucuronidase and the glycoconjugate mannose-BSA by rat alveolar macrophages is inhibited by chloroquine and ammonium chloride. We have previously reported that these drugs cause a loss of cell surface binding activity and that they do not inhibit internalization of receptor ligand complexes when incubated with cells at 37 degrees C. In this report we more clearly delineate the intracellular site of weak base inhibition of receptor recycling and the mechanism of that inhibition. From our analysis of the kinetics of ligand transport we conclude that there are two functionally distinct intracellular pools of receptor. One of these, the cycling pool, is not sensitive to the presence of weak bases, and receptor-ligand complexes return from this pool to the cell surface intact. The second pool is responsible for the time-dependent intracellular delivery of ligand to acid vesicles, which is inhibited by weak bases. Chloroquine and ammonium chloride appear to inhibit the dissociation of receptor-ligand complexed in this second pool and thereby the production of free receptors for the continuation of receptor-mediated endocytosis. We examine the internalization and binding of ligand in normal and paraformaldehyde-treated cells and find that these are strongly affected by pH. In particular, the dissociation rate of receptor ligand complexes is enhanced greater than 7.5 fold by lowering the medium pH from 7 to 6. From these results we propose that weak bases raise the pH of acid intracellular compartments, slowing the rate of receptor-ligand dissociation and thereby reducing the cellular pool of free receptors available for further uptake of ligand. In addition, we demonstrate that receptor-ligand complexes cannot return to the cell surface from the amine-sensitive (acid) intracellular pool that led us to call this the nonreleasable pool. This final observation indicates that receptor movements through these two pools are functionally distinct processes.     

Surface immunoglobulin on rabbit lymphoid cells. I. Ultrastructural distribution and endocytosis of b4 allotypic determinants on peripheral blood lymphocytes

Immunoglobulin (Ig) b4 allotypic determinants are detected on the surface membrane of rabbit peripheral blood lymphocytes by an indirect immunoferritin labeling technique. Cells coated with antiallotype antibodies are labelled with soluble complexes of ferritin and rabbit antiferritin of a given allotype. At 0 °C a patchy distribution of labeled surface immunoglobulin is visualized on 80% of the lymphocytes examined. Warming of the cells for 1–5 min at 37 °C causes rapid endocytosis of surface label in a perinuclear fashion. Cap formation is not observed. Cross-linking of immunoferritin labelled surface determinants with sheep anti-rabbit Ig (SARG) inhibits endocytosis and promotes aggregation of small surface patches. Indirect evidence suggests that sloughing and/or stripping of labelled surface Ig can occur after this aggregation. These surface changes may be the first step in the induction of lymphocyte activation.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.