The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.     

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.     

Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study

125I-human growth hormone (125I-hGH) binds specifically to receptors on cultures human lymphocytes (IM-9). When this process is studied by use of quantitative EM radioautography, under conditions of incubation at 15 degrees C for 5 min, the ligand is localized to the plasma membrane of the cell. At 30 degrees and 37 degrees C, however, 125I-hGH is progressively internalized by the cell as a function of time. The internalized ligand is found predominantly in the Golgi region of the cells, with a five-fold preferential localization to membrane-bounded structures with the morphological and cytochemical characteristics of lysosomes. Up to 59% of these lysosome-like structures are positive for the acid phosphatase reaction under the conditions of incubation at 37 degrees C for 120 min. When the cell associated radioactivity after 15- 120 min of incubation at 37 degrees C is extracted in 1 M acetic acid and filtered on a Sephadex G-100 column, 58-73% of the material elutes as intact hGH. When cells are incubated with 125I-hGH at 37 degrees C for 15-120 min, separated from the incubation medium, and washed and diluted 100-fold, the percent 125I-hGH dissociable decreases as a function of increasing time of incubation. When cells are incubated with 125I-hGH for 15 min at 37 degrees C and the radioactivity that dissociates from the cells during 15-90 min is studied, the labeled material appearing in the incubation medium is progressively degraded as a function of time of incubation. When the dissociation process is studied radioautographically, grains are found both in plasma membrane and intracelluar compartments after 30 min of association, but after 30 and 120 min of dissociation a higher proportion of grains are in the intracellular compartment. After 120 min of association, there is less dissociation from either compartment and a preferential increase of grains in the intracellular compartment. These data suggest that receptor-linked internalization of a polypeptide hormone provides a mechanism that couples degradation of the ligand with loss of the cell surface receptor.     

Stage-specific assays for coated pit formation and coated vesicle budding in vitro

Internalization of biotin-S-S-125I-transferrin (125I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated coated pits were measured by inaccessibility of 125I-BSST to exogenously added antibodies. Later events involving coated vesicle budding and membrane fission were measured by resistance of 125I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated 125I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated 125I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for 125I-BSST internalization in intact cells with no activity being observed below 10 degrees C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via coated pits and coated vesicles and resulted in delivery of ligand to endosomal structures.     

Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin

It has been recently shown (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson, 1983, Cell, 33:273-285) that after a hypotonic shock followed by incubation in a K+-free medium, human fibroblasts arrest their coated pit formation and therefore arrest receptor-mediated endocytosis of low density lipoprotein. We have used this technique to study the endocytosis of transferrin, diphtheria toxin, and ricin toxin by three cell lines (Vero, Wi38/SV40, and Hep2 cells). Only Hep2 cells totally arrested internalization of [125I]transferrin, a ligand transported by coated pits and coated vesicles, after intracellular K+ depletion. Immunofluorescence studies using anti-clathrin antibodies showed that clathrin associated with the plasma membrane disappeared in Hep2 cells when the level of intracellular K+ was low. In the absence of functional coated pits, diphtheria toxin was unable to intoxicate Hep2 cells but the activity of ricin toxin was unaffected by this treatment. By measuring the rate of internalization of [125I]ricin toxin by Hep2 cells, with and without functional coated pits, we have shown that this labeled ligand was transported in both cases inside the cells. Hep2 cells with active coated pits internalized twice as much [125I]ricin toxin as Hep2 cells without coated pits. Entry of ricin toxin inside the cells was a slow process (8% of the bound toxin per 10 min at 37 degrees C) when compared to transferrin internalization (50% of the bound transferrin per 10 min at 37 degrees C). Using the indirect immunofluorescence technique on permeabilized cells, we have shown that Hep2 cells depleted in intracellular K+ accumulated ricin toxin in compartments that were predominantly localized around the cell nucleus. Our study indicates that in addition to the pathway of coated pits and coated vesicles used by diphtheria toxin and transferrin, another system of endocytosis for receptor-bound molecules takes place at the level of the cell membrane and is used by ricin toxin to enter the cytosol.     

Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.     

Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.     

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

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

Distribution of ferritin receptors and coated pits on giant HeLa cells.

HeLa cells bind horse spleen ferritin when the two are incubated at 0 degrees C. Since the majority of this bound ferritin is located in coated pits, we conclude that the ferritin binds to a specific receptor which takes part in an endocytic cycle. When substrate-attached and well-spread giant HeLa cells are briefly labelled at 0 degrees C with ferritin, ferritin particles are found to be concentrated towards the cell periphery, where they exist largely outside coated pits. This peripheral concentration is a property of circulating (and not just newly synthesized) receptors because it is not affected by prior incubation of giant cells in cycloheximide. However, coated pits are themselves roughly uniformly distributed over the surface of these cells. These results provide evidence that the membrane internalised by coated pits on these cells is returned to the cell surface at the leading edge of the cell. Because of this separation of the sites of endocytosis and exocytosis, a flow of membrane must occur across the cell surface. This flow is composed of lipid plus receptors. The implications of this for capping and for cell spreading are discussed.     

Galactose-specific recognition system of mammalian liver: receptor distribution on the hepatocyte cell surface

An isolated perfused liver system was used to study the distribution of asialoglycoprotein (ASGP) binding sites on rat hepatocyte cell surfaces. The number of surface receptors was quantitated by monitoring clearance of 125I-labeled ligands from the perfusate medium under two conditions that blocked their internalization: low temperature (less than 5 degrees C) or brief formaldehyde fixation. The cell surface distribution of binding sites was visualized in the electron microscope with either asialoorosomucoid covalently coupled to horseradish peroxidase (ASOR-HRP) or lactosaminated ferritin (Lac-Fer), both of which were bound with similar kinetics and to similar extents as ASOR itself. At low temperature or after prefixation, ASGP binding sites were present over much of the sinusoidal cell surface, but were concentrated most heavily over coated pits. Quantitation of ligand distribution at 4 degrees C with Lac-Fer gave an approximately 70-fold greater density of ferritin particles over coated membrane than over uncoated regions. We obtained no evidence for gradual movement of ASGP receptors into or out of coated pits within the time-course of our experiments. Finally, the number and distribution of cell surface binding sites was unaffected by previous exposure to ASOR or by inhibition of endocytic vesicle-lysosome fusion and ASOR degradation at 16 degrees C.     

Recycling of epidermal growth factor in A431 cells

The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.     

Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).     

Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42- kdalton component that comigrates with rabbit muscle actin and a 18.5- kdalton minor component that comigrates with calmodulin as well as 110- , 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.     

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells

Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.     

Binding and degradation of125I-insulin by isolated rat renal brush border membranes: Evidence for low affinity,high capacity insulin recognition sites

Summary The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane.¹²⁵I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time-and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of¹²⁵I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane assoicated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of¹²⁵I-insulin by BBV, but these processes were not appreciably afected by the insulin-like growth factors IGF-I and IGF-II or by cytochromec and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of¹²⁵I-insulin with BBV was studied at various medium osmolarities (300–1100 mosm) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of¹²⁵I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink¹²⁵I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an¹²⁵I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of¹²⁵I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10^–5 m. These results indicate the presence of low-affinity, high-capacity binding sites for¹²⁵I-insulin on renal brush border membranes which can clearly distinguish insulin from the insulin-like growth factors and other low molecular weight proteins and polypeptides, but which do not differentiate insulin from its analogues ad do the biological receptors for the hormone. The properties and location of these binding sites make them attractive candidates for the sites at which insulin is reabsorbed in the renal tubule.     

Initial steps in receptor-mediated endocytosis : The influence of temperature on the shape and distribution of plasma membrane clathrin-coated pits in cultured mammalian cells

We have examined the shape and distribution of clathrin-coated pits in Swiss 3T3 cells at 4 or 37 degrees C using electron microscopy with serial sections and immunofluorescence light microscopy. Both groups were fixed in glutaraldehyde and preserved further using a membrane contrast enhancement technique consisting of sequential osmium-ferrocyanide, thiocarbohydrazide and osmium-ferrocyanide treatment in situ. Concanavalin A-horseradish peroxidase (conA-HRP) was used to identify these structures participating in endocytosis. Two hundred twenty-two clathrin-coated structures were analysed; 126 from cells fixed at 4 degrees C, and 96 from cells fixed after a 3 min warm-up to 37 degrees C. All coated structures labeled with conA-HRP had demonstrable connections to the plasma membrane. These coated structures were morphologically classified into three categories: (a) flat pits; (b) curved pits; and (c) pits with narrow-neck connections to the plasma membrane. At 37 degrees C, 27% of coated pits had narrow neck connections to the plasma membrane whereas at 4 degrees C only 1% had such connections. Receptosomes (endosomes) labeled with conA-HRP were found only after incubation at 37 degrees C, indicating that active endocytosis was occurring in cells at 37 degrees C, but not at 4 degrees C. Immunofluorescence with anti-clathrin antibody was used to quantitate the number of clathrin-coated pits in Swiss 3T3 cells incubated at 4 and 37 degrees C prior to fixation. No difference was detected. There were 426 +/- 122 pits per cell at 37 degrees C and 441 +/- 106 at 4 degrees C. These results support the hypothesis that formation of a narrow neck connected a coated pit to the cell surface is an early step in the mechanism of receptor-mediated endocytosis.     

Effect of preferential insertion of LDL receptors near coated pits

Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10, plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37 degrees C.     

Biochimica et biophysica acta,vol. 646 (1981)

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for d-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, galactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Dissé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2–5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Hepatic receptor for asialo-glycoproteins. Ultrastructural demonstration of ligand-induced microaggregation of receptors

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for D-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, glactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Disé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2-5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

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

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