Receptor-mediated endocytosis and nuclear transport of human interleukin 1 alpha.

In this study we demonstrate that 125I-labelled interleukin (IL) 1 alpha binds specifically to its receptor on the surface of EL4 6.1 cells and is subsequently endocytosed and translocated from the cell membrane to the nucleus, where it progressively accumulates. Two-dimensional polyacrylamide-gel electrophoresis revealed that the internalized 125I-IL1 alpha associated with the nucleus was intact, with negligible breakdown products present. Specific and saturable binding of 125I-IL1 alpha was demonstrated on purified nuclei isolated from these cells. Binding of the radiolabelled ligand showed similar kinetics to that of the plasma-membrane receptor, and was inhibited by both unlabelled IL1 alpha and IL1 beta. Equilibrium binding studies on isolated nuclei revealed a single high-affinity binding site, with a Kd of 17 +/- 2 pM, and 79 +/- 12 binding sites per nucleus. These studies demonstrate that receptor-mediated endocytosis of IL1 results in its accumulation in the nucleus, and this mechanism may play an important role in mediating some of the actions of IL1.     

Receptor-mediated endocytosis and cytoskeleton

Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.     

Mathematical modeling of intracellular membrane transport: Receptor-mediated endocytosis and degradation of low-density lipoproteins

Receptor-mediated endocytosis of low-density lipoproteins, their transport within endosomes, and subsequent degradation in lysosomes are essential components of the molecular system for cholesterol homeostasis in vertebrate cells. The system under study is also an example of clathrin-mediated endocytosis, a possible way of cell communication with the environment. Construction of a detailed mathematical model of this system would allow comprehensive study of mechanisms and kinetics of molecular processes and evaluation of the effect of various mutations, disorders, and environmental changes on the system operation. Receptor-mediated endocytosis of low-density lipoprotein particles and their subsequent degradation in the cell have been modeled. A network of mono-and bimolecular reactions best describing the system has been proposed. The results of calculation of kinetic parameters of the molecular system obtained with the model are in agreement with experimental evidence.     

Mathematical modeling of intracellular membrane transport: Receptor-mediated endocytosis and degradation of low-density lipoproteins

Receptor-mediated endocytosis of low-density lipoproteins, their transport within endosomes, and subsequent degradation in lysosomes are essential components of the molecular system for cholesterol homeostasis in vertebrate cells. The system under study is also an example of clathrin-mediated endocytosis, a possible way of cell communication with the environment. Construction of a detailed mathematical model of this system would allow comprehensive study of mechanisms and kinetics of molecular processes and evaluation of the effect of various mutations, disorders, and environmental changes on the system operation. Receptor-mediated endocytosis of low-density lipoprotein particles and their subsequent degradation in the cell have been modeled. A network of mono-and bimolecular reactions best describing the system has been proposed. The results of calculation of kinetic parameters of the molecular system obtained with the model are in agreement with experimental evidence.

     

Receptor-mediated endocytosis: An overview of a dynamic process

The decade of the 70’s was remarkable for the insights that rapidly accumulated to provide us with an understanding of one of the fundamental processes of animal cell metabolism, namely, how mammalian cells ingest a host of extracellular substances to satisfy their various metabolic needs. It has long been appreciated that the surfaces of mammalian cells are in a continual state of flux. Surface membranes often fold inward and pinch of in a vesicular form trapping some of the contents of the extracellular material which are thus transported into the cell. This process is called endocytosis (reviewed in Silversteinet al., 1977). When extracellular fluids are taken up in this manner, the process is called fluid-phase endocytosis or pinocytosis. When solids are ingested, the process is called phagocytosis. Although quantitatively important over the long run, these modes of uptake are slow, non-specific and dependent on the concentration of the substance in the extracellular medium. In recent years it has been recognized that animal cells have developed a specialized form of this vesicular transport system to selectively retrieve and assimilate macromolecules from the extracellular milieu with high efficiency. This process is called receptor-mediated endocytosis. In this review an attempt is made to collate and correlate the evidence establishing receptor-mediated endocytosis as a dynamic process that routes cell surface receptors and ligands through multiple intracellular compartments to their ultimate destination.     

Receptor-mediated endocytosis of fucosylated neoglycoprotein by macrophages

The characteristics of the recognition system involved in the receptor mediated endocytosis of the neoglycoprotein, fucose human serum albumin (HSA) were studied. It was found that (i) fucose-HSA showed strong affinity binding and uptake by various macrophages; (ii) binding was specific for L-fucose and D-mannose; (iii) binding was found to be inhibited by oxidant like H₂O₂ and swainsonine whereas it was elevated by dexamethasone; (iv) clearance of¹²⁵I-fucose-HSA was rapid and strongly inhibited by unlabelled fucose-HSA. Greater than 70% of fucose-HSA was found in liver and more than 60% of this was found in liver lysosomes; (v) uptake of fucose-HSA was thirty-fold more efficient in liver macrophages (Kupffer cells) than in hepatocytes; (vi) moreover, mannose-HSA and ovalbumin which are potent inhibitors of mannose/N-acetylglucosamine receptors inhibited clearance and uptake of fucose-HSA by liver as well as by isolated Kupffer cells suggesting the involvement of both fucose and mannose receptors or a single type of receptor having greater affinity for fucose-HSA than for mannose-HSA. These results emphasize the important role of fucose-terminated glycoproteins in site-specific drug targeting.     

Receptor-mediated endocytosis involves tyrosine phosphorylation of cortactin

Efficient internalization of cell surface receptors requires actin polymerization mediated by Arp2/3 complex and cortactin, a prominent substrate of the protein-tyrosine kinase Src. However, the significance of cortactin tyrosine phosphorylation in endocytosis is unknown. We found that overexpression of a cortactin mutant deficient in tyrosine phosphorylation decreased transferrin uptake. Suppression of cortactin expression by RNA interference also reduced transferrin internalization. Such inhibition was effectively rescued by overexpressing wild-type cortactin but not a cortactin mutant deficient in tyrosine phosphorylation or a mutant with deletion of the Src homology 3 domain. Likewise, purified phosphorylation-null cortactin failed to restore the formation of clathrin-coated vesicles in a cortactin-depleted cell extract. In vitro analysis revealed that Src-mediated phosphorylation enhanced the association of cortactin with dynamin-2 in a tyrosine phosphorylation-dependent manner. Quantitative analysis demonstrated that Src enhances the affinity of cortactin for dynamin-2 by more than 3-fold. On the other hand, Src-treated dynamin-2 had no effect on its interaction with cortactin. These data indicate that Src kinase is implicated in clathrin-mediated endocytosis by phosphorylation of cortactin.     

Receptor-mediated endocytosis of tuftsin by macrophage cells

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N epsilon-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37 degrees C, using a concentration of 200 nM and 2 microM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.     

Receptor-mediated endocytosis of transferrin in K562 cells

Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.     

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.     

Receptor-mediated endocytosis of fibroblast β-glucuronidase by peritoneal macrophages

β-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of β-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Receptor-mediated endocytosis of aldehyde-modified proteins by sinusoidal liver cells

Formaldehyde-treated serum albumin (f-Alb) is known to be endocytosed by sinusoidal lever cells via a receptor-mediated mechanism. The receptor purified from rat livers exhibited a molecular weight of 125,000, consisting of two glycoprotein components with molecular weights of 53,000 and 30,000, respectively. Experiments using antireceptor antibody demonstrated that the f-Alb receptor is distinct from the receptor that mediates endocytotic uptake of acetylated low-density lipoprotein, but they share a common property of being inhibited by several polyanions, suggesting that polyanion-sensitivity might play an important role in the scavenger function of simusoidal liver cells. Studies on the ligand specificity of this receptor revealed that a covalent modification by formaldehyde of a limited number of lysine residues in albumin has led to the formation of a receptor-recognition domain(s). Furthermore, in addition to formaldehyde, the ligand activity was also generated with albumin modified by other aliphatic aldehydes, such as glycoaldehyde and glyceraldehyde. This phenomenon was extended to several proteins other than albumin. These data suggest therefore that the f-Alb receptor originally described as being specific for albumin modified by formaldehyde may play a general role as a scavenger receptor for aldehyde-modified proteins.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.     

Receptor-mediated endocytosis in the procyclic form of Trypanosoma brucei

In Trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, a deep invagination of the plasma membrane where the flagellum extends from the cell. Both bloodstream and procyclic trypanosomes are capable of internalizing macromolecules. However, structures resembling coated vesicles were only identified in bloodstream form and not in procyclic form trypanosomes. Due to the apparent absence of coated vesicles in procyclics, the significance of receptor-mediated endocytosis in procyclic trypanosomes has been considered of minimal importance. We show that the flagellar pocket associated cysteine-rich acidic transmembrane protein (CRAM) may function as an high density lipoprotein receptor in the procyclic form trypanosome. Using anti-CRAM IgG we have characterized the process of CRAM-mediated endocytosis in procyclic form trypanosomes. The wild type procyclic trypanosome binds and internalizes anti-CRAM IgG but not the non-immune IgG in a saturable and time-dependent manner; the binding and uptake of (125)I-labeled anti-CRAM IgG are inhibited by excess unlabeled anti-CRAM IgG. Uptake and degradation of anti-CRAM IgG do not occur at 4 degrees C. At 28 degrees C, the internalized anti-CRAM IgG were efficiently degraded through a process that is inhibited by incubation at 4 degrees C and sensitive to the presence of chloroquine. The uptake and degradation of anti-CRAM IgG does not occur in the CRAM null mutant cell line. These results suggested that the uptake of anti-CRAM IgG in the wild type procyclics occurs via receptor-mediated endocytosis of the CRAM protein. Deletion of the cytoplasmic extension of CRAM drastically reduced the degradation but not the binding of anti-CRAM IgG. This result indicated that potential internalization signals may be present in the cytoplasmic extension of CRAM. This is the first time that the importance of receptor-mediated endocytosis in procyclic form trypanosomes has been demonstrated.     

Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei

The uptake of various host plasma proteins by the bloodstream form of Trypanosoma brucei was studied both biochemically, using radiolabeled proteins, and with the electron microscope, using colloidal gold particles as molecular tracers onto which plasma proteins had been adsorbed. Total plasma proteins and serum albumin were taken up by a mechanism of fluid endocytosis with low clearance (0.1 microliter [mg cell protein]-1 h-1), while low-density lipoprotein (LDL) and transferrin were taken up by a receptor-mediated process with a clearance of two to three orders of magnitude higher than that of serum albumin. Binding prior to uptake of LDL and transferrin was saturable, depended on the presence of Ca2+, and the labeled ligand could be displaced by the homologous but not by heterologous protein. Binding of gold-labeled proteins was seen only to the membrane of the flagellar pocket and not elsewhere on the plasma membrane. After 1 h of incubation at 30 degrees C with gold-labeled LDL and transferrin, labeled cellular structures represented respectively half and one-third of the total volume of all single-membrane bounded endocytotic and electron-dense vacuoles within the cell.     

Receptor-mediated endocytosis of insulin by cultured endothelial cells.

Label-fracture immunochemistry and pre-embedding indirect immunocytochemistry were applied to investigate insulin uptake by endothelial cells. Freeze fracture replicas showed that a small percentage of native insulin receptors are associated with non-coated pits (4%) and coated pits (2%). After warming, receptor bound insulin became increasingly associated with such endocytotic vesicles. After 2 min the percentage of detectable insulin associated with non-coated and coated pits increased to 16% and 8%, respectively. Pre-embedding immunocytochemical localization of insulin gave results consistent with those obtained from the label-fracture studies. Both non-coated and coated vesicles appeared labelled after 5 min of warming. Non-coated vesicles contained 25% of the cell associated insulin while 9% was associated with coated pits and vesicles. After 10 min of warming, 9% of label was located in non-coated vesicles and 7% in coated vesicles. A large proportion (29%) of the label was found in tubular-vesicular endosomes at this time. After 15 min of warming, 30% of the remaining cell-associated gold label was found in multivesicular bodies. These experiments demonstrate that insulin uptake by endothelium is mediated by both coated and non-coated vesicles and that, once internalized, insulin is routed through endosomal pathways that primarily result in transcytosis.     

Receptor-mediated endocytosis and exocytosis of transferrin in Concanavalin A-stimulated rat lymphoblasts

Iron-loaded transferrin has been shown to be necessary for the support of cell proliferation in culture. This function depends upon interaction of transferrin with a specific high-affinity cell surface receptor. The present report is directed toward determining the consequences of the interaction of transferrin with this receptor on Concanavalin A-stimulated rat lymphocytes. Three specific questions have been posed: (a) Is transferrin endocytosed following binding to its specific receptor in a temperature-dependent fashion? (b) Following endocytosis, is the carrier protein released from the cell in a structurally and functionally intact form? and (c) Is the cell surface transferrin receptor also endocytosed following ligand binding? The results provide affirmative answers to all questions. Using two independent probes of the cell surface versus intracellular location of transferrin we observed that cell-bound transferrin moved from the cell surface to the inside of the cell and subsequently back to the medium. This process occurred in a temperature-dependent fashion. When cells containing only intracellular transferrin were further incubated at 37°C approximately 80% of cell-bound transferrin was released to the medium. Nearly all of this material retained reactivity with antibody to transferrin. In addition, exocytosed transferrin exhibited qualitatively and quantitatively equivalent binding reactivity with the transferrin receptor and showed identical electophoretic mobility on SDS gel electrophoresis. Finally, using similar methodology to that employed with transferrin itself, we provide evidence that the specific receptor is also endocytosed.     

Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor

A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.