Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells (original) (raw)
Related papers
Journal of Cell Science, 1985
Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell h e . There are about 1.6 x lo6 binding sites per cell surface, exhibiting a KO of about lo-' M. Upon warming cells to 37 O C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 "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-l. In the absence of externally added ligand only 60% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-'. 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 (~5 0 % ) occupancy this loss is monotonic with the extent of internalization.
Endothelial binding of transferrin in fractionated liver cell suspensions
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1985
Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that bepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125Ior SgFe-labeled transferrin led to exclusive binding to endothelial cells but not bepatocytes nor Kupffer cells. Kinetic analysis demonstrated K d of 1.9" 10-7 M, B,,ax of 3.1 pmol/106 cells with receptor numbers estimated as 1.8. 106/cell. The rate of binding was at least 28 ng (0.35 pmoi) protein/106 cells per min, corresponding to 2.1 • 105 molecules/cell per min. At 4°C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.
The Journal of Cell Biology, 1987
After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'-diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both liga...
Receptor-mediated endocytosis of human transferrin and its cell surface receptor
Journal of Cellular Physiology, 1985
We have studied the process of transferrin endocytosis in human erythromyeloid cell line K562 using fluorescein (FL) and rhodamine (RD) labeled ironsaturated transferrin (FeTF), and a fluorescein labeled monoclonal antibody to the transferrin receptor (FL-mAB). Because the antireceptor antibody and FeTF bind to different sites on the TF receptor molecule, it is possible to simuitaneously and independently follow receptor and ligand. We have measured the relative amounts of transferrin or antireceptor antibody bound in the presence or absence of proteolytic enzymes using a cell sorter. At 4OC almost all of the FL-TF and the FL-mAB is surface bound in a diffuse pattern. Within minutes of elevating the temperature to 37OC surface aggregates form and the FL-TF is internalized. At this time about one sixth of the transferrin is still surface bound and accessible to papain digestion. The remainder localizes in a perinuclear cluster of vesicles. Monoclonal antibody binds to the cell surface transferrin receptor but is not internalized at 4OC or 37OC. When unlabeled diferric transferrin is added, it promotes the uptake of the FI-mAB. The addition of goat anti-mouse immunoglobulin also promotes FL-mAB uptake. These studies support the concept that a specific trigger is required for transferrin receptor endocytosis.
Synthesis and Secretion of Transferrin by Cultured Mouse Hepatoma Cells
Differentiation, 1978
The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transfemns. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [3Hlleucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transfemn synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.
Identification of transferrin receptors on the surface of human cultured cells
Proceedings of the National Academy of Sciences, 1979
We have examined the binding of human transferrin to cultured human choriocarcinoma cell lines and to detergent extracts of such cells. The results indicate the presence of a high-affinity saturable binding site (Ka = 4.25 x 10(8) M-1) that is specific for transferrin. This receptor has also been detected on three other human cell lines of different phenotypic origin, including Wil-2 (splenic lymphocytes of B-cell origin), RPMI-2650 (a quasi-diploid nasopharyngeal carcinoma), and WI-38 (embryonic lung fibroblasts). By using anti-human transferrin antiserum to immunoprecipitate the receptor-transferrin complex from detergent extracts of cells containing saturating levels of transferrin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single polypeptide of 90,000 daltons has been identified as a subunit of the putative transferrin receptor. The protein shows immunochemical identity and coelectrophoreses in sodium dodecyl sulfate gels with a cell surface glycopr...
The Journal of cell biology, 1983
The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid comp...
Journal of Biological Chemistry, 1997
Cell-surface receptors that undergo clathrin-mediated endocytosis contain short amino acid sequences in their cytoplasmic domain that serve as internalization signals. Interactions between these sequences and components of the endocytic machinery should become limiting upon overexpression of the constitutively recycling transferrin receptor (TfR). A tetracycline-responsive system was used to induce overexpression of the TfR up to 20-fold in HeLa cells. Internalization assays indicate the rate of 125 I-transferrin uptake per surface TfR is reduced by a factor of 4 in induced cells. Consistent with endocytosis being the rate-limiting step, TfRs shift from an endosomal to more of a plasma membrane distribution with TfR overexpression. The clathrin-associated protein AP-2 has been proposed to interact directly with the cytoplasmic domain of many receptors, yet no changes in the amount or distribution of AP-2 were detected in induced cells. The internalization rate for the epidermal growth factor receptor was also measured, with or without induction of TfR expression. Even though endocytosis of the TfR is saturated in induced cells, 125 I-labeled epidermal growth factor continues to be internalized at a rate identical to that seen in uninduced cells. We propose that there are different limiting steps for the endocytosis of these two receptors.
Intracellular interactions of transferrin and its receptor during biosynthesis
The Journal of Cell Biology, 1990
The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfidelinked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and