Jos van Renswoude

The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number (approximately equal to 150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.

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.

At physiological temperature, the Fe-carrier transferrin is taken up by K562 human erythroleukemia cells through receptor-mediated endocytosis. Both ligand (now minus Fe) and receptor recycle back to the cell surface where the receptor is rapidly reutilized. After endocytosis, transferrin becomes transiently lodged within an acidic compartment inside the cell, as judged by the changed spectral characteristics and quantum yield of fluorescein isothiocyanate-labeled transferrin that is cell-associated at 37 degrees C. Upon binding to transferrin, anti-fluorescein antibody strongly quenches the emission of the fluorescein-labeled residues on the protein and is used to assess whether the transferrin is at the cell surface (incubation at 0 degrees C) or mainly internalized into the cell (incubation at 37 degrees C). Using Percoll gradient fractionation of postnuclear supernatants, we show that the acidic compartment is not the lysosomal compartment.

Treatment of human K562 cells with 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in an approximately 50% reduction in cell surface transferrin receptors within 30-45 min as judged by binding of both ligand and anti-receptor antibody. The affinity of the remaining surface receptors for diferric transferrin appeared to be unaltered. The time-dependent loss in transferrin receptors was also dependent upon PMA concentration, with a half-maximal effect observed at approximately 1 nM. The kinetic parameters for the binding, internalization, intracellular residency, and recycling of 125I-labeled transferrin were unchanged by PMA treatment, as were the rate and extent of internalization of anti-receptor antibody. Moreover, despite the decrease in surface receptors, uptake of 59Fe from transferrin proceeded at a rate comparable to that seen in untreated cells. Accounting for this observation was the fact that ligand induced a reduction in surface receptors in untreated but not PMA-treated cells. Quantitative immunoprecipitation of transferrin receptors from surface-iodinated K562 cells revealed that little receptor internalization occurred in untreated cells in the absence of ligand, but internalization of ligand-occupied receptors in these cells was readily detected. In contrast, PMA treatment resulted in the rapid internalization of surface receptors irrespective of occupancy. Thus, binding of ligand appeared to trigger the internalization of receptors that were relatively static in their unoccupied state, and a signal for receptor internalization was also provided by PMA treatment. The possibility that this signal involves phosphorylation of the transferrin receptor is discussed.

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.