scholarly journals Calmodulin dependence of transferrin receptor recycling in rat reticulocytes

1990 ◽  
Vol 266 (1) ◽  
pp. 261-272 ◽  
Author(s):  
J A Grasso ◽  
M Bruno ◽  
A A Yates ◽  
L Wei ◽  
P M Epstein
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Iron Uptake ◽  
Regulatory Control ◽  
Receptor Recycling ◽  
Calmodulin Antagonists ◽  
Single Class ◽  
Functional Integrity ◽  
Iron Delivery ◽  
Rat Reticulocytes

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.

Menadione-induced oxidative stress leads to a rapid down-modulation of transferrin receptor recycling

1993 ◽  
Vol 106 (1) ◽  
pp. 309-318
Author(s):  
W. Malorni ◽  
F. Iosi ◽  
M.T. Santini ◽  
U. Testa
Keyword(s):  
Oxidative Stress ◽  
Cell Surface ◽  
Transferrin Receptor ◽  
Thiol Group ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
General Mechanism ◽  
Receptor Recycling ◽  
Ultrastructural Studies ◽  
Oxidative Balance

It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.

scholarly journals Iterative endocytosis of transferrin by K562 cells

1994 ◽  
Vol 298 (1) ◽  
pp. 165-170 ◽  
Author(s):  
S P Young ◽  
A Bomford
Keyword(s):  
Cell Surface ◽  
Iron Uptake ◽  
Protein A ◽  
K562 Cells ◽  
Iron Chelator ◽  
Receptor Recycling ◽  
Weak Base ◽  
Uptake Process ◽  
The Difference ◽  
Diferric Transferrin

The effect of iron on the exocytosis of transferrin by K562 cells was studied by first allowing the cells to endocytose apotransferrin or diferric transferrin. Subsequent release of the apotransferrin was very rapid with a t 1/2 of 3.01 min, compared with 5.5 min for diferric transferrin. Release of apotransferrin was slowed by the weak base methylamine, t 1/2 8.0 min, but the effect of this agent was substantially greater when iron-transferrin was used, t 1/2 18.65 min, suggesting that methylamine affects both iron removal and receptor recycling. Release of iron-transferrin could be accelerated to a rate comparable with that of apotransferrin by addition of the permeant iron-chelator desferrioxamine. The difference in the rates of release of different forms of the protein could be explained by the re-endocytosis of the iron-rich protein, a process detected by the accelerated release of transferrin when the cells were washed in medium at pH 5.5 containing an iron-chelator or treated with a protease-containing medium to digest transferrin accessible at the cell surface. It appears that in cells incubated under control conditions, re-endocytosis of transferrin, which is incompletely depleted of iron, occurs and that a transferrin molecule may make two passes through the cell before all the iron is removed. This mechanism helps to explain why very little iron-transferrin is released from cells and why the efficiency of the iron uptake process is so high.

Intracellular Recycling and Iron Donating Ability of Transferrin Receptor 2α Protein Expressed in Transferrin Receptor-Deficient Chinese Hamster Ovary Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2671-2671
Author(s):  
Katsuya Ikuta ◽  
Junko Jimbo ◽  
Takaaki Hosoki ◽  
Motohiro Shindo ◽  
Kazuya Sato ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Cell Surface ◽  
Iron Metabolism ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Iron Uptake ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Hepcidin Expression

Abstract [Introduction] Transferrin receptor 2 (TfR2), a homologue of the transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like the classical TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes as homodimer on their cell surface, but the β form lacks intracellular and transmembrane portions and therefore is likely to be an intracellular protein. Although the main physiological functions of these two isoforms are still not fully understood, TfR2α binds transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. Disruption of TfR2 leads to hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. However, TfR1 have remarkably high binding affinity to diferric Tf and only little expression on the cell surface of hepatocytes so that TfR1 must be fully saturated under physiological conditions. Besides, TfR2 is upregulated in some conditions in that iron accumulation was observed, for instance in the liver of the patients of hepatitis C, indicating that TfR2 might also function as the iron donator for the hepatocytes. The aim of the present study was to investigate the interactions of TfR2α with Tf, and to elucidate whether TfR2α have the ability of iron donation to the cells. [Methods] To investigate the functional properties of TfR2α, we expressed TfR2α protein with FLAG-tagging in transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). The stably transfected cells expressing TfR2α-FLAG were applied for the 125I -labeled Tf (125I -Tf) binding study at 4 °C. The cells were applied for Tf and iron uptake study using 125I -Tf and 59Fe -loaded at 37 °C. To determine the fate of Tf internalized into the cells, the release of internalized Tf were then investigated. Degradation of released Tf was also investigated by precipitation using trichloroacetic acid/phosphotungustic acid. [Results] The association constant for binding of 125I-Tf to TfR2α was calculated to be 5.6 × 106 M−1 from non-linear least squares curve fitting to a saturable binding isotherm, which is about 50 times lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 °C, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, confirming that the existence of Tf internalization via TfR2α. After internalization, efflux of Tf, without substantial degradation, is also confirmed. Thus, Tf internalized by TfR2α, and presumably recycles, like TfR1 pathway. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1. [Discussion and Conclusions] TfR2α binds to Tf although its affinity for Tf is low, and TfR2α possesses iron donating ability to the cells when TfR2α is over-expressed in our study. Although TfR2α has been reported to be involved in iron sensing and regulation of hepcidin expression, the involvement of the Tf binding and iron donating properties of TfR2α should be considered when the pathophysiology of iron metabolism is investigated, especially in the conditions that TfR2α is upregulated, for instance in hepatitis C.

Characterization of the Interaction Between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy (AFM).

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1853-1853
Author(s):  
Katsuya Ikuta ◽  
Alexandre Yersin ◽  
Junko Jimbo ◽  
Takaaki Hosoki ◽  
Motohiro Shindo ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Cell Surface ◽  
Iron Metabolism ◽  
Transferrin Receptor ◽  
Iron Uptake ◽  
Force Microscopy ◽  
Functional Assays ◽  
Atomic Force ◽  
The Difference ◽  
Diferric Transferrin

Abstract [Introduction and aim] Transferrin receptor 2 (TfR2) is a homologue of the classical transferrin receptor 1 (TfR1), and TfR2 has two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes on their cell surface, but the β form lacks intracellular and transmembrane portions and therefore is likely to be an intracellular protein. Although the main physiological functions of these two isoforms are still not fully understood, TfR2α binds diferric transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. In fact, mutations of TfR2 cause hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. The aim of the present study was to investigate the interaction of TfR2α with Tf by functional assays and atomic force microscopy (AFM), which would be a powerful tool for investigation of the interaction between ligand and receptor on living cell surface. [Methods] To investigate the functional properties of TfR2α, we established the stable clone that expresses TfR2α protein with FLAG-tagging from transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). That clone was applied for the 125I –labeled Tf (125I –Tf) binding study at 4 oC. The cells were applied for Tf and iron uptake study using 125I –Tf and 59Fe –loaded Tf at 37 oC. AFM is the method that can investigate the unbinding force between receptor and ligand at single molecule level when their binding is physically detached, and we applied this method for determine the interaction between Tf and TfR2α using the transiently transfected human hepatome-derived HLF cells with TfR2α-expression vector. [Results] The association constant for binding of 125I-Tf to TfR2α was calculated to be 5.6 x 106 M−1 from non-linear least squares curve fitting to a saturable binding isotherm, which is much lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 oC, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, We also confirmied the existence of internalized Tf into cells via TfR2α. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1. The interaction between Tf and TfR2α was also confirmed by AFM, but the unbinding force was different from that between Tf and TfR1. This report might be the first that showed the binding and unbinding properties between Tf and TfR2α by AFM. [Discussion and conclusions] TfR2α binds to Tf although its affinity for Tf is low, and TfR2α possesses iron donating ability to the cells when TfR2α is over-expressed in our functional assays. AFM study also showed the binding between Tf and TfR2α on cell surface, but the properties of unbinding between them were different from those between Tf and TfR1. In conclusion, TfRα binds to Tf at cell surface and functions as iron donator to the cells like TfR1, but there should be the difference in the properties of binding with Tf between TfR1 and TfR2α, implying that the difference makes them having different physiological role in living organisms.

scholarly journals Transferrin receptor 1-mediated iron uptake plays an essential role in hematopoiesis

Haematologica ◽  
2019 ◽  
Vol 105 (8) ◽  
pp. 2071-2082 ◽  
Author(s):  
Shufen Wang ◽  
Xuyan He ◽  
Qian Wu ◽  
Li Jiang ◽  
Liyun Chen ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Iron Uptake ◽  
Essential Role ◽  
Transferrin Receptor 1

scholarly journals Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

1988 ◽  
Vol 106 (4) ◽  
pp. 1061-1066 ◽  
Author(s):  
T E McGraw ◽  
K W Dunn ◽  
F R Maxfield
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Phosphorylation Site ◽  
Major Protein ◽  
Kinase C ◽  
Recycling Pathway

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

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