scholarly journals Local clustering of transferrin receptors promotes clathrin-coated pit initiation

2010 ◽  
Vol 191 (7) ◽  
pp. 1381-1393 ◽  
Author(s):  
Allen P. Liu ◽  
François Aguet ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Major Pathway ◽  
Receptor Ligand ◽  
Local Clustering ◽  
Pit Initiation

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor–ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

scholarly journals Rapid endocytosis of the transferrin receptor in the absence of bound transferrin.

1985 ◽  
Vol 100 (2) ◽  
pp. 633-637 ◽  
Author(s):  
C Watts
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Protein A ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Prolonged Incubation ◽  
Surface Receptors ◽  
Recycling Pathway

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.

scholarly journals A novel class of clathrin-coated vesicles budding from endosomes.

1996 ◽  
Vol 132 (1) ◽  
pp. 21-33 ◽  
Author(s):  
W Stoorvogel ◽  
V Oorschot ◽  
H J Geuze
Keyword(s):  
Plasma Membrane ◽  
Microscopic Examination ◽  
Transferrin Receptor ◽  
Brefeldin A ◽  
Coated Vesicles ◽  
Integral Membrane Proteins ◽  
Transferrin Receptors ◽  
Receptor Recycling ◽  
Coated Pits ◽  
Novel Technique

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.

scholarly journals Combined radiolabel-binding and immunocytochemical evaluation of receptor–ligand interactions. Studies of transferrin receptors on activated lymphocytes

1981 ◽  
Vol 200 (1) ◽  
pp. 173-176 ◽  
Author(s):  
Gillian M. P. Galbraith ◽  
Robert M. Galbraith
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Receptor Ligand ◽  
Activated Lymphocytes ◽  
Sources Of Error ◽  
Composite Approach ◽  
Receptor Interactions ◽  
Potential Sources ◽  
Ligand Interactions

A protocol that involved both immunohistological and radiolabel-binding procedures was devised for the study of transferrin–receptor interactions. This composite approach yielded considerably more information than did either technique used alone, and also provided a simple means for exclusion of several common potential sources of error.

scholarly journals Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.

1983 ◽  
Vol 97 (2) ◽  
pp. 508-521 ◽  
Author(s):  
C R Hopkins ◽  
I S Trowbridge
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Gold Colloid ◽  
A431 Cells ◽  
Coated Pits ◽  
Gold Complexes ◽  
Thin Sections ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Peripheral Cytoplasm

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 complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

scholarly journals Transcriptional regulation by iron of the gene for the transferrin receptor.

1986 ◽  
Vol 6 (1) ◽  
pp. 236-240 ◽  
Author(s):  
K Rao ◽  
J B Harford ◽  
T Rouault ◽  
A McClelland ◽  
F H Ruddle ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
K562 Cells ◽  
Iron Chelator ◽  
Transcriptional Level ◽  
Transferrin Receptors ◽  
Intracellular Iron ◽  
Human Transferrin Receptor ◽  
Diferric Transferrin ◽  
Permeable Iron

Treatment of K562 cells with desferrioxamine, a permeable iron chelator, led to an increase in the number of transferrin receptors. Increasing intracellular iron levels by treatment of cells with either human diferric transferrin or hemin lowered the level of the transferrin receptors. By using a cDNA clone of the human transferrin receptor, we showed that the changes in the levels of the receptor by iron were accompanied by alterations in the levels of the mRNA for the receptor. The rapidity of these changes indicated that the mRNA had a very short half-life. By using an in vitro transcriptional assay with isolated nuclei, we obtained evidence that this regulation occurred at the transcriptional level.

scholarly journals Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size

2011 ◽  
Vol 22 (14) ◽  
pp. 2588-2600 ◽  
Author(s):  
Costin N. Antonescu ◽  
François Aguet ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Mammalian Cells ◽  
Lipid Binding ◽  
Coated Pits ◽  
Inositol Phosphatase ◽  
Major Mechanism ◽  
Synaptojanin 1 ◽  
Phosphatidylinositol 4 ◽  
Sirna Knockdown ◽  
Interfering Rna ◽  
Pit Initiation

Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.

scholarly journals Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis

2014 ◽  
Vol 25 (19) ◽  
pp. 3070-3080 ◽  
Author(s):  
Marko Lampe ◽  
Fabienne Pierre ◽  
Suleiman Al-Sabah ◽  
Cornelius Krasel ◽  
Christien J. Merrifield
Keyword(s):  
Opioid Receptor ◽  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Mu Opioid Receptor ◽  
Event Analysis ◽  
Coated Pits ◽  
Mu Opioid ◽  
Dynamic Relationship ◽  
Β2 Adrenergic Receptor ◽  
Endocytic Vesicles

The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein–coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)–based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

scholarly journals Selective inhibition of the growth of human erythroid bursts by monoclonal antibodies against transferrin or the transferrin receptor

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1631-1638 ◽  
Author(s):  
KM Shannon ◽  
JW Larrick ◽  
SA Fulcher ◽  
KB Burck ◽  
J Pacely ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transferrin Receptor ◽  
Thymidine Incorporation ◽  
Selective Inhibition ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Transferrin Receptors ◽  
Human Erythropoietin ◽  
Surface Receptor ◽  
Transferrin Receptor Expression

Abstract The relative requirements of colonies derived from erythroid (BFU-E) and myeloid (CFU-c) progenitors for transferrin were examined using monoclonal antibodies directed against the transferrin molecule (TF-6) or its cell surface receptor (TFR-A12, TFR1–2B). Growth of erythroid bursts was profoundly reduced at concentrations of all three antibodies that had no effect on CFU-c-derived colonies. When TFR1–2B was layered over cultures established one to seven days previously, further burst development was inhibited, and degeneration of early erythroid colonies was observed. Addition of erythropoietin augmented transferrin receptor expression on cells harvested after 1 to 2 weeks in culture and analyzed by flow cytometry. Recombinant human erythropoietin gave results comparable to those obtained in experiments using human urinary erythropoietin. Analysis of erythroblasts plucked directly from culture plates confirmed the presence of transferrin receptors on BFU-E-derived colonies. Thymidine incorporation was maximal early in the second week of culture and coincided with high transferrin receptor expression. These data demonstrate that transferrin must be available into the second week of culture to support the growth and differentiation of BFU- E-derived erythroid bursts, that the generation of erythroid colonies from BFU-E is more dependent on transferrin than myeloid colony formation from CFU-c, and that erythropoietin modulates the expression of transferrin receptors on growing bursts.

scholarly journals Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

Effect of iron on transferrin receptor expression by human placental syncytiotrophoblast cells

1997 ◽  
Vol 9 (6) ◽  
pp. 609 ◽  
Author(s):  
Martha L. Kennedy ◽  
Gordon C. Douglas ◽  
Barry F. King
Keyword(s):  
Transferrin Receptor ◽  
Iron Status ◽  
Primary Cultures ◽  
Receptor Expression ◽  
Transferrin Receptors ◽  
Iron Salts ◽  
Intracellular Receptor ◽  
Cytotrophoblast Cells ◽  
Diferric Transferrin ◽  
Transferrin Receptor Expression

Transferrin receptor expression has been examined in primary cultures of morphologically differentiated placental syncytiotrophoblast cells. More than 90% of the cells were multinucleated. Incubation of syncytiotrophoblast for 4 days in the presence of iron salts had no effect on receptor expression assessed by measuring the binding of 125I-labelled transferrin. However, incubation of cells in the presence of human diferric transferrin (10-100 µM) led to a 50% decrease in surface and intracellular receptor expression. This down-regulation was not accompanied by a signicant decrease in receptor synthesis. In contrast to syncytiotrophoblast, expression of intracellular transferrin receptors in non-differentiated cytotrophoblast cells decreased when cells were cultured with iron salts; this was accompanied by decreased receptor synthesis. Addition of diferric transferrin to cytotrophoblast cells led to a 50% reduction in surface and intracellular receptor expression, similar to that seen in the syncytiotrophoblast. This reduction was accompanied by a decrease in receptor synthesis. In contrast to that of most cell types, the expression and distribution of trophoblast transferrin receptors were not altered by insulin, epidermal growth factor or hydrocortisone. These characteristics of syncytiotrophoblast transferrin receptor expression may assist in ensuring a supply of iron to the fetus regardless of the maternal iron status.

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