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 In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella.

1994 ◽  
Vol 125 (6) ◽  
pp. 1265-1274 ◽  
Author(s):  
C R Hopkins ◽  
A Gibson ◽  
M Shipman ◽  
D K Strickland ◽  
I S Trowbridge
Keyword(s):  
Plasma Membrane ◽  
Chick Embryo ◽  
Cell Surface ◽  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Intracellular Processing ◽  
Recycling Pathway ◽  
Embryo Fibroblasts ◽  
Migrating Cells

By following the intracellular processing of recycling transferrin receptors and the selective sorting of a-2 macroglobulin in chick embryo fibroblasts, we have shown that the concentration of 60 nm diam tubules which surrounds the centrioles represents a distal compartment on the recycling pathway. In migrating cells transferrin receptor tracers can be loaded into this compartment and then chased to the cell surface. When they emerge the recycling transferrin receptors are distributed over the surface of the leading lamella.

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.

scholarly journals Regulation of cell-surface receptors for human interferon-γ on the human histiocytic lymphoma cell line U937

1991 ◽  
Vol 274 (3) ◽  
pp. 775-780 ◽  
Author(s):  
D S Finbloom
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Specific Radioactivity ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Cell Surface Receptors ◽  
Minor Role ◽  
Ifn Gamma ◽  
Surface Receptors ◽  
High Specific Radioactivity

Interferon-gamma (IFN gamma) binds to high-affinity receptors on monocytes and is rapidly internalized. This study investigates the ability of the human monocyte-like cell line, U937, to regulate the cell-surface expression of the IFN gamma receptor (IFN gamma R) during endocytosis of ligand. Recombinant IFN gamma was radiolabelled to high specific radioactivity with Bolton-Hunter reagent and used to enumerate IFN gamma R on treated U937 cells. Cells which had internalized IFN gamma for up to 3 h displayed maximal levels of IFN gamma R at all time points tested after all unlabelled IFN gamma had been acid-stripped from the cell at pH 2.78. Therefore there was no evidence of down-modulation of the receptor. After trypsin treatment of the IFN gamma R, the cells were able to synthesize and insert into the cell membrane up to 1000 IFN gamma R molecules/h after a 60 min lag. Since biosynthesis played a minor role during the first 30 min of endocytosis, I examined other possibilities to explain the lack of down-modulation of the receptor. A solubilized-receptor assay revealed the presence of an intracellular pool of receptors equal to about 25% of the number of cell surface receptors. Using trypsin to differentiate between intracellular and surface receptors, I observed that 43% of those receptors that were internalized after a 30 min exposure to IFN gamma (580 molecules) could be recycled back to the plasma membrane. In addition, equal rates of receptor decay (t1/2 = 5 h) were observed in the presence of cycloheximide with or without IFN gamma. All the data taken together suggest that during the first 30 min of endocytosis both the expression of an intracellular source of receptor and recycling of internalized receptors contribute to maintain optimal receptor expression.

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 Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.

1986 ◽  
Vol 102 (4) ◽  
pp. 1271-1283 ◽  
Author(s):  
M G Roth ◽  
C Doyle ◽  
J Sambrook ◽  
M J Gething
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Dna Sequences ◽  
Acquired Resistance ◽  
Endocytic Pathway ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Coated Pits ◽  
Herpes Simplex Virus 1 ◽  
Surface Receptors

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

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.

scholarly journals Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.

1983 ◽  
Vol 97 (2) ◽  
pp. 329-339 ◽  
Author(s):  
C Harding ◽  
J Heuser ◽  
P Stahl
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Intracellular Membrane ◽  
Coated Pits ◽  
Surface Receptors ◽  
Receptor Mediated Endocytosis ◽  
Freeze Dried ◽  
Inner Surface ◽  
Multivesicular Endosomes ◽  
Rat Reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin-resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.

In Vivo Internalization of the Somatostatin sst2A Receptor in Rat Brain: Evidence for Translocation of Cell-Surface Receptors into the Endosomal Recycling Pathway

2001 ◽  
Vol 17 (4) ◽  
pp. 646-661 ◽  
Author(s):  
Zsolt Csaba ◽  
Véronique Bernard ◽  
Lone Helboe ◽  
Marie-Thérèse Bluet-Pajot ◽  
Bertrand Bloch ◽  
...  
Keyword(s):  
Cell Surface ◽  
Rat Brain ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Endosomal Recycling ◽  
Recycling Pathway

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

2012 ◽  
Vol 24 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Abo Bakr Abdel Shakor ◽  
Mona Mohamed Atia ◽  
Katarzyna Kwiatkowska ◽  
Andrzej Sobota
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Coated Pits

scholarly journals Intracellular interactions of transferrin and its receptor during biosynthesis.

1990 ◽  
Vol 111 (4) ◽  
pp. 1383-1392 ◽  
Author(s):  
J J Neefjes ◽  
T Hengeveld ◽  
O Tol ◽  
H L Ploegh
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line ◽  
Hepatoma Cell Line Hepg2 ◽  
Acidic Compartment

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 disulfide-linked 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 is ultimately delivered to an acidic compartment, where Tf releases its Fe3+. We did not observe conversion of Tf to apoTf in the TGR, showing that the part of the TGR passed by secreted Tf has a pH higher than 5.5. We conclude that when a ligand-receptor combination is synthesized by one and the same cell, ligand and receptor can interact during biosynthesis and be transported to the cell surface.

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