scholarly journals A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis

1990 ◽  
Vol 267 (1) ◽  
pp. 31-35 ◽  
Author(s):  
E Alvarez ◽  
N Gironès ◽  
R J Davis
Keyword(s):  
Transferrin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Cytoplasmic Domain ◽  
Cysteine Residue ◽  
Transferrin Receptors ◽  
Human Transferrin ◽  
Human Transferrin Receptor ◽  
Significant Difference ◽  
The Difference ◽  
Human Receptor

The rate of receptor-mediated endocytosis of diferric 125I-transferrin by Chinese-hamster ovary cells expressing human transferrin receptors was compared with the rate measured for cells expressing hamster transferrin receptors. It was observed that the rate of endocytosis of the human transferrin receptor was significantly higher than that for the hamster receptor. In order to examine the molecular basis for the difference between the observed rates of endocytosis, a cDNA clone corresponding to the cytoplasmic domain of the hamster receptor was isolated. The predicted primary sequence of the cytoplasmic domain of the hamster transferrin receptor is identical with that of the human receptor, except at position 20, where a tyrosine residue in the human sequence is replaced with a cysteine residue. To test the hypothesis that this structural change in the receptor is related to the difference in the rate of internalization, we used site-directed mutagenesis to examine the effect of the replacement of tyrosine-20 with a cysteine residue in the human transferrin receptor. It was observed that the substitution of tyrosine-20 with cysteine caused a 60% inhibition of the rate of iron accumulation by cells incubated with [59Fe]diferric transferrin. No significant difference between the rate of internalization of the mutant (cysteine-20) human receptor and the hamster receptor was observed. Thus the substitution of tyrosine-20 with a cysteine residue can account for the difference between the rate of endocytosis of the human and hamster transferrin receptors.

scholarly journals Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization.

1990 ◽  
Vol 110 (2) ◽  
pp. 283-294 ◽  
Author(s):  
S Q Jing ◽  
T Spencer ◽  
K Miller ◽  
C Hopkins ◽  
I S Trowbridge
Keyword(s):  
Amino Acid ◽  
Transferrin Receptor ◽  
High Efficiency ◽  
Signal Sequence ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Human Transferrin ◽  
Amino Acid Region ◽  
Human Transferrin Receptor ◽  
Mutant Receptors

Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.

scholarly journals The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

1994 ◽  
Vol 124 (5) ◽  
pp. 705-715 ◽  
Author(s):  
RJ Garippa ◽  
TW Judge ◽  
DE James ◽  
TE McGraw
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Amino Terminus ◽  
Ovary Cells ◽  
Major Function ◽  
Amino Terminal ◽  
Human Transferrin Receptor

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.

Expression and changing distribution of the human transferrin receptor in developing Drosophila oocytes and embryos

1996 ◽  
Vol 109 (13) ◽  
pp. 3113-3119 ◽  
Author(s):  
M.S. Bretscher
Keyword(s):  
Transferrin Receptor ◽  
Messenger Rna ◽  
Plasma Membranes ◽  
Posterior Pole ◽  
Transferrin Receptors ◽  
Human Transferrin ◽  
Human Transferrin Receptor ◽  
Cell Plasma ◽  
Cytoplasmic Vesicles ◽  
Ring Canals

In order to understand better the membrane systems in a developing Drosophila oocyte, the human transferrin receptor has been expressed there. This was achieved using the armadillo promoter combined with K10 or oskar trailer sequences; these enable the messenger RNA to be transcribed in nurse cells and then transported to, and translated in, oocytes. This is the first exogenous protein to be expressed in oocytes. At stage 8, the transferrin receptors are mainly concentrated towards the posterior pole of the oocyte and are associated with large cytoplasmic vesicles; when combined with the shibire mutation the transferrin receptors are transferred to the oolemma, demonstrating that they participate in an endocytic cycle. At stage 10, the transferrin receptors are localised either to the anterior margin of the oocyte or to the posterior pole, depending on where the mRNA is located. In newly laid eggs, all the transferrin receptors are found in large cytoplasmic vesicles. The results reveal remarkable sorting processes which occur as oocytes mature and show that ring canals, which separate the oolemma from nurse cell plasma membranes, act as barriers to prevent components in these two compartments from intermixing.

scholarly journals Structural model of phospholipid-reconstituted human transferrin receptor derived by electron microscopy

Structure ◽  
1998 ◽  
Vol 6 (10) ◽  
pp. 1235-1243 ◽  
Author(s):  
Hendrik Fuchs ◽  
Uwe Lücken ◽  
Rudolf Tauber ◽  
Andreas Engel ◽  
Reinhard Geßner
Keyword(s):  
Electron Microscopy ◽  
Transferrin Receptor ◽  
Structural Model ◽  
Human Transferrin ◽  
Human Transferrin Receptor

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.

The Promoter Region of the Human Transferrin Receptor Gene

1988 ◽  
Vol 526 (1 Hemochromatos) ◽  
pp. 54-64 ◽  
Author(s):  
John L. Casey ◽  
Bruno Jeso ◽  
Krishnamurthy Rao ◽  
Tracey A. Rouault ◽  
Richard D. Klausner ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Promoter Region ◽  
Receptor Gene ◽  
Human Transferrin ◽  
Human Transferrin Receptor

scholarly journals Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain.

1990 ◽  
Vol 1 (4) ◽  
pp. 369-377 ◽  
Author(s):  
T E McGraw ◽  
F R Maxfield
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rate Constant ◽  
Transferrin Receptor ◽  
Aromatic Amino Acid ◽  
Opposite Polarity ◽  
Receptor Internalization ◽  
Aromatic Side Chain ◽  
Human Transferrin ◽  
Human Transferrin Receptor

The objective of this work is to identify the elements of the human transferrin receptor that are involved in receptor internalization, intracellular sorting, and recycling. We have found that an aromatic side chain at position 20 on the cytoplasmic portion of the human transferrin receptor is required for efficient internalization. The wild-type human transferrin receptor has a tyrosine at this position. Replacement of the Tyr-20 with an aromatic amino acid does not alter the rate constant of internalization, whereas substitution with the nonaromatic amino acids serine, leucine, or cysteine reduces the internalization rate constant approximately three-fold. These results are consistent with similar studies of other receptor systems that have also documented the requirement for a tyrosine in rapid internalization. The amino terminus of the transferrin receptor is cytoplasmic, with the tyrosine 41 amino acids from the membrane. These two features distinguish the transferrin receptor from the other membrane proteins for which the role of tyrosine in internalization has been examined, because these proteins have the opposite polarity with respect to the membrane and because the tyrosines are located closer to the membrane (within 25 amino acids). The externalization rate for the recycling of the transferrin receptor is not altered by any of these substitutions, demonstrating that the aromatic amino acid internalization signal is not required for the efficient exocytosis of internalized receptor.

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