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

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.

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

Cell Regulation ◽

10.1091/mbc.1.4.369 ◽

1990 ◽

Vol 1 (4) ◽

pp. 369-377 ◽

Cited By ~ 77

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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Structural requirements for high efficiency endocytosis of the human transferrin receptor

Journal of Inorganic Biochemistry ◽

10.1016/0162-0134(92)84066-v ◽

1992 ◽

Vol 47 (1) ◽

pp. 209-217 ◽

Cited By ~ 9

Author(s):

Ian S. Trowbridge ◽

James F. Collawn

Keyword(s):

Transferrin Receptor ◽

High Efficiency ◽

Structural Requirements ◽

Human Transferrin ◽

Human Transferrin Receptor

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Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽

10.1182/blood.v83.2.580.bloodjournal832580 ◽

1994 ◽

Vol 83 (2) ◽

pp. 580-586 ◽

Cited By ~ 2

Author(s):

EA Rutledge ◽

BJ Root ◽

JJ Lucas ◽

CA Enns

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Transferrin Receptor ◽

Transmembrane Domain ◽

Glycosylation Site ◽

Soluble Form ◽

Protein Sequence Analysis ◽

Wild Type ◽

Human Transferrin Receptor ◽

Intracellular Compartments

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

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

Biochemical Journal ◽

10.1042/bj2670031 ◽

1990 ◽

Vol 267 (1) ◽

pp. 31-35 ◽

Cited By ~ 29

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.

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Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽

10.1182/blood.v83.2.580.580 ◽

1994 ◽

Vol 83 (2) ◽

pp. 580-586 ◽

Cited By ~ 42

Author(s):

EA Rutledge ◽

BJ Root ◽

JJ Lucas ◽

CA Enns

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Transferrin Receptor ◽

Transmembrane Domain ◽

Glycosylation Site ◽

Soluble Form ◽

Protein Sequence Analysis ◽

Wild Type ◽

Human Transferrin Receptor ◽

Intracellular Compartments

Abstract The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

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Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.853 ◽

1991 ◽

Vol 112 (5) ◽

pp. 853-861 ◽

Cited By ~ 31

Author(s):

T E McGraw ◽

B Pytowski ◽

J Arzt ◽

C Ferrone

Keyword(s):

Amino Acid ◽

Transferrin Receptor ◽

Aromatic Amino Acids ◽

Site Mutation ◽

Site Specific ◽

Aromatic Residues ◽

Human Transferrin ◽

Human Transferrin Receptor ◽

Internalization Rate ◽

Native Context

Site-specific mutagenesis has been used to define the sequences required for efficient internalization of the human transferrin receptor. It has previously been shown that the sole cytoplasmic tyrosine, at position 20, is required for efficient internalization. When two other cytoplasmic aromatic residues, the phenylalanines at positions 13 and 23, are substituted with alanines internalization is also reduced. The phenylalanine 23 mutation decreases the internalization rate constant approximately threefold, and mutation of phenylalanine 13 decreases it by approximately twofold. The mutation at position 23 has as serious an effect on internalization as substitution with a nonaromatic amino acid for the single tyrosine. These results demonstrate the importance of several aromatic amino acids in maintaining efficient internalization of the transferrin receptor. Substitution of a tyrosine at a second site, for a serine at position 34, within the cytoplasmic domain of a transferrin receptor with a nonaromatic amino acid at position 20, results in a complete reversion of the internalization-defective phenotype. This reversion is completely dependent upon a tyrosine, as phenylalanine substituted at position 34 does not revert the internalization-defective phenotype. This result demonstrates that a tyrosine placed outside of its native context can still function in the internalization of the transferrin receptor, suggesting a flexibility in surrounding sequences required for efficient internalization.

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Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

The Journal of Cell Biology ◽

10.1083/jcb.139.4.917 ◽

1997 ◽

Vol 139 (4) ◽

pp. 917-927 ◽

Cited By ~ 39

Author(s):

Anne E. West ◽

Rachael L. Neve ◽

Kathleen M. Buckley

Keyword(s):

Amino Acid ◽

Synaptic Vesicle ◽

Transferrin Receptor ◽

Synaptic Vesicles ◽

Hippocampal Neurons ◽

Expression System ◽

Amino Acid Sequences ◽

Amino Acid Region ◽

Human Transferrin Receptor ◽

Cultured Hippocampal Neurons

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.

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