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

Abstract

Abstract. 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 *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 •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.

MHC class II presentation of endogenously expressed antigens by transfected dendritic cells

“…24 The cytosolic domain of TfR contains tyrosine motifs that upon internalization direct trafficking of the receptor and its cargo to early endosomes. 26,27 This sorting signal is present in TfR-OVA and thus we expect the fusion protein to be transported to these endocytic vesicles. Early and late endocytic compartments were shown to contribute peptides for MHC class II presentation.…”

The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Multiple Signals that Regulate its Cell Surface Expression and Endocytosis

“…For the constructs used in this study (see Figure 1), the chimeras were made using a unique Hind III restriction site engineered into CD4 at nt 1351 such that four residues from the CD4 cytoplasmic domain (-RCRH) were included in the constructs. These residues act as a spacer to place the SIV Env membrane proximal endocytosis signal a requisite distance from the membrane for functional activity (40). This spacer compensates for the IVQMLA sequence in SIV Env (see Table 2) that we have argued is part of the cytoplasmic domain of the protein (13).…”

The major histocompatibility complex-encoded class I-like HFE abrogates endocytosis of transferrin receptor by inducing receptor phosphorylation

“…To measure the kinetics of internalization and recycling of 125 I-Tf, we followed the procedure described by Jing et al 46 Briefly, cells were incubated at 4°C for 1 h in prewarmed serum-free DMEM supplemented with 4 g/ml of 125 I-Tf (NEN, MA, USA) and then incubated for various times at 37°C in pre-warmed BSA-DMEM containing 50 g/ml of unlabeled diferric Tf. For the zero time point, ice-cold medium was added to these wells and then removed immediately.…”

“…48 Excess of radio-iron was removed by filtration through a PD-10 column (Pharmacia, Sweden). Iron uptake measurements were performed according to the protocol described by Jing et al 46 Briefly, cells were incubated in BSA-DMEM containing 20 g/ml 59 Fe-Tf at 37°C for 0, 1, 2, 3, and 4 h. After incubation, cells were removed in 0.5 ml of 1 N NaOH, and radioactivity was counted in a gamma counter. The relative levels of TfR expressed by the different cells were pre-determined for each experiment, as previously described.…”