Internalization Sequence Research Articles

Endocytosis and Lysosomal Targeting of Epidermal Growth Factor Receptors Are Mediated by Distinct Sequences Independent of the Tyrosine Kinase Domain

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research.

The EGF receptor (EGFR) and HER2 are members of a growth factor receptor family. Overexpression of either protein in advanced breast cancer correlates with poor prognosis. EGF stimulates growth by binding to EGFR, activating the receptor's intracellular tyrosine kinase. The initial consequence is phosphorylation of specific tyrosine-containing sequences in the receptor's carboxyl terminus. These phosphotyrosines serves as high affinity recognition sites for proteins that, in turn, transmit the growth signal inside the cell. Mechanistic studies suggest that EGF binds to a single EGFR, triggering dimerization with another like receptor molecule. This dimerization is thought to initiate the tyrosine kinase activation. The EGF receptor family was recently expanded with the sequencing of HER3 and HER4. Each of the four family members was postulated to regulate a unique growth or differentiation signaling repertoire when activated by a receptor-specific ligand. However, new data from numerous laboratories suggest that EGFR family members may play a complex and ultimately more flexible role in signaling by forming heterodimers between family members, e.g. EGFR:HER2 or HER4:HER2. These heterodimers may form even when only one member of the pair binds its ligand. This review summarizes current work on heterodimerization and attempts to predict the consequences for downstream signaling. In brief, when compared to ligand-dependent receptor homodimers comprised of two proteins with the same internalization sequence and phosphorylated tyrosine residues, heterodimers are likely to: i) expand substrate selection and downstream signaling pathway activation; ii) promote interaction between sets of substrates in the mixed receptor complexes that would not ordinarily be physically juxtaposed; iii) alter the duration of receptor signaling by changing rates of receptor internalization, ligand loss, kinase inactivation, recycling, etc.; and iv) alter rates of receptor and substrate dephosphorylation. In addition to understanding interactions of heterodimers with the internalization machinery, identification of receptor-specific substrates and binding proteins for each EGFR family member will be necessary to explicate the role of heterodimers in growth and differentiation.

Characteristics of the internalization signal in the Y543 influenza virus hemagglutinin suggest a model for recognition of internalization signals containing tyrosine.

Several proteins, including the hemagglutinin (HA)-Y543 mutant influenza virus hemagglutinin, are internalized by clathrin-coated pits but do not have a sequence that fits a recently proposed consensus for internalization signals containing tyrosine. To determine whether or not the HA-543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and mannose 6-phosphate/insulin-like growth factor (IGF) II receptor, we have mutated amino acid positions of HA-Y543 shown to be important for internalization of the two receptors. Our results indicate that the HA-Y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, low density lipoprotein, and mannose 6-phosphate/IGFII. However, amino acids with side chains having very different chemical properties functioned well in positions that are important for the internalization signal. The variety of amino acid side chains found in known internalization sequences suggests that atoms of the polypeptide chain backbone may contribute important interactions for binding proteins to clathrin coats, with many of the side chains serving mainly to permit these interactions, a situation similar to that observed for the binding of peptides by histocompatibility proteins.

Clathrin-coated pit-mediated receptor internalization. Role of internalization signals and receptor mobility.

Most signals controlling receptor-mediated endocytosis have been identified by alteration of sequences present in receptors normally internalized via clathrin-coated pits. In the present work we have reconsidered the factors that control internalization the other way around: i.e. by introducing potential internalization sequences in complement receptor 1 (CR1) which does not preferentially associate with clathrin-coated pits. The analysis of the internalization efficiency of NPxY related motifs generated by substituting His2010 and/or Glu2015 by either Phe or Tyr indicates that FxNPxY is the stronger promoter of endocytosis and that the signal efficiency depends on the presence of aromatic residues (including a tyrosine) at both ends of the -xNPx- motif. Moreover, CR1-tyr (substitution of Glu2015 for Tyr) internalization was superposable to that of a receptor composed of the extracellular and transmembrane domains of CR1 fused to the intracytoplasmic tail of the low density lipoprotein (LDL) receptor (including the FxNPxY motif) (CR1-LDL). When analyzed by fluorescence recovery after photobleaching, the surface mobility of CR1-LDL was decreased as compared with that of either CR1-tyr or CR1-wt, despite a similar association with clathrin-coated pits. The role of receptor mobility in internalization was confirmed by the observation that CR1-tl, with a deletion of the cytoplasmic tail, was more mobile and more efficiently internalized than CR1-wt.

The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.

Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence.

Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor.

The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.

The receptor for yolk lipoprotein deposition in the chicken oocyte.

The final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically synthesized yolk precursor proteins from the plasma. The two major yolk-forming components, very low density lipoprotein (VLDL) and vitellogenin (VTG), have been shown to interact with a 95-kDa protein present in detergent extracts of ovarian membranes; this protein is absent in hens of a mutant nonlaying chicken strain (Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398). Here, we have purified the 95-kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor-mediated endocytosis by ultrastructural immunolocalization, structural, and functional studies. The receptor was visualized exclusively in the oocyte proper and was absent from somatic cells, in agreement with the previously reported expression of two different lipoprotein receptors in somatic cells and oocytes, respectively, of laying hens (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Amino acid sequences of tryptic fragments of the oocyte receptor were obtained, and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains. In particular, the chicken receptor's internalization sequence, Phe-Asp-Asn-Pro-Val-Tyr, is identical with that in low density lipoprotein receptors from mammals as well as Xenopus laevis. The ligand-binding properties, specificity, and kinetic parameters of the oocyte receptor were characterized in filtration assays employing pure ligands and receptor. In conjunction with ligand-blotting experiments following limited protease digestion of the receptor, the binding assay data suggest that VTG recognizes a substructure of the VLDL-binding site. These studies establish that a cell-specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays a key role in control of oocyte growth.

Endocytosis and signals for internalization

The most important recent advance in the field of endocytosis has been the identification of the internalization signals of several constitutively recycling receptors. Common structural features and chemistry of internalization sequences have been defined, and an exposed tight turn has been implicated as the conformational recognition motif for endocytosis.

Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis

Using detailed functional studies on 24 human transferrin receptor mutants, we identified YXRF as the internalization sequence. Provided that at least 7 residues separate this tetrapeptide from the transmembrane region, changing the tetrapeptide position within the TR cytoplasmic domain does not reduce internalization activity. Thus, any conformational determinant for internalization must be localized to the YXRF sequence. Twenty-eight tetrapeptide analogs of YXRF, found by an unbiased search of all known three-dimensional protein structures, significantly favored tight turns similar to a type I turn. Of the ten tetrapeptides most closely related to YXRF, eight were surface exposed and had tight-turn conformations, as were four of five tetrapeptides with sequences related to the low density lipoprotein receptor internalization motif, NPXY. The internalization sequences of both receptors contain aromatic residues with intervening hydrogen-bonding residues. Thus, two distinct internalization sequences favor a common structural chemistry and implicate an exposed tight turn as the recognition motif for high efficiency endocytosis.