ReviewEndocytosis and intracellular trafficking of ErbBs
Introduction
The discovery of EGF and its receptor was immediately followed by the investigation of the pathways and mechanisms of EGFR endocytosis. Such an interest in understanding EGFR endocytic trafficking has been driven by the recognition of the important role that this trafficking has in the regulation of signaling processes triggered by receptor tyrosine kinases (RTKs). In addition, the availability of EGF, antibodies to EGFR and other experimental tools has been and still remains to be the key factor that helps to sustain the ever increasing number of publications on endocytosis of this receptor.
The first comprehensive study of the EGF endocytosis, in which many of the key concepts of internalization and lysosomal degradation of EGF have been established, was published by Carpenter and Cohen [1]. This and other early studies by Cohen's group remain the basis of the current understanding of EGFR endocytosis. EGFR endocytosis is one of the most well characterized models for studying the morphology, kinetics and mechanisms of endocytic pathways, and is a prototypic model for the endocytosis of other RTKs. Studies of endocytosis of other ErbBs have been trailing the EGFR research because the natural ligands to ErbB3 and ErbB4 were discovered much later than EGF, and because the experimental tools to study these receptors and ErbB2 are only now becoming available.
EGFR is also the most popular model used to study the cross-talks between endocytosis and signaling. After internalization, EGF and EGFR are efficiently degraded, which results in the dramatic decrease in the half-life (t1/2) of the EGFR protein [2]. Accelerated internalization and degradation of activated EGFR lead to the decreased number of receptors at the cell surface, a phenomenon referred to as EGF-induced down-regulation of EGFR. Thus, the process of EGFR down-regulation and degradation is the major negative feedback regulatory mechanism that controls the intensity and duration of receptor signaling [3]. On the other hand, EGF-receptor complexes remain to be active in endosomes and continue to signal after internalization. Therefore, endocytosis has both “positive” and “negative” effects on the signaling network. The complex role of endocytosis in the regulation of EGFR signaling has been discussed in detail in several recent review articles [4], [5], [6], [7]. Hence this chapter will focus on the most recent advances in understanding the molecular mechanisms of the endocytic trafficking of EGFR and other ErbBs.
Section snippets
Turnover and trafficking of ErbBs in the absence of activation
In cultured cells expressing low or moderate levels of EGFR (< 200,000/cell), receptors turn over with t1/2 in the range of 6–10 h, whereas in cells overexpressing EGFR, such as human epidermoid carcinoma A-431, t1/2 could be 24 h or longer [2], [8], [9]. The turnover rate of ErbB2 expressed in NR6 cells was similar to that rate of unstimulated EGFR [10]. The t1/2 of endogenous ErbB3 in MCF-7 cells was 2.4 h [11]. ErbB4 expressed in COS cells had a half-life of about 5–7 h [12]. The general
Ligand-induced endocytosis of EGFR
ErbBs are activated by ligand-induced dimerization. There are more than 20 ligands of EGFR, ErbB3 and ErbB4, whereas ErbB2 does not have its own soluble ligand and is activated through heterodimerization with other ligand-occupied ErbBs [24]. The ligand-induced endocytic trafficking of activated EGFR is the best characterized among ErbBs. Therefore, in the following sections we will describe endocytosis and post-endocytic sorting of activated EGFR as the basic model, and subsequently discuss
Pathways through endosomes
Clathrin-coated vesicles containing EGF-receptor complexes rapidly release their coat and fuse with early endosomes, compartments of a heterogeneous morphology consisting of vesicular and tubular membranes and located at the periphery of the cell [78], [79], [80], [81] (Fig. 2). The accumulation of EGF and EGFR in early endosomes is evident after 2–5 min of endocytosis at 37 °C. Early endosomes are highly dynamic and tend to rapidly recycle the cargo, or fuse with each other leading to the
Proteins modulating endocytosis and sorting of EGFR
Whereas Grb2 and Cbl directly and specifically determine the rates of EGFR turnover, internalization and degradation, a number of other proteins have been identified that can have a modulatory effect on the rate of EGF-induced EGFR down-regulation. Interestingly, many of such proteins function by targeting Cbl activity [119]. For example, the Sprouty 2 protein is capable of binding to the RING and TKB domains of Cbl, thus inhibiting Cbl E3 ligase activity and reducing EGFR ubiquitination [120],
Trafficking of activated ErbB2
Because ErbB2 does not have a natural soluble ligand, the early studies of ErbB2 internalization utilized the chimeric proteins consisting of the extracellular domain of EGFR and the intracellular domain of ErbB2 and used labeled EGF to follow internalization of these chimeric receptors [10]. In these studies the EGFR-ErbB2 chimera was internalized several times slower than EGFR despite activation of the ErbB2 kinase and phosphorylation of the ErbB2 intracellular domain. It was concluded that
Trafficking of ErbB3 and ErbB4
ErbB3 and ErbB4 are activated by a common family of soluble ligands called heregulins or neuregulins. Unlike other ErbBs, ErbB3 has inactive kinase and is tyrosine phosphorylated by the heterodimeric partner. Ligand binding typically causes down-regulation of these receptors [11], [12], [163], albeit to an extent that is significantly lower than that observed with EGFR down-regulation [164], [165], [166]. The neuregulin-induced internalization of ErbB3 and ErbB4 has been observed in various
Conclusions and outstanding questions
Despite an effort of many research groups, many key aspects of the molecular mechanisms of EGFR endocytic trafficking are not understood. It has become clear that the process of EGFR endocytosis is very robust as it relies on several redundant mechanisms, e. g. Grb2-, Cbl- and ubiquitin-dependent and independent, and therefore, is difficult to dissect. Our knowledge about the endocytosis of EGFR under physiological conditions in cell culture and in vivo is especially lapsing. For example, most
Acknowledgments
We thank Melissa Adams for her critical reading of the manuscript. This work was supported by NCI grant CA08915 and ACS grant RSG-00-247-04-CSM.
References (170)
Trafficking of the ErbB receptors and its influence on signaling
Exp. Cell Res.
(2003)- et al.
Endocytosis conducts the cell signaling orchestra
Cell
(2006) - et al.
The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues
J. Biol. Chem.
(1985) - et al.
Polar expression of ErbB-2/HER2 in epithelia. Bimodal regulation by Lin-7
Dev. Cell
(2003) - et al.
The LIN-2/LIN-7/LIN-10 complex mediates basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial cells
Cell
(1998) - et al.
The role of tyrosine kinase activity in endocytosis, compartmentalization and down-regulation of the epidermal growth factor receptor
J. Biol. Chem.
(1991) - et al.
Ligand-induced internalization of the epidermal growth factor receptor is mediated by multiple endocytic codes analogous to the tyrosine motif found in constitutively internalized receptors
J. Biol. Chem.
(1993) - et al.
An integrated model of epidermal growth factor receptor trafficking and signal transduction
Biophys. J.
(2003) - et al.
Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes
Cell
(1984) - et al.
Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor
J. Biol. Chem.
(2002)
Tightly regulated and inducible expression of dominant interfering dynamin mutant in stably transformed HeLa cells
Methods Enzymol.
Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference
J. Biol. Chem.
Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation
Cell
Multiple autophosphorylation site mutations of the epidermal growth factor receptor
J. Biol. Chem.
Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing
Cell
Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation
J. Biol. Chem.
Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase
J. Biol. Chem.
Association of epidermal growth factor receptors with coated pit adaptins via a tyrosine phosphorylation-regulated mechanism
J. Biol. Chem.
Epidermal growth factor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif
J. Biol. Chem.
Stoichiometric interaction of the epidermal growth factor receptor with the clathrin-associated protein complex AP-2
J. Biol. Chem.
Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking
J. Biol. Chem.
Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1 [in process citation]
Mol. Cell
RING finger mutations that abolish c-Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation
Mol. Cell
Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain
Mol. Cell
Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis
J. Biol. Chem.
Differential effects of Cbl isoforms on Egfr signaling in Drosophila
Mechanisms of development
Membrane transport in the endocytic pathway
Curr. Opin. Cell Biol.
The endocytosis of epidermal growth factor in A431 cells: a pH of microenvironment and the dynamics of receptor complexes dissociation
Exp. Cell Res.
Dimerization of internalized growth factor receptors
J. Biol. Chem.
Selective membrane protein trafficking: vectorial flow and filter [see comments]
Trends Biochem. Sci.
Endosomal and non-endosomal functions of ESCRT proteins
Trends Cell Biol.
125I-Labeled human epidermal growth factor: binding internalization, and degradation in human fibroblasts
J. Cell Biol
“Down-regulation” of EGF receptors: direct demonstration of receptor degradation in human fibroblasts
J. Cell Biol.
Ligand-induced transformation by a noninternalizing epidermal growth factor receptor
Science
Signal transduction and endocytosis: close encounters of many kinds
Nat. Rev. Mol. Cell Biol.
Signaling on the endocytic pathway
Curr. Opin. Cell Biol.
Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes
Proc. Natl. Acad. Sci. U. S. A.
Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells
J. Cell Physiology
The carboxyl terminus of epidermal growth factor receptor/erbB-2 chimerae is internalization impaired
Oncogene
Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1
Mol. Cell Biol.
Isoform-specific monoubiquitination, endocytosis, and degradation of alternatively spliced ErbB4 isoforms
Proc. Natl. Acad. Sci. U. S. A.
Epidermal growth factor: the receptor and its function
BioFactors
Apical epidermal growth factor receptor signaling: regulation of stretch-dependent exocytosis in bladder umbrella cells
Mol. Biol. Cell
Basolateral localization of the Caenorhabditis elegans epidermal growth factor receptor in epithelial cells by the PDZ protein LIN-10
Mol. Biol. Cell
Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development
Embo. J.
Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin
Mol. Biol. Cell
Untangling the ErbB signalling network
Nat. Rev. Mol. Cell Biol.
Epidermal growth factor: morphological demonstration of binding internalization and lydosomal association in human fibroblasts
Proc.Nat. Acad. Sci. U. S. A.
Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts
J. Cell Biol.
Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits
Mol. Biol. Cell
Cited by (435)
Insights into the role of derailed endocytic trafficking pathway in cancer: From the perspective of cancer hallmarks
2024, Pharmacological ResearchIrreversible tyrosine kinase inhibitors induce the endocytosis and downregulation of ErbB2
2023, Biochemistry and Biophysics ReportsExploring the next generation of antibody–drug conjugates
2024, Nature Reviews Clinical Oncology