Endocytosis and intracellular trafficking of ErbBs

doi:10.1016/j.yexcr.2008.07.029

Experimental Cell Research

Volume 315, Issue 4, 15 February 2009, Pages 683-696

https://doi.org/10.1016/j.yexcr.2008.07.029 Get rights and content

Abstract

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.

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 (t_1/2) 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 t_1/2 in the range of 6–10 h, whereas in cells overexpressing EGFR, such as human epidermoid carcinoma A-431, t_1/2 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 t_1/2 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.

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ReviewEndocytosis and intracellular trafficking of ErbBs

Abstract

Introduction

Section snippets

Turnover and trafficking of ErbBs in the absence of activation

Ligand-induced endocytosis of EGFR

Pathways through endosomes

Proteins modulating endocytosis and sorting of EGFR

Trafficking of activated ErbB2

Trafficking of ErbB3 and ErbB4

Conclusions and outstanding questions

Acknowledgments

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Cell

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Cell

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell

Mol. Cell

Mol. Cell

J. Biol. Chem.

Mechanisms of development

Curr. Opin. Cell Biol.

Exp. Cell Res.

J. Biol. Chem.

Trends Biochem. Sci.

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

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Nat. Rev. Mol. Cell Biol.

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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

Review
Endocytosis and intracellular trafficking of ErbBs

¹²⁵I-Labeled human epidermal growth factor: binding internalization, and degradation in human fibroblasts

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