expression of the oncoproteins ErbB2 and ErbB3
By
Markus Dietrich
Laboratory for Molecular and Cellular Cancer Research, Institute of Clinical Medicine, Faculty of Medicine,
University of Oslo, Norway
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-161-9
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
The presented work was funded by The South-Eastern Norway Regional Health Authority, to whom I am very grateful for the given opportunity.
I would also like to express my gratitude to several persons that supported me during the time of this thesis.
I would like to thank my supervisor Dr. Espen Stang for giving me the opportunity to work as a PhD student. Thank you for being an excellent supervisor and for the introduction to the field of molecular cancer research. I am very grateful that you were always available for discussion of results and even in challenging phases never lost the positive attitude. Your great knowledge and never ending new ideas for experiments to try really helped me in the completion of my thesis.
Thank you for the attention to detail and for being very patient and supportive.
A big thank you also goes to my co-supervisor Dr. Vibeke Bertelsen. I am very grateful for your feedback over the last years and also very glad that even though you left the group one year ago you were still always available for discussion of results and feedback. Also thank you for your attention to details that for sure raised the quality of the work done in the lab.
Prof. Henrik Huitfeldt, thank you for being a co-supervisor and for providing good mood and enthusiasm in our meetings. Your thinking out of the box for new experimental approaches and your critical questions on results were always appreciated.
All present and former members of the Stang group deserve a big thank you. Marianne Rødland, thank you for the experimental support to my projects, especially the countless microscopy stainings. Thank you to Salman Malik and Filip Nikolaysen for great experimental support, especially while I was writing my thesis. Your contribution was highly appreciated. Anne Marthe Fosdahl, thank you for being a great office mate and co-author throughout my PhD. I really appreciate your hard work and your contributions to our successful projects.
Thank you to Monika Szymanska for being a great co-worker, co-author and office mate. I really appreciate all your contributions to our projects and that you always helped to solve problems even after you had already moved to a new work group. Thank you to you and Tara Sarin for always pleasant lunch and coffee breaks that helped to come back to work with full motivation.
You two are really good friends and we will stay in contact also when we cannot spend lunch breaks together anymore.
I am very grateful for all the co-authors that helped in my projects and a big thank you goes to all of you and I want to thank all my colleagues at the Department of Pathology at Rikshospitalet for an inspiring, friendly and very comfortable work environment.
I wish to express my deep felt gratitude to my family and friends that always supported and motivated me throughout these years. Your constant encouragement helped me over the past years. I could not have done that without you.
Finally, I want to thank Laura for always being there for me. Your patience, support, motivation and love helped throughout the good and bad times in the last four years.
Oslo, October 2017 Markus Dietrich
Table ofContents
Abbreviations ... I Papers Included ... IV
Introduction ... 1
Receptor Tyrosine Kinase (RTK) ... 2
The ErbB protein family ... 2
Structure and function ... 3
ErbB ligands ... 4
Phosphorylation and ubiquitination... 6
ErbB Activation and Signaling ... 7
Maturation and trafficking of ErbB proteins ... 9
Endocytosis ... 9
Clathrin-mediated endocytosis ... 10
Adaptors of clathrin-mediated endocytosis ... 13
Clathrin-independent endocytosis ... 16
Macropinocytosis ... 16
Caveolin-dependent endocytosis ... 16
Clathrin- and caveolin-independent pathways ... 17
Endosomal sorting ... 18
Endocytic recycling ... 20
Endocytic degradation ... 21
Endocytosis and endosomal sorting of EGFR ... 22
Endocytosis and endosomal sorting of ErbB2 ... 23
Endocytosis and endosomal sorting of ErbB3 ... 25
ErbB proteins in cancer ... 26
Protein kinase C (PKC) ... 27
PKC structure ... 27
PKC activation ... 28
Growth factor receptors and PKC signaling ... 30
Aims of the Study ... 33
Summary of Papers ... 34
Paper I ... 34
Paper II ... 35
Paper III... 36
Paper IV ... 37
Methodological Considerations ... 38
Cell culture ... 38
Chemical inhibitors and activators ... 39
Transient transfections ... 40
RNA interference ... 41
High-throughput screening ... 42
Immunological detection ... 42
Western blotting ... 43
Immunoprecipitation (IP) and co-IP ... 43
Wide-field fluorescent microscopy and confocal microscopy ... 44
Immuno-electron microscopy ... 45
Flow cytometry ... 46
Internalization of radioactive transferrin (125I-Tf) ... 46
General Discussion ... 47
Internalization and intracellular sorting of ErbB3... 47
Effects of PKC activity on ErbB2 and ErbB3 ... 50
Hsp90 and ErbB2 in endocytosis resistance ... 53
References ... 55
I
Abbreviations
17-AAG 17-N-allylamino-17-demethoxygeldanamycin AP(s) Accessory protein(s)
AP-2 Adaptor protein 2
Arf6 ADP-ribosylation factor 6
ARH Autosomal recessive hypercholesterolemia Cbl Casitas B-lineage lymphoma
BAR Bin-Amphiphysin-Rvs CCP Clathrin-coated pit CCV Clathrin-coated vesicle
CHIP C-terminus of Hsc70-interacting protein CHC Clathrin heavy chain
CHX Cycloheximide
CIE Clathrin- independent endocytosis CLASP(s) Clathrin Associated Sorting Protein(s) CME Clathrin- mediated endocytosis
CUL5 Cullin-5
DAB2 Disabled-2
DAG Diacylglycerol
EEA1 Early endosomal antigen 1 EGF Epidermal growth factor
EGFP Enhanced green fluorescent protein EGFR Epidermal growth factor receptor
EHD1 Eps15-homologydomain protein
ENTH Epsin N-terminal homology
Eps15 Epidermal growth factor receptor substrate 15 ER Endoplasmic reticulum
ERC Endocytic recycling compartment ERAD ER-associated degradation
ESCRT Endosomal sorting complexes required for transport FEME Fast endophilin-mediated endocytosis
GA Geldanamycin
II
GAK Cyclin G-associated kinase GPCR G-protein-coupled receptor
GPI-AP Glycosyl phosphatidylinositol-anchored proteins Grb2 Growth factor receptor-bound protein 2
HRG Heregulin
HRP Horseradish peroxidase
Hrs Hepatocyte growth factor regulated tyrosine kinase substrate
Hsc70 Heat shock cognate 70 Hsp Heat shock protein
IGF1 Insulin-like growth factor 1 IL-2R-β Interleukin-2 receptor-β-chain ILVs Intraluminal vesicles
Immuno- EM
Immuno-electron microscopy
IP Immunoprecipitation
IP3 Inositol trisphosphate
Lamp1 Lysosomal-associated membrane protein 1 MAPK Mitogen-activated protein kinase
mRNA Messenger RNA
mTORC2 Mammalian target of rapamycin complex 2 MVBs Multivesicular bodies
Nedd4 Neural precursor cell-expressed developmentally downregulated protein 4
Nrdp1 Neuregulin receptor degradation pathway protein 1 PAE Porcine Aortic Endothelial
PDK1 Phosphoinositide-dependent protein kinase 1 PI3K Phosphatidylinositol 3-kinase
PI3P Phosphatidylinositol-3-phosphate PIP2 Phosphatidylinositol 4,5-bisphosphate PIP3 Phosphatidylinositol 3,4,5-trisphosphate PKC Protein kinase C
PLCγ Phospholipase C-γ
III
PMA Phorbol 12-myristate 13-acetate PS Phosphatidylserine
Rab Ras-associated binding
RNAi RNA interference
RTK Receptor tyrosine kinase
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SH2 Src homology 2
siRNA Short interfering RNA Sos Son of sevenless
STAT Signal transducer and activator of transcription
Tf Transferrin
TfR Transferrin receptor
TGFα Transforming growth factor alpha TGN Trans-Golgi network
Tsg101 Tumor susceptibility gene 101 UIM Ubiquitin interacting motif
IV
Papers Included
Paper I
Szymanska, M., Fosdahl, A.M., Raiborg, C., Dietrich, M., Liestøl, K., Stang, E. and Bertelsen, V.
Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3.
Biochim Biophys Acta. 2016 Volume 1863, Issue 6, Part A, June 2016, Pages 1179-1188.
doi:10.1016/j.bbamcr.2016.03.011
Paper II
Fosdahl, A.M., Dietrich, M., Schink, K.O., Malik, M.S., Skeie, M., Bertelsen, V. and Stang, E.
ErbB3 interacts with Hrs and is sorted to lysosomes for degradation
Biochim Biophys Acta. 2017 Volume 1864, Issue 12, December 2017, Pages 2241-2252.
doi.org/10.1016/j.bbamcr.2017.08.011
Paper III
Dietrich, M., Bertelsen V. and Stang, E.
Protein kinase C regulates ErbB3 turnover
Manuscript
Paper IV
Dietrich, M., Malik, M.S., Nikolaysen, F. and Stang, E.
Protein kinase C induced internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation
Manuscript
1
Introduction
Cellular signaling is one of the basic molecular mechanisms of life. It helps cells to receive and transmit signals that control fundamental processes such as proliferation, migration and survival. Plasma membrane expressed receptors function in this context as transmitters of extracellular signals in the form of ligands to the intracellular environment. Among the variety of receptor families the epidermal growth factor receptor (EGFR) or ErbB family of receptor tyrosine kinases (RTKs) plays a very important role.
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Receptor Tyrosine Kinase (RTK)
Receptor tyrosine kinases are primary mediators of information from the extracellular environment into the cells. The family of RTKs consists of 58 members (Roskoski 2014).
RTKs are involved in various cellular processes, such as proliferation and differentiation, cell survival and metabolism, as well as cell migration and cell- cycle control (Ullrich and Schlessinger 1990, Blume-Jensen and Hunter 2001).
Mutations in RTKs and the resulting aberrant activation of intracellular signaling pathways are often linked to the development of among others cancer, diabetes, inflammation and arteriosclerosis.
RTKs function as cell surface allosteric enzymes and their basic structure consists of a single transmembrane domain that connects the extracellular ligand-binding domain with the intracellular kinase domain. In most cases activation occurs after ligand-binding followed by homo- or heterodimerization.
However, a subset of RTKs dimerizes also in the absence of ligand like the insulin receptor and insulin-like growth factor 1 (IGF1) receptor (Ward, Lawrence et al.
2007). Activation of RTKs leads to recruitment of target proteins to the intracellular domain that in turn initiates complex signaling cascades.
The ErbB protein family
The EGFR or ErbB protein family consists of the four members EGFR (also known as ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 and ErbB4. This family of receptors is ubiquitously expressed in epithelial, mesenchymal and neuronal cells. The EGFR family is involved in multiple complex and tightly controlled signaling pathways involved in various cellular functions such as cell proliferation and organ development (Casalini, Iorio et al. 2004, Tebbutt, Pedersen et al. 2013).
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Structure and function
The receptors in the EGFR family are composed of an extracellular domain, a single hydrophobic transmembrane segment and an intracellular protein kinase domain (Roskoski 2014). The ligand-binding extracellular domain consists of four subdomains. Domain I and III participate in ligand binding while the cysteine rich domains II and IV facilitate disulfide bond formation and especially domain II, which often is referred to as the dimerization arm, is involved in homo- and heterodimer formation (Roskoski 2014) (Figure 1). The transmembrane segment consists of 19-25 amino acids and spans the plasma membrane to connect extra- and intracellular domain. The intracellular domain is responsible for interactions with intracellular signaling molecules. It consists of a juxtamembrane segment, a protein kinase domain and a carboxyterminal tail. The four ErbB family members share approximately 40-45% sequence homology but have become functionally specialized (Stein and Staros 2000).
Except for ErbB2 the receptors exist in a tethered conformation in the absence of ligand. The β-hairpin loop of domain II interacts with domain IV which sequesters the dimerization arm. The dimerization domain is therefore, not available for interaction with other ErbB family members (Baselga and Swain 2009) (Figure 1). Binding of ligand to the ectodomain at domain I and III leads to a conformational change. The receptor adopts an open conformation in which the dimerization loop gets exposed and therefore, allows for interaction of receptor ectodomains (Figure 1). The crystal structure for ErbB2 revealed that the receptor is locked in the open conformation poised to constantly interact with other receptors (Garrett, McKern et al. 2003). This conformation also explains the inability to identify a high-affinity ligand for ErbB2 as the potential ligand-binding domains I and III are fixed in close proximity.
Ligand binding to the extracellular domain of ErbB proteins and dimerization subsequently induces a conformational change in the intracellular tyrosine kinase domain leading to autophosphorylation of the receptor. For activation of the tyrosine kinase the N-lobe of one tyrosine kinase domain interacts with the C-lobe of its dimerization partner (Zhang, Gureasko et al. 2006) (Figure 1).
4
Different expression patterns of the different receptors and ligands reflect cell type specific involvement of the receptors in growth mediation and differentiation (Olayioye, Neve et al. 2000).
Figure 1. The ErbB protein family and their ligands. There are four members of the ErbB family. The receptors share a similar structure and are composed of an extracellular domain, a transmembrane α-helix and an intracellular tyrosine kinase domain. The extracellular sub- domains I and III are important for ligand-binding, while sub-domains II and IV are facilitating dimerization. ErbB2 has no known soluble ligand but exists in a constitutively open conformation. The other ErbB family members exist in a closed conformation in the absence of ligands. ErbB3 has only a marginal kinase activity compared to the other ErbB receptors.
Abbreviations: epidermal growth factor (EGF), transforming growth factor alpha (TGFα), amphiregulin (AR), epigen (EPG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EPR), heregulin (HRG). Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer (Baselga and Swain 2009). The figure and the figure legend are modified.
ErbB ligands
Activating ligands of the ErbB family are produced from transmembrane precursors and have an EGF-like domain for high affinity binding. Expression and processing of the precursors is strictly regulated to control activation of the receptors (reviewed in Yarden and Sliwkowski 2001). Specifically to mention in this context is ErbB2 which so far has no known ligand but due to its open conformation serves as a preferred dimerization partner for the other ErbB
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proteins. Binding of ligands to the extracellular domain of EGFR, ErbB3 or ErbB4 induces the formation of kinase active receptor dimers.
Seven different ligands bind to and activate mammalian EGFR. The ligands can be classified into high-affinity ligands including epidermal growth factor (EGF), transforming growth factor-alpha (TGFα), heparin-binding EGF-like growth factor and betacellulin and low-affinity ligands such as amphiregulin, epiregulin and epigen (Singh, Carpenter et al. 2016). All EGFR ligands are initially produced as transmembrane precursor proteins that have to undergo extracellular domain cleavage to release soluble ligands. These soluble ligands can then bind and activate EGFR.
Heregulins, or also called neuregulins, are a large family of EGF-like signaling molecules that trigger activation of ErbB3 and ErbB4. They are involved in cell- cell communication during development but also in adults. Heregulins are ErbB- specific ligands which all share an EGF-like motif of 45-55 amino acids including six cysteine residues that covalently interact and form three loops. The EGF domain alone is sufficient for activation of ErbB receptor tyrosine kinases. The heregulin family comprises four family members HRG-1, HRG-2, HRG-3 and HRG-4 (Holmes, Sliwkowski et al. 1992, Carraway, Weber et al. 1997, Zhang, Sliwkowski et al. 1997, Harari, Tzahar et al. 1999). Nevertheless, the variety of ligands is much bigger due to alternative splicing and thereby, creation of at least 26 different isoforms in different species (Breuleux 2007). While heregulin isoforms encoded by the genes HRG-1 and HRG-2 activate both ErbB3 and ErbB4, isoforms encoded by HRG-3 and HRG-4 are exclusively inducing activation of ErbB4. For this study of most interest is the splice variant Type I HRG-1 hereafter generally referred to as heregulin (Hrg) as it is the main activating ligand for ErbB3.
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Phosphorylation and ubiquitination
In general, activation of receptor tyrosine kinases including the ErbB family happens via activating ligands or growth factor binding to the ectodomains of two receptors and subsequently inducing an activated dimerization state (Lemmon and Schlessinger 2010). The induced post translational modifications are mainly phosphorylation and ubiquitination. The kinase domains catalyze phosphorylation of various tyrosine residues that function as docking sites for adaptor proteins or enzymes necessary for downstream signaling. The phosphorylation happens in trans, meaning the first member of the dimer phosphorylates the second member and vice versa.
Ubiquitin is a small regulatory protein and ubiquitination represents a universal and major way to affect stability, interaction with other proteins, enzymatic activity and subcellular localization of target proteins (Acconcia, Sigismund et al.
2009). Ubiquitination is facilitated by a sequential activation cascade mediated by three classes of enzymes. Ubiquitin- activating enzymes, also known as E1 enzymes, bind and activate ubiquitin and pass it on to the ubiquitin-conjugating enzymes (E2 enzymes). These enzymes conjugate the activated ubiquitin with ubiquitin ligases (E3 enzymes) that facilitate the covalent attachment of ubiqutin to the ε-amino group of a lysine residue of the target protein via an isopeptide bond (Acconcia, Sigismund et al. 2009). While there are only two E1 enzymes encoded in the human genome, there are more than 40 E2 enzymes. The number of E3 enzymes is constantly increasing and outnumbering the other two enzyme classes (Clague, Heride et al. 2015). The variety of ubiquitin-conjugating and ubiquitin-activating enzymes gives rise to a variety of ubiquitination reactions. A substrate can become both mono- and polyubiquitinated. Ubiquitin does in itself contain seven lysine residues and a C-terminal methionine residue which all can be ubiquitinated Polyubiquitination occurs by linking several ubiquitin molecules together in a chain on the same substrate protein. The function of the polyubiquitin chain depends on the lysine where it becomes linked. While linkage of ubiquitin via lysine 48 is a classical signal for proteasome-mediated degradation, linkage of ubiquitin via lysine 63 is often associated with lysosomal degradation (reviewed in Haglund and Dikic 2012).
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ErbB Activation and Signaling
Most ErbB receptors resemble a simple growth factor receptor signaling pathway characterized by binding of ligand to a monomeric receptor which promotes receptor dimerization, activation of the cytoplasmic catalytic domain and auto-phosphorylation of tyrosine residues. Nevertheless, in higher eukaryotes the simple signaling pathway has evolved to a multilayer signaling network with combinatorial expression of receptors. Furthermore, it involves a range of second messengers, protein-protein interactions, protein-lipid interactions and post translational modifications.
Activation of ErbB receptors by its ligands can induce homo- or heterodimerization (Figure 2). Monomeric receptors that do not have bound ligand are under physiological conditions not capable of signaling. The ErbB proteins including splice variants of ErbB4 can form 28 different homo- and heterodimers. ErbB2 has no identified ligand and, even though there have been reports on homodimerization when ErbB2 is overexpressed (Hu, Sun et al.
2015), it most likely has to rely on heterodimerization with other ErbB proteins for activation and downstream signaling. Likewise does ErbB3, which has a very weak tyrosine kinase activity, to a high degree depend on heterodimerization for full activation and phosphorylation.
Dimerization induces transphosphorylation of the dimer and subsequently activation of a variety of intracellular pathways such as the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the phospholipase C-γ (PLCγ) pathway and the signal transducer and activator of transcription (STAT) pathway (reviewed in Yarden and Pines 2012).
As MAPK and PI3K pathways are the major pathways activated downstream of ErbB proteins they will be described in more detail.
Tyrosine phosphorylation of EGFR and other ErbBs create binding sites for growth factor receptor-bound protein 2 (Grb2) and Src homology 2 (SH2). This leads to recruitment of son of sevenless (Sos), which activates the MAPK pathway consisting of Ras, Raf, MEK and MAPK/ERK before induction of transcription of target proteins.
With respect to the PI3K pathway ErbB3 plays a major role. Even though ErbB2 can potently activate MAPK/ERK signaling it needs ErbB3 as dimerization
8
partner for binding of the p85 regulatory subunit of PI3K. ErbB2/ErbB3 heterodimers signal predominantly via the PI3K/Akt pathway. The regulatory subunit (p85) of PI3K binds to a wide variety of phosphotyrosines in ErbB3 and lead in turn to the activation of PI3K (Roskoski 2014). PI3K further catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphat- idylinositol 3,4,5-trisphosphate (PIP3). This conversion attracts the protein- serine/threonine kinase Akt which further gets phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2). Biphosphorylated Akt catalyzes the phosphorylation and activation of mTOR and from there a variety of further downstream pathways get activated (Engelman 2009, Vanhaesebroeck, Stephens et al. 2012).
In addition, ligand-independent transactivation of ErbB proteins can add another layer of signaling complexity as it has been shown that the receptors and downstream pathways can be recruited by G-protein, Wnt, integrin and other growth factor pathways.
Figure 2. Activation of the ErbB proteins. Ligand binding to the extracellular domain of the receptor induces conformational changes leading to exposure of sub-domain II, which functions as dimerization arm. Subsequently, the receptors dimerize, the tyrosine kinase domain becomes activated and tyrosine residues in the C-terminal tail become phosphorylated. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer (Baselga and Swain 2009).
The figure and the figure legend are modified.
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Maturation and trafficking of ErbB proteins
Generally, after translation ErbB proteins translocate through the endoplasmic reticulum membrane to the Golgi apparatus where post translational modifications like glycosylation of the extracellular domain occur. From there the receptors are delivered to the plasma membrane (Sorkin and Goh 2009). All ErbB receptors are synthesized and routed to specific cellular locations in the absence of ligand. The distribution can be influenced with respect to internal and external stimuli like cell polarization.
The ErbB family members have a ligand-independent turnover that strongly varies between the receptors and also partly correlates to the expression level.
While the turnover of EGFR and ErbB2 is in the range of t1/2 6-10h (Beguinot, Lyall et al. 1984), the ErbB3 turnover rate was 2.4h (Cao, Wu et al. 2007). Thus, an average receptor may potentially cycle through the endocytic pathway dozens of times during its life span, without being targeted for degradation (Wiley 2003).
Endocytosis
Endocytosis is a basic cellular process used by cells for the internalization of a variety of molecules. Signaling receptors undergo endocytosis to downregulate them from the plasma membrane. This process limits the sensitivity of a cell to receptor-activating ligands. Once internalized the receptor can be recycled back to the cell surface or be targeted for degradation. In general there is a distinction between clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) (Figure 3).
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Figure 3. Endocytic pathways. The variety of endocytic activities executed by cells includes phagocytosis, for uptake of large particles, macropinocytosis, for larger uptake of fluid and plasma membrane, and a variety of pinocytic mechanisms. Pinocytosis is characterized by uptake of smaller particles via invagination of the plasma membrane leading to the formation of vesicles. The various pinocytic mechanisms are classified by their cellular requirements for dynamin and actin. Cargo uptake can be clathrin- and dynamin-dependent, clathrin- and/or dynamin-independent, caveolin- and dynamin-dependent or independent of clathrin, caveolin and dynamin. The different endocytic uptake mechanisms result in the formation of intracellular vesicles that will fuse with early endosomes. Abbreviations: GEEC, glycosyl phosphatidylinositol-anchored protein enriched early endosomal compartments; CLIC, clathrin- independent carrier. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology (Mayor and Pagano 2007). The figure legend is modified.
Clathrin-mediated endocytosis
Clathrin-mediated endocytosis describes the mechanism by which the proteins involved in the process recruit their cargo into clathrin-coated pits (CCPs) and subsequently form clathrin-coated vesicles (CCVs). CME serves a range of different functions including the regulation of surface expression of proteins;
sampling the cells environment for growth and guidance cues; nutrient uptake into the cell; control of signaling pathway activation and turnover of membrane compartments by targeting them for degradation. CME is used by all known eukaryotic cells. Clathrin coats also form on endosomes and clathrin-coated vesicles are formed at the trans-Golgi network (TGN). The term clathrin- mediated endocytosis is, however, used to refer to vesicles formed from the plasma membrane. The most common implicated cargoes for CME are receptor tyrosine kinases, G-protein-coupled receptors (GPCRs), the transferrin receptor
11
(TfR) and the anthrax toxin (McMahon and Boucrot 2011). This chapter will focus on the endocytosis of plasma membrane localized receptors. The endocytosis of cargo receptors can be stimulated by ligand binding (for example, EGFR), but it has also been shown that some receptors are constitutively internalized such as the TfR (Hopkins, Miller et al. 1985) and ErbB3 (Sak, Breen et al. 2012). The CCV cycle consists of five steps: nucleation, cargo selection, coat assembly, scission and uncoating (Figure 4). After uncoating, the vesicles fuse with endosomes where cargo is sorted and either recycled back to the plasma membrane or targeted to more mature endosomes and later compartments such as multivesicular bodies (MVBs) and lysosomes (Grant and Donaldson 2009).
The main and name-giving player in CME is clathrin. It was purified and identified already in 1975 by Barbara Pearse, who named it clathrin (from the Latin clatratus, meaning “like a lattice”) (Pearse 1975, Pearse 1976). It forms a triskelion structure consisting of three clathrin heavy chains (CHCs) and three clathrin light chains. Interaction of the triskelia leads to formation of a polyhedral lattice that coats the forming vesicle. As clathrin does not bind directly to the plasma membrane or cargo receptors, the connection occurs via adaptor and accessory proteins such as adaptor protein 2 (AP-2) which will be closer described in the next chapter (Kelly, McCoy et al. 2008).
A wide range of plasma membrane accessory adaptor proteins has been identified that can be cell type as well as cargo specific and bind to different receptors (reviewed in Traub 2009). When cargo is finally selected and bound by AP-2 or other cargo-specific adaptor proteins the clathrin coat needs to be assembled. Soluble clathrin triskelia, which are directly recruited from the cytosol, start polymerizing into hexagons and pentagons. Clathrin is nucleating at sites of the plasma membrane that are destined for internalization and often characterized by an accumulation of adaptor proteins. The nucleation promotes the further polymerization of clathrin and the creation of curved lattices. The subsequent displacement of cargo accessory adaptor proteins and curvature effectors, such as epidermal growth factor receptor substrate 15 (Eps15), promotes the curvature formation of the plasma membrane (Tebar, Sorkina et al.
1996, Saffarian, Cocucci et al. 2009)(Figure 4). This consequently stabilizes the deformation of the attached membrane and leads into the formation and
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constriction of a vesicle neck. Dynamin is a GTPase that has been shown to be responsible for the pinching off of the CCP from the plasma membrane (Chen, Obar et al. 1991, van der Bliek and Meyerowitz 1991). Dynamin is recruited by Bin-Amphiphysin-Rvs (BAR) domain containing proteins such as amphiphysin or endophilin, that have a preference to accumulate at the curvature of the vesicle neck (Wigge, Kohler et al. 1997, Ferguson, Raimondi et al. 2009, Sundborger, Soderblom et al. 2011). After formation of the CCP, dynamin assembles into ring- like structures (Hinshaw and Schmid 1995) around the constricted neck of deeply invaginated CCPs and upon hydrolysis of GTP it mediates the fission of the vesicle from the plasma membrane (Praefcke and McMahon 2004) (Figure 4). Nevertheless, the exact mechanism of the scission is not yet fully understood, but it is known that the protein undergoes a GTP hydrolysis- dependent conformational change which is thought to support the scission (Sweitzer and Hinshaw 1998, Roux, Uyhazi et al. 2006). After detachment from the plasma membrane and formation of the CCV, the clathrin coat is removed from the vesicle with the help of the ATPase heat shock cognate 70 (hsc70) and its cofactor auxilin or cyclin G-associated kinase (GAK) (Schlossman, Schmid et al. 1984, Ungewickell, Ungewickell et al. 1995) (Figure 4). The uncoated vesicle can travel to and fuse with target endosomes while the released clathrin machinery can be reused for another cycle of CCP formation.
Figure 4. Formation of clathrin-coated vesicles (CCV). The formation of CCVs is a multi-step process. The nucleation is mediated by clathrin-associated sorting proteins (CLASPs) and association of clathrin. The clathrin coated pit (CCP) begins to mature, more adaptor and scaffolding proteins are recruited and the clathrin coat gets assembled. The GTPase dynamin is recruited to the neck of the forming vesicle and mediates the scission of the vesicle. The formed vesicle is released into the cytoplasm and rapidly uncoated. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology (McMahon and Boucrot 2011).
The figure and the figure legend are modified.
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Adaptors of clathrin-mediated endocytosis
A wide range of adaptors necessary for the formation of clathrin-coated pits and subsequently vesicles at the plasma membrane have been identified. Endocytic adaptors are divided in two main groups: multimeric adaptor proteins and monomeric non-classic adaptor proteins also known as clathrin-associated sorting proteins (CLASPs), which recognize certain motifs of target proteins (Table 1). Nevertheless, it is worth mentioning that not all the adaptors are always used or required for CME, their requirement may differ between cell types and/or different cargoes. Cargo likely plays a driving role in CCP formation as it is expected that empty CCVs will not have a useful function. A group of researchers around David Drubin (University of California, Berkeley) suggested that the wide range of more than 40 proteins involved in CME can be grouped into four different functional modules that mediate coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission (Kaksonen, Toret et al. 2005). The following paragraphs will take a closer look on some of the key adaptors relevant for this study.
Table 1. Endocytic signals and adaptors
Signals or domains Adaptors Adaptor subunits or domains
YXXØ AP-2 µ2
[DE]XXXL[LI] AP-2 α-σ2
[YF]XNPX[YF] ARH; Dab2; Idol; SNX17, 27, and 31
PTB domain
NPFX(1,2)D Sla1p SLA1 homology domain
Ubiquitin Eps15, Epsin 1 and 2 UIM domain
GPCR phosphorylation β-arrestin 1 and 2 Amino terminus Synaptotagmin I C2A (C2B) domain Stonin 2 μHD domain
Mid2p cytosolic domain Syp1p μHD domain
Alk8 cytosolic domain Fcho1 μHD domain
VAMP 7 longin domain Hrb, AP-3 C-terminal unstructured domain VAMP 2, VAMP 3, VAMP 8
SNARE motifs
CALM ANTH domain
Abbreviations: ARH, autosomal recessive hypercholesterolemia; PTB, phosphotyrosine-binding;
UIM, ubiquitin-interacting motifs; GPCR, G-protein-coupled receptor; CALM, clathrin assembly lymphoid myeloid leukemia. Adapted with permissionCold Spring Harbor Laboratory Press: CSH Perspectives in Biology (Traub and Bonifacino 2013).
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“Assembly” or “accessory” proteins (APs) were isolated and identified (Zaremba and Keen 1983, Pearse and Robinson 1984, Ahle and Ungewickell 1986) as the connecting partner between the clathrin coat and the plasma membrane, where they act as adaptors (Vigers, Crowther et al. 1986). Among the first identified adaptors was the heterotetrameric AP-2 complex (Pearse and Robinson 1984). It was initially believed to be always involved in CME but it has been later shown that other adaptors can compensate the AP-2-mediated processes in CME. The complex consists of two large “adaptin” subunits (α and β2), one medium sized subunit (µ2), and one small subunit (σ2). For AP-2 the internalization sequences that are recognized by different protein subunits have been identified (reviewed in Traub and Bonifacino 2013). A simple tyrosine motif (YXXØ) present in cytoplasmic tails of receptors such as the TfR is recognized by the AP-2 µ2- subunit (Ohno, Stewart et al. 1995) and the acidic dileucine [DE]XXXL[LIM]
sequence is recognized by the α-σ2 hemicomplex (Kelly, McCoy et al. 2008).
Furthermore, AP-2 binds to the plasma membrane-specific lipid PIP2 via the α- subunit (Honing, Ricotta et al. 2005). When AP-2 gets recruited to the plasma membrane with the help of the large subunits (α and β2), it undergoes a conformational change that enables the protein to simultaneously bind to cargo- sorting signals and clathrin (Kelly, Graham et al. 2014). The interaction with the cytoplasmic tail of transmembrane receptors occurs directly via the µ-subunit and σ-subunit, and indirectly via cargo using its appendage domains to bind AP-2 (Collins, McCoy et al. 2002, Kelly, McCoy et al. 2008). Since AP-2 is not only binding clathrin but also most of the accessory proteins, it is considered to be the major hub of interactions in the maturing CCP.
The CLASPs are mono- and dimeric and vary in structure and binding properties.
Cargo-specific adaptors recognize a single transmembrane receptor or a small family of receptors. Known adaptors are Numb, autosomal recessive hypercholesterolemia (ARH) or disabled-2 (DAB2). These proteins contain an N- terminal phosphotyrosine binding domain that recognizes [YF]XNPX[YF] cargo- sorting motifs (Uhlik, Temple et al. 2005). DAB2 is a known adaptor involved in CME of megalin (Gallagher, Oleinikov et al. 2004). Arrestins are crucial in trafficking of signaling receptors. They regulate the inactivation, internalization and signaling of GPCRs (reviewed in Gurevich and Gurevich 2006). β-arrestins
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help control the strength and duration of GPCR signaling by binding to phosphorylated GPCRs. This attenuates the signaling and blocks the receptor interaction with G-proteins (Lohse, Benovic et al. 1990). Furthermore, β- arrestins promote GPCR endocytosis by interaction with clathrin and other adaptor proteins (Goodman, Krupnick et al. 1996). There are also a few proteins that bind cargo but do not contain any clathrin-binding motif. These proteins include Eps15 that selects ubiquitinated cargo and stonins that bind to and sort cargos of the synaptic vesicle. Stonins bind AP-2 with their N-terminal domain, while the C-terminal domain is essential for selecting cargos of the synaptic vesicle.
For this study of greatest interest is the epsin family of adaptor proteins. The epsin family is a conserved family of endocytic adaptor proteins. They are essential for embryonic development in higher eukaryotes. Epsins recognize ubiquitinated cargo and contribute to membrane bending. Three members of the epsin family epsin-1, -2 and -3 have been identified as plasma membrane localized. The structure of epsin proteins is comprised of an approximately 150 amino acid long epsin N-terminal homology (ENTH) domain, which is responsible for binding to PIP2, a lipid enriched at endocytic sites in the plasma membrane (Itoh, Koshiba et al. 2001). In addition, the ENTH domain promotes membrane curvature (Ford, Mills et al. 2002). The ENTH domain is followed by ubiquitin interacting motifs (UIMs). Most epsins contain two to three UIM copies for mediating interaction with ubiquitinated cargo (Polo, Sigismund et al. 2002, Shih, Katzmann et al. 2002). The region between the UIMs and the C-terminal domain displays high divergence between species but contain some conserved motifs for binding of various components of the endocytic machinery such as clathrin and AP-2. Epsin-1 has also been suggested to be involved in CIE of EGFR when the receptor is stimulated with high concentrations of EGF (Sigismund, Woelk et al. 2005).
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Clathrin-independent endocytosis
Several endocytic pathways do not utilize clathrin for the internalization of cargo into the cell. Some of these pathways occur constitutively while others are triggered by specific signals or even hijacked by pathogens. The main differences in the clathrin-independent pathways of endocytosis are in their mechanisms and kinetics of vesicle formation, the molecular machinery involved and the final destination of the incorporated cargo. There is a variety of different pathways characterized of which some of them will be shortly explained below.
Macropinocytosis
Macropinocytosis does not appear to have specific cargoes or markers and is therefore only characterized by morphology. Large ruffles or blebs of plasma membrane are forming initially before collapsing back onto the plasma membrane. Thereby large irregular shaped vacuoles get formed, called macropinosomes. This leads to a nonselective engulfment of membrane portions containing, among others, activated receptors. Furthermore, this pathway can facilitate a nonspecific uptake of fluids and solutes. Macropinocytosis can be triggered by overstimulation by stimuli such as growth factors, phosphatidylserine (PS)-containing apoptotic cell remnants, viruses and bacteria (Kerr and Teasdale 2009). Due to the unspecific uptake of membrane molecules the sorting of the receptors happens after uptake. Inactive receptors are usually recycled back to the plasma membrane while activated receptors are sorted by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation.
Caveolin-dependent endocytosis
Endocytosis mediated by caveolae is an endocytic pathway for uptake of small molecules and belongs to the best studied CIE. Caveolae are cholesterol- and sphingolipid-rich (Simons and Ikonen 1997), 50-80nm flask-shaped invaginations of the plasma membrane whose expression is associated with a member of the caveolin protein family (reviewed in Nabi and Le 2003). Another
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important assembly partner is glycosyl phosphatidylinositol-anchored proteins (GPI-APs) (Aboulaich, Vainonen et al. 2004). The main molecules taken up via this pathway include sphingolipids and sphingolipid binding toxins such as cholera and shiga toxin, endothelin, growth hormones and bacteria (Duncan, Shin et al. 2002, Pelkmans and Helenius 2002). Caveolae-dependent endocytosis is characterized by sensitivity to dynamin inhibition and cholesterol depletion (Henley, Krueger et al. 1998). The internalization of caveolae is facilitated by disruption of the actin cytoskeleton (reviewed in Nabi and Le 2003) and caveolin-1 functions as a stabilizer of the caveolae. The budding of the caveolae occurs in a dynamin-dependent manner and the forming caveolar vesicles are then further transported to their intracellular destination.
Clathrin- and caveolin-independent pathways
Among the variety of clathrin- and caveolin-independent pathways the most common ones are regulated by small GTPases that regulate internalization among them RhoA, CDC42 and ADP-ribosylation factor 6 (Arf6).
RhoA-dependent endocytosis is best studied for the internalization of the β-chain of the interleukin-2 receptor (IL-2R-β). After ligand binding IL-2R-β sorts into detergent-resistant membranes, which are frequently found to be involved in CIE. RhoA and dynamin inhibition potently inhibits this endocytic route (Lamaze, Dujeancourt et al. 2001).
CDC42-mediated endocytosis facilitates the main clathrin-independent and caveolin-independent endocytic route for the uptake of cholera toxin B and the plant protein ricin (Llorente, Rapak et al. 1998, Lajoie, Kojic et al. 2009). The invaginations of the plasma membrane in CDC42-regulated endocytosis are longer and have a relatively wide surface in contrast to the clathrin- and caveolin-regulated pathways (Kirkham, Fujita et al. 2005). This leads to a co- internalization of large volumes of fluid phases.
In addition, a role for the GTPase Arf6 has been suggested but it is still unclear whether this can be considered an own pathway or just contributing to other internalization pathways by actin remodeling. Furthermore, most of the effects
18
of Arf6 appeared to be cell type specific (Naslavsky, Weigert et al. 2004, Kalia, Kumari et al. 2006).
Some CIE events might be too fast to be recorded by classical methods to study endocytosis. However, in recent years there have been major advances revealing fast and ultrafast clathrin-independent endocytic pathways. One of the recently discovered pathways is fast endophilin-mediated endocytosis (FEME) (Boucrot, Ferreira et al. 2015). This pathway is mediated by endophilin and induces a rapid formation of endocytic vesicles upon certain stimuli. It is triggered by receptor activation and specific to certain receptors. It has so far been identified for some GPCRs, interleukin-2 receptor and certain receptor tyrosine kinases (Boucrot, Ferreira et al. 2015).
Endosomal sorting
After internalization of molecules through an endocytic pathway, they are sorted and traffic through a variety of tubulovesicular compartments, collectively termed endosomes (Elkin, Lakoduk et al. 2016). The endosomal network is a dynamic and interconnected system that allows trafficking and transfer of cargoes between compartments in the cell. After entering the cell the endocytic vesicles undergo several cycles of homotypic fusion to finally form early endosomes (Salzman and Maxfield 1988). Within early endosomes the fate of the internalized cargo gets determined by the sorting into different endosomes. A key protein family involved in most membrane transport processes is the Ras- associated binding (Rab) protein family. It consists of more than 60 small GTPases and they exhibit various functions in the endosomal system. Rab proteins are involved in cargo budding, selection, coating, vesicle transport, vesicle uncoating, tethering and vesicle fusion (reviewed in Seabra, Mules et al.
2002). As these processes are facilitated by different Rab family members, Rab proteins are also commonly used as microscopic markers for identification of the different intracellular sorting compartments. The initial cargo sorting station is the early endosome. Cargo can be routed from early endosomes to late endosomes and lysosomes for degradation, to recycling endosomal carriers that transport cargo directly back to the plasma membrane, to the endocytic recycling
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compartment (ERC), or to the TGN (Figure 5). Early endosomes receive the incoming material from primary vesicles that are internalized from the plasma membrane. Rab5, early endosome antigen (EEA1) and phosphatidylinositol-3- phosphate (PI3P) mark the early endosome and are important regulators for early endosomes. The pH in the early endosomes gradually decreases during fusion events and as a consequence of the low pH in the lumen of the endosome, many ligands are released from their receptors (Maxfield and McGraw 2004).
This step can already determine the further sorting of the internalized proteins for recycling or degradation.
Figure 5. Endosomal compart- ments. After release of the CCP in the cytoplasm it fuses with early endosomes, where internalized cargo is either sorted back to the plasma membrane in recycling endosomes or further sorted into ILV of MVBs.
Cargo can also undergo retrograde transport from endosomes to the Golgi and ER. MVBs may fuse with or mature to lysosomes where cargo may be degraded. MVBs can be released into the extracellular space by fusion of the MVBs with the plasma membrane as exosomes.
Abbreviations: CCP, clathrin coated pit; ILV, intraluminal vesicle; MVB, multivesicular body; ER, endoplasmic reticulum. Adapted by permission from Macmillan Publishers Ltd:
Nature Reviews Drug Discovery (Rajendran, Knolker et al. 2010). The figure legend is modified.
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Endocytic recycling
Endosomal recycling pathways balance the removal of internalized extracellular material by returning a large part of internalized proteins and lipids back to the plasma membrane. This helps in maintaining the composition of the plasma membrane. The recycling pathways are generally classified in a rapid recycling route and a slow recycling route. The rapid recycling route that recycle proteins directly back to the plasma membrane from early endosomes. Even an earlier stage has been documented for the TfR (Mayor, Presley et al. 1993) and glycosphingolipids (Choudhury, Sharma et al. 2004). The two Rab proteins Rab4 and Rab35 have been identified as key components in mediating the rapid recycling.
The slow recycling route requires transport of cargo from the early endosome to the ERC and from there back to the plasma membrane. In many tissues the ERC, is located in close proximity to the Golgi complex and it is molecularly defined by the presence of Rab11 and/or Eps15-homology-domain protein (EHD1). It evolves from extended tubules of early endosomes that loose Rab5 and acquire Rab11 (Sonnichsen, De Renzis et al. 2000). From the ERC internalized proteins can be sorted for transport back to the plasma membrane or to the TGN. An example for the latter is sorting of the protein TGN38 which is mainly expressed in the TGN but a small percentage is also found on the plasma membrane.
Membrane downregulated TGN38 can be sorted back to the TGN via the ERC (Ghosh, Mallet et al. 1998). The transport from the ERC back to the plasma membrane can probably occur by several distinct mechanisms. These include mechanisms that are dependent on Arf6, Rab11, Rab 22a or Rab8a (reviewed in Grant and Donaldson 2009). The transport from the ERC to the plasma membrane is mediated by recycling carriers that are released from the ERC and that finally fuse with the plasma membrane.
Another mechanism of recycling is the formation of exosomes. Exosomes are small vesicles which are released from cells when specialized MVBs fuse back with the plasma membrane and release their internal vesicles and thus their endosomal packed content into the extracellular space. This process is mediated by Rab27a, the ESCRT-III complex and syndecans (Baietti, Zhang et al. 2012).
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Endocytic degradation
The endosomal compartments undergo a maturation process, from so called early to late endosomes/lysosomes, in which the luminal pH decreases, key phosphatidylinositol lipids get altered and different Rab-family GTPases get recruited (Elkin, Lakoduk et al. 2016). Late endosomes/lysosomes are characterized by certain markers such as lysosomal-associated membrane protein 1 (Lamp1) and CD63. Early endosomal sorting depends initially on acidification. The acidification occurs with the help of a v-type vacuolar H+ ATPase, which pumps hydrogen ions into the endosomal lumen and decreases the pH (Mellman, Fuchs et al. 1986, Fuchs, Schmid et al. 1989). Maturing early endosomes can accumulate intraluminal vesicles (ILVs) in their vacuolar portions. These ILVs increase as the endosomes mature and finally develop into late endosomes/MVBs.
One of the most common sorting signals for downregulation and degradation of surface proteins is ubiquitin which gets attached to the cytoplasmic domain of the protein. The recognition of ubiquitin and sorting of cargo into ILVs is mediated by the ESCRT machinery. This machinery is composed of four different protein complexes called ESCRTs -0, -I, -II, -III. The ESCRT complexes exist as cytoplasmic complexes which get recruited to the limiting membrane of endosomes during the formation of MVBs. The different ESCRT subunits can recognize a variety of cellular components including ubiquitinated cargo, coat proteins such as clathrin and endosomal lipids including PI3P (Raiborg and Stenmark 2009). These complexes retain ubiquitinated cargo in the membrane of early endosomes and hinder receptor recycling (Raiborg, Bache et al. 2002).
The ESCRT-0 complex consists of the subunits hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) and STAM. These proteins interact preferably with polyubiquitin chains of cargo via their UIMs (Polo, Sigismund et al. 2002). Hrs has the ability to bind PI3P via a FYVE domain which recruits ESCRT-0 to endosomal membranes (Raiborg, Bremnes et al. 2001). Hrs also interacts with the tumor susceptibility gene 101 (Tsg101) which is part of ESCRT-I. This leads to recruitment of ESCRT-I and subsequent recruitment of further ESCRT complexes. Vps4 is responsible to disassemble the ESCRTs and complete the process of ILV biogenesis (reviewed in Henne, Stenmark et al.
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2013). After fusion of the late endosomes/MVBs with lysosomes the ILVS are exposed to and degraded by lysosomal hydrolysis.
Endocytosis and endosomal sorting of EGFR
The endocytic downregulation of EGFR is the best studied endocytic pathway for the ErbB proteins. The protein half life of EGFR is normally between 6 and 10 hours but can be much longer when EGFR is overexpressed. The protein half life does however, strongly decrease when EGF binds to EGFR leading to an activated receptor that becomes rapidly internalized and degraded (reviewed in Sorkin and Goh 2009). Acceleration of internalization and degradation should prevent excessive signaling of activated receptors. Endocytosis via CME is the main pathway of activated EGFR under physiological conditions, although several examples of clathrin-independent EGFR endocytosis have been reported (Yamazaki, Zaal et al. 2002, Sigismund, Woelk et al. 2005, Orth, Krueger et al.
2006). Sigismund et al. as an example showed that increasing EGF concentrations for receptor stimulation shift the internalization of EGFR from CME to clathrin-independent pathways. Furthermore, they reported that low doses of EGF do not induce EGFR ubiquitination and that non-ubiquitinated EGFR is endocytosed via CME. Sigismund et al. also suggest that while low concentrations of EGF give rise to prolonged/sustained signaling, high concentrations of EGF lead to degradation (Sigismund, Argenzio et al. 2008).
Nevertheless, several studies demonstrated that EGFR is ubiquitinated also at low concentrations of EGF and undergoes CME also at high doses of EGF (Kazazic, Bertelsen et al. 2009, Sorkin and Goh 2009, Sousa, Lax et al. 2012, Fortian, Dionne et al. 2015). Several mechanisms and adaptor proteins that regulate ligand-induced CME of EGFR have been identified among them interaction with AP-2 and ubiquitination (Sorkin and Carpenter 1993, Goh, Huang et al. 2010). Studies have shown that ubiquitination is sufficient for internalization of EGFR, as a truncated EGFR with ubiquitin fused to the C-terminal tail (Haglund, Sigismund et al. 2003) or a chimeric EGFR with four connected ubiquitin molecules (Bertelsen, Sak et al. 2011), were constitutively internalized. The ubiquitin ligase casitas B-lineage lymphoma (Cbl) is recruited
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to activated EGFR either directly to pY 1045 or indirectly via the SH3 domain of Grb2 (Meisner, Conway et al. 1995, Huang and Sorkin 2005). Ubiquitination of EGFR via Cbl occurs at the plasma membrane. This leads to internalization and sorting of EGFR in ILVs of MVBs and therefore, the entry into the lysosomal degradative pathway (reviewed in Katzmann, Odorizzi et al. 2002). It has been shown that a decrease in ubiquitination of EGFR for example by mutation of critical tyrosine residues leads to degradation impaired receptors as they get not sorted to ILVs (Grovdal, Stang et al. 2004). It has also been shown that Cbl mutants displaying impaired activity block EGFR degradation but instead sort the receptors for recycling (Peschard and Park 2003).
The interaction with both epsin and Eps15 which are found in CCPs (Stang, Blystad et al. 2004, Kazazic, Bertelsen et al. 2009), are important for CME of ubiquitinated EGFR (Stang, Blystad et al. 2004, Haglund and Dikic 2012, Fortian, Dionne et al. 2015). Internalized EGFR gets initially sorted in early endosomes. A known sorting signal for EGFR is ubiquitin.
Furthermore, the ligand which binds to EGFR is determining the fate of the receptor. While EGF-activated EGFR gets sorted for degradation in lysosomes, binding of TGFα leads to internalization of the receptor followed by recycling.
One reason for this is that, unlike EGF, TGFα dissociates from the receptor in the mildly acidic environment of the early endosomes, leading to dephosphorylation and deubiquitination of the receptor (Longva, Blystad et al. 2002, Roepstorff, Grandal et al. 2009).
In addition, it has been suggested that the localization of EGFR influences the signaling mediated by EGFR. While EGFR at the plasma membrane mediates proliferative signaling, it has been shown that EGFR signaling from endosomes can induce apoptosis (Burke, Schooler et al. 2001, Hyatt and Ceresa 2008, Rush, Quinalty et al. 2012).
Endocytosis and endosomal sorting of ErbB2
The behavior of ErbB2 regarding endocytic downregulation differs significantly from that of EGFR. ErbB2 is considered to be strongly endocytosis-deficient as it is not internalized. This was suggested to occur due to a lack of common
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endocytosis signals like interaction with AP-2 (Baulida, Kraus et al. 1996), or because of possible endocytosis inhibitory signals (Sorkin, Di Fiore et al. 1993).
Studies of ErbB2 localization revealed that it is strictly located to membrane protrusions at the plasma membrane, apart the newly synthesized ErbB2 which can also be found in the endoplasmic reticulum/Golgi region (Hommelgaard, Lerdrup et al. 2004). The lack of ErbB2 specific ligands makes it impossible to study ligand-induced internalization of ErbB2 homodimers, but it has been shown that heterodimerization of ErbB2 with EGFR inhibits downregulation of EGFR (Wang, Zhang et al. 1999, Haslekas, Breen et al. 2005). This supports the notion that ErbB2 is resistant to downregulation. Nevertheless, other studies suggested that EbB2 is internalized but very fast and efficiently recycled back to the plasma membrane (Harari and Yarden 2000, Austin, De Maziere et al. 2004).
The best studied mechanism to induce downregulation and subsequent endosomal sorting of ErbB2 is inhibition of heat shock protein (Hsp) 90. Hsp90 is an ATPase and a chaperone which is associated with several client proteins during maturation. However, it can remain bound to clients also after maturation as this is the case for ErbB2 and Akt. ErbB2 is thus an Hsp90 client and Hsp90 has been suggested to actively stabilize ErbB2 at the plasma membrane (Austin, De Maziere et al. 2004). Hsp90 is bound to ErbB2 on a loop within the N-lobe of the kinase domain (Tikhomirov and Carpenter 2003, Xu, Yuan et al. 2005). A recent study suggested that interaction of ErbB2 with flotillins (Pust, Klokk et al.
2013) and/or Erbin (Asp, Kvalvaag et al. 2016), either directly or through Hsp90, keeps ErbB2 at the plasma membrane and it was shown that depletion of flotillins cause internalization and degradation of ErbB2. Hsp90 inhibitors, such as Geldanamycin (GA) or 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) induce ubiquitination of ErbB2 which can serve as an internalization signal and/or signal for proteasome-mediated degradation of the C-terminus (Lerdrup, Bruun et al. 2007, Pedersen, Madshus et al. 2008) and thereby potentially cleave a region containing a plasma membrane retention signal (Tikhomirov and Carpenter 2000). Inhibition of Hsp90 with GA or 17-AAG results in interference with ATP-binding and dissociation of Hsp90 accompanied by recruitment of Hsp70 (Xu, Mimnaugh et al. 2001) and the ubiquitin ligase C-terminus of Hsc70- interacting protein (CHIP) (Xu, Marcu et al. 2002, Zhou, Fernandes et al. 2003)
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and Cullin-5 (CUL5) (Ehrlich, Wang et al. 2009, Samant, Clarke et al. 2014).
Which ErbB2 lysine residues become ubiquitinated upon Hsp90 inhibition is still unknown, but it has been shown that Hsp90 inhibition results in both K48- and K63-linked polyubiquitination of ErbB2 (Marx, Held et al. 2010, Vuong, Berger et al. 2013). Therefore, ErbB2 internalization could be mediated in two different ways, where ubiqutin either serves as internalization signal itself, or mediates proteasome mediated cleavage of an intracellular region leading to ErbB2 internalization.
Upon internalization of ErbB2, mediated by Hsp90 inhibitors, ErbB2 localizes to early endosomes. From there it is further sorted to ILVs of MVBs (Pedersen, Madshus et al. 2008). The involvement of the ESCRT machinery in endosomal sorting of ErbB2 is not yet known, but a possible involvement of ubiquitin in sorting of ErbB2 to ILVs has been suggested (Vuong, Berger et al. 2013).
Endocytosis and endosomal sorting of ErbB3
Remarkably little has been known about the downregulation of ErbB3 compared to EGFR and ErbB2. ErbB3 was for a long time considered to be endocytosis- resistant as it does not interact with AP-2 due to a lack of sorting signals (Baulida, Kraus et al. 1996), but it was shown that it undergoes a slow ligand- dependent internalization (Baulida, Kraus et al. 1996). A recent study also showed that ErbB3 is constitutively internalized in a clathrin-dependent manner and it is constitutively degraded (Sak, Breen et al. 2012). The ubiqutin ligases neuregulin receptor degradation protein-1 (Nrdp1) (Diamonti, Guy et al. 2002, Qiu and Goldberg 2002) and neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4) (Huang, Choi et al. 2015) have been reported to be involved in ubiquitination and proteasomal degradation of ErbB3. Other studies have also shown that ErbB3 may undergo lysosomal degradation as lysosomal inhibitors reduced the ligand-induced degradation of ErbB3 (Cao, Wu et al. 2007). Furthermore, it has been shown that ErbB3 expression is regulated by a quantity control mechanism involving the ER-associated degradation (ERAD) pathway (Fry, Simion et al. 2011).
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ErbB proteins in cancer
ErbB family receptors are associated with a range of diseases like psoriasis, heart disease and Alzheimer’s disease, but are best studied for their involvement in cancer development. Cancer is often referred to as a pathophysiological condition of abnormal signaling. The potent cell proliferation signals induced by the ErbB network are therefore utilized by cancer cells in the form of oncogenic mutations leading to strong clonal expansion. EGFR was the first cell-surface receptor linked to cancer (Todaro, De Larco et al. 1976). In the 40 years since this discovery, other ErbB family members have been found dysregulated in various cancers. The oncogenic potential of ErbBs arise from mutation (Humphrey, Gangarosa et al. 1991), overexpression of the receptor (Leahy 2004), or as a downstream effect of inappropriate ligand expression (Sizeland and Burgess 1992).
The central role of the ErbB network in tumor development makes it a very attractive target for pharmacological intervention. Due to the possibility of extracellular manipulation and the detailed understanding of the underlying intracellular biochemistry huge progress has been made in the development of drugs and therapeutic antibodies targeting ErbB family members, especially EGFR and ErbB2. Nevertheless, when ErbB family members are targeted with drugs the receptor blockade can be by-passed by increased expression of other family members, especially ErbB3 (Frolov, Schuller et al. 2007). Another by- passing mechanism is the activation of other receptor tyrosine kinases such as c-met. This can again lead to an increased expression of ErbB3 (Engelman, Zejnullahu et al. 2007). The biggest problem with this signaling diversion is different pathway activation and subsequently a loss of treatment efficiency.
