Recent Advances in the Importance of Ubiquitylation for Receptor Internalization and Signaling

Abstract

Receptor tyrosine kinases are activated by binding to their ligands, which trigger modifications in their cytoplasmic domains to initiate signal transduction. Control mechanisms to modulate the signaling of growth factor receptors are essential for proper signaling and require several levels of regulation. Post-translational modifications play crucial roles in intracellular trafficking through mechanisms that are not fully understood. Ubiquitylation is recognized as an essential signal in establishing molecular networks controlling receptor internalization and trafficking at the membrane and in sorting endosomes. In turn, receptor trafficking influences how the signaling networks are activated. Recent advances show how receptor targeting to clathrin-coated pits and internalization influences signaling by allowing specific target activation. At the same time, progress has been made in showing how membrane proteins are organized to facilitate the recruitment of activated receptors to clathrin-coated pits and how this whole process depends on the ubiquitylation of the receptors and endocytosis accessory proteins. Here, we review recent advances in the role of ubiquitylation in receptor internalization and trafficking.

1. Introduction

Receptor internalization is an essential process in regulating cell signaling, as the uptake of activated receptors and routing into the endocytic pathway regulates downstream cell behaviors. Signals from the plasma membrane may trigger different cellular outcomes than signaling from endosomes [1]. For example, activation of the epidermal growth factor receptor (EGFR) transduces several signaling pathways including Ras-Raf-mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2 (MAPK/ERK1/2), phosphoinositide 3kinase (PI3K), protein kinase B (PKB, referred to as Akt), Src-family kinases (SFKs), STATs, phospholipase Cg1, Rho family GTPases and other pathways [2]. The quality, amplitude and duration of this signaling are tightly regulated by the compartmentalization and trafficking of the receptor along the endocytic pathway (reviewed in [3]).

The EGFR is one of the most studied human proteins, notably because of its involvement in several types of cancer [4]. This receptor tyrosine kinase (RTK) has seven ligands (EGF, TGFA, HBEGF, BTC, AREG, EREG and EPGN) that typically trigger a pro-survival and proliferation response within the cell [5]. This must be tightly regulated through feedback loops to prevent overactivation of the receptor. One of the ways this is done for EGFR and many other RTKs is internalization of the activated receptor followed by its degradation in lysosomes [6]. There are several endocytic processes available in a human cell, and each require many proteins for different levels of regulation.

Clathrin-mediated endocytosis (CME) is well known for its participation in EGFR regulation [7]. It has previously been reported that under an EGF concentration of 5 ng/ml CME is the only internalization pathway triggered, and physiological concentration of EGF in human serum is usually below 1 ng/ml [2]. CME initiation begins immediately after activation of the EGFR through the recruitment of endocytic proteins that form the clathrin coat, mainly Fcho1/2, AP2, Eps15, clathrin, CALM, EPSIN and HIP1R. Next, membrane bending occurs with N-WASP, ARP2/3 and actin until a clathrin-coated pit (CCP) containing the activated receptors is formed. Detachment of the vesicle requires amphiphysins, endophilin and dynamin, which complete the scission and allow for vesicle internalization [8]. The clathrin coat is then quickly dismantled, and the receptor will once again undergo modifications to continue signaling. Fusion of the internalized vesicles with endosomes provides a new platform for EGFR signaling by allowing new interactions with cytoplasmic proteins [9].

EGFR dimerization, activation and internalization require conformational changes, ubiquitylation and phosphorylation, as well as the recruitment of many accessory proteins to an extent that is not yet fully understood. However, many studies have shown a requirement for ubiquitylation throughout the process, whether it be directly related to the receptor or to interacting proteins. Ubiquitylation is a post-translational modification requiring an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Together, these enzymes catalyze a series of reactions to transfer ubiquitin to a target protein. The specificity of the reaction relies on E3 ubiquitin ligases and their ability to interact with their targets [10].

This review aims to provide an understanding of recent advances in the importance of ubiquitylation for receptor internalization and signaling, with a focus on the epidermal growth factor receptor upon EGF stimulation.

2. RTK activation leads to the activation of ubiquitin ligases and ubiquitylation of the receptor

The first step required for EGFR activation after binding of a ligand is the dimerization of the receptor. Ligand binding triggers a conformational change that allows an EGF receptor to form a homo or heterodimer with another RTK such as ErbB2 [11]. It has been demonstrated that the interaction between EGFR and ErbB2 is better stabilized upon EGF binding than the EGFR/EGFR interaction. The seven EGFR ligands will each preferentially induce either homodimers or heterodimers, and this dimerization also depends on the availability of receptors at the cell membrane [12]. This mechanism, with the variety of EGFR ligands, provides a platform for signal modulation.

Ligand binding and dimerization of the receptor leads to the activation of the tyrosine kinase in the intracellular domain as well as its phosphorylation [3]. While this is the typical process described to initiate signal transduction, the receptor also undergoes other modifications such as ubiquitylation. It has been established that ubiquitylation of the EGFR regulates its endocytosis (Figure 1) and endosomal sorting (Figure 2) (reviewed in [13]). The most common E3 ubiquitin ligases known to interact with EGFR directly and through adaptors are Casitas B-lineage lymphoma (Cbl) and its isoform Cbl-b [14]. These E3s are part of the RING-domain family, and a third isoform, Cbl-c, is present in mammals, though it is not directly involved in RTK ubiquitylation [15].

Figure 1.

Ubiquitylation is essential for EGFR internalization. 1: ubiquitin participates in the formation of protein lattices at nascent endocytic sites. 2: ubiquitylation of the receptor and accessory proteins is necessary for clathrin-coated pit (CCP) formation and recruitment of activated receptors to CCPs. 3: E3 ubiquitin ligases add ubiquitin to target proteins. Deubiquitinating enzymes (DUBs) remove ubiquitin. Cycles of ubiquitylation and deubiquitylation regulate receptor internalization (created with BioRender).

Figure 2.

Ubiquitylation of EGFR post-internalization regulates interactions and modulates EGFR degradation and recycling. After internalization and fusion with endosomes, ubiquitylation of the receptor is necessary for interactions with endosomal sorting complexes required for transport (ESCRT) proteins and sorting to multivesicular bodies (MVBs). E3 ubiquitin ligases add ubiquitin to target proteins. Deubiquitinating enzymes (DUBs) remove ubiquitin. Cycles of ubiquitylation and deubiquitylation regulate receptor fate (created with BioRender).

EGFR ubiquitylation by Cbl and Cbl-b is well established, but it was only recently that their independent binding and regulation modes were clarified [14]. Cbl and Cbl-b mutants that cannot ubiquitylate their substrates were used to demonstrate that their recruitment to EGFR after EGF activation does not entirely rely on redundant mechanisms, as Grb2 depletion affected Cbl binding to a much greater extent than Cbl-b. In contrast, mutation of EGFR Y1045 affected the binding of Cbl-b more than Cbl, and without affecting the EGFR CME-dependent internalization [14]. This coincides with previous findings that alterations in Cbl recruitment and EGFR ubiquitylation do not affect its internalization, but rather it is sorting past the early endosomes and, therefore, its degradation [16].

Following the understanding of Cbl and Cbl-b interactions with EGFR, their role in this receptor’s degradation was reassessed. While overexpression of WT Cbl and Cbl-b accelerated degradation, expression of either dominant-negative mutants did not significantly affect the half-life of EGFR in HSC3 (human tongue squamous carcinoma) cells [13]. The HSC3 cell line is typically used to study metastasis and tumour invasion due to its ability to produce high levels of growth factors and to promote angiogenesis [17]. In these cells, the ubiquitylation of the receptor was never fully inhibited despite Cbl mutations and inactivation of Grb2 [14]. Previous studies on Cbl and Cbl-b in HeLa led to Cbl-b’s role in EGFR regulation being dismissed; this discrepancy might be due to HSC3 having endogenous levels of Cbl-b ~ fivefold higher than HeLa [18]. The use of cell lines from different organs gives models with contrasting results due to variable levels of protein expression.

As depletion of Cbl and Cbl-b does not prevent internalization and ubiquitylation is never fully inhibited, a number of E3s may compensate and act in early and late endosomes to ubiquitylate the receptor [14]. RNF126 and Rabring7 are RING-domain E3s that associate with the EGFR later in the endocytic process. These two E3s play a role in receptor ubiquitylation and endosomal sorting complexes required for transport (ESCRT)-II stabilization. Indeed, their depletion leads to inefficient degradation of EGFR, a reduced number of multivesicular bodies (MVBs) and its retention in late endocytic compartments, placing them as regulators downstream of Cbl and Cbl-b [19]. Sorting from late endosomes to lysosomes also relies on ZNRF1—a RING-domain E3—that mediates EGFR ubiquitylation on residues separate from those affected by Cbl [20].

HECT-domain E3s such as NEDD4 are also involved in modulating EGFR signaling and regulating its degradation. NEDD4 monoubiquitylates Eps15, a process requiring its own ubiquitylation [21]. Interestingly, NEDD4 is recruited to EGFR-loaded endosomes and associates with EGFR to regulate its transport to MVBs and lysosomes. Knockdown of NEDD4 results in impaired lysosomal degradation of EGFR. However, the exact mechanism through which this occurs is unclear due to interactions of NEDD4 with proteins implicated in CME regulation. NEDD4 binds and ubiquitylates ACK1, a kinase involved in the regulation of ligand-induced EGFR degradation [22].

3. Ubiquitylation is essential for receptor internalization and modulates signaling

The number of E3s activated by EGFR and involved in its ubiquitylation provides a platform to modulate its transport and signaling, but direct interaction with EGFR is not the only mechanism for E3s to mediate those processes. Their binding and ubiquitylation to activate or degrade proteins involved in the chain of events triggered by EGFR dimerization must also be considered.

The first step of CME is the recruitment of endocytic proteins to create a network involved in clathrin pit formation. The initiation of endocytosis occurs on membrane patches delimited by weak liquid-like interactions between Eps15 and Fcho1/2 that concentrate downstream components [23]. The specific conditions allowing these events were characterized only recently. Ede1—the yeast homolog of Eps15—is a key promoter of the formation of endocytosis initiation sites. In Ede-1 mutants, the aggregation of early endocytic protein at the membrane fails to occur, abrogating the formation of clathrin-coated pits and CME. Ede-1 forms condensates at the plasma membrane that recruit other endocytic proteins in liquid-like droplets. The central domain of Ede-1 contains coil-coiled and prion-like regions that are both essential for the formation of the condensates [24].

Similar results were obtained in mammalian cells, pointing to the central role of Eps15 in the formation of protein condensates both in vitro and in cellulo [25]. It was further shown that the addition of polyubiquitin stabilized Eps15 condensates. Moreover, the knockout of Eps15 blocked the initiation of endocytosis, and neither the expression of Eps15 lacking the ubiquitin-interacting motif nor of Eps15 fused to a deubiquitinating enzyme could rescue it. These data lead to the conclusion that ubiquitin is essential for a stable initiation of CME by participating in the assembly of a flexible protein network (Figure 1) [25].

Other proteins that participate in CME must be recruited to initiation sites; the evolutionarily conserved epsin N-terminal homology (ENTH) domain can be found in many proteins recruited to nascent clathrin pits. A unique module named AP180 N-terminal homology (ANTH) domain was identified in proteins previously described as containing an ENTH domain. Both ENTH and ANTH domains can bind inositol phospholipids and proteins to participate in the formation of clathrin coats [26]. The ANTH domain of endocytic proteins has recently been shown to bind to ubiquitin through its C-terminal region, which is not present in ENTH domains. Specifically, CALM, HIP1R and Sla2 were found to bind ubiquitin via their ANTH domains. Further experiments demonstrated that the loss of the ubiquitin-binding domains in components of the endocytic machinery impeded internalization of GPCRs relying on ubiquitylation for internalization. The ability of ANTH to bind ubiquitin is thus required to recognize ubiquitin as an internalization signal [27].

The crucial role of ubiquitin in nascent endocytic sites implies ubiquitin ligases and deubiquitinating enzymes as potentially important regulators of endocytosis. ITCH is a notable HECT-domain E3 implicated in EGFR internalization. ITCH is part of the Nedd4 family of HECT E3s but stands out with a unique proline-rich region (PRR) that has been shown to interact with Src homology 3 (SH3) domains of other proteins [28]. While ITCH can associate with several SH3-domain proteins, its preferential proteins for complexes are endophilins, amphyphisins and pacsins [29]. This suggests a role in regulation of endocytic proteins involved in EGFR internalization. While no direct interactions between ITCH and EGFR have been demonstrated, the knockout of ITCH in HeLa cells caused a delay in EGFR internalization through CME [30]. Expression of wild-type ITCH in the knockout cells rescued the internalization phenotype, which validated its role in the endocytic process. Moreover, a mutant that rendered the HECT-domain catalytically inactive and a mutant with a modified PRR preventing binding to SH3 domain-containing proteins could not rescue this phenotype [30]. This is especially interesting as ITCH is often studied as a WW domain E3 similar to NEDD4 and WWP2, which are highly homologous but lack the PRR. The absence of a PRR in other E3s of this family places ITCH as a critical and unique regulator of EGF-dependent EGFR CME.

4. Ubiquitylation modulates signaling

The delay in EGFR endocytosis in the absence of ITCH leads to questions about its consequences on signal transduction and the need to understand the link between protein activation and the endocytic process. The Ras/MAPK pathway is the main pathway activated by EGFR upon EGF binding. The activation of MAPK through this mechanism leads to the recruitment of membrane fusion proteins to early endosomes to initiate the formation of signaling endosomes (reviewed in [31]). RTKs in endosomes retain their ability to signal, and their transport along microtubules enables interactions with new proteins to modulate signaling.

Therefore, while signal transduction occurs from the cell membrane, it has been established that EGFR signaling continues in endosomes and undergoes new levels of modulation. Akt (also known as PKB) exists in three isoforms (Akt1,2,3) and is known to be activated by EGF binding to EGFR through PI3K activation. Canonically, this activation occurs at the plasma membrane [3], though evidence of a requirement for CME in the activation of Akt has been found and establishes a role for CCPs in the modulation of this protein’s activation. Perturbation of clathrin using siRNA has demonstrated a defect in Akt phosphorylation following EGF stimulation, but perturbation of dynamin2 did not affect Akt activation. These findings lead to clathrin scaffolds being established as microdomains required in some signaling pathways [32]. To understand the impact of CCPs on signaling, the adaptors TOM1L1 and the Src-family kinase Fyn were investigated for their role in Akt signaling. Interestingly, while EGFR activation leads to activation of all three Akt isoforms, the perturbation of either TOM1L1 or Fyn led to a diminution of Akt2 phosphorylation but did not affect Akt1 [33]. As PI3K is required for Akt phosphorylation, its localization can provide a better understanding of its function. The microtubule-associated scaffolding molecule MAP4 participates in endosomal vesicle trafficking along microtubules and associates with the catalytic subunit P110α of PI3K, suggesting Akt signaling from endosomes rather than the plasma membrane [34]. These findings both suggest a role for CME in Akt signaling, and lead to the question of how Akt activation is affected by non-clathrin-dependent endocytic pathways.

5. DUBs are necessary for signaling networks and interactions to evolve post internalization

The ubiquitylation status of proteins is the balance between the activity of ubiquitin ligases and ubiquitin proteases, often referred to as deubiquitylating enzymes (DUBs). As the essential role of ubiquitylation of several proteins is critical in EGFR trafficking, DUBS must also be involved. However, the DUBs identified to this day have mostly been found to affect receptor degradation rather than its internalization, and many remain to be characterized in RTK signaling. Indeed, a genome-wide siRNA screen, targeting 92 active DUBs to identify those impacting the regulation of EGFR degradation showed that at least 15 of these enzymes either damper or accelerate EGFR internalization rate [35]. Only a few of these have been studied closely.

USP9X has been identified as a DUB influencing EGFR signaling and fate. The absence of USP9X causes both a defect in internalization and early to late endosome trafficking of EGF bound EGFR, thus delaying its degradation. This could be due to its interactions with Eps15 under normal conditions [35]. Interestingly, USP9X also interacts with the ubiquitin ligase ITCH and protects it from degradation triggered by its auto-ubiquitylation [36]. As the absence of ITCH in HeLa cells delayed EGFR internalization and therefore degradation, the loss of USP9X could lead to ITCH degradation and give a similar phenotype, though this remains to be elucidated. The regulatory effects of DUBs through ubiquitin ligases are also exemplified by USP25, another DUB implicated in EGFR regulation. USP25 controls Cbl association with the receptor and therefore its ubiquitylation levels post ligand binding [37].

Other DUBs can affect EGFR degradation, and their presence either increases or decreases it. The associated molecule with a Src homology 3 domain of signal transducing adaptor molecule (AMSH) is part of the JAB1/MPN/MOV34 (JAMM) family. This metalloprotease was first characterized as an important regulator of the ESCRT and can regulate ubiquitin signaling in EGFR trafficking (Figure 2). Its knockdown has been shown to enhance the degradation of the receptor, which would facilitate the transport along the ESCRT complex towards internalization in multivesicular bodies [38]. Other DUBs, like OTUD7 (Cezanne-1) and Usp2, contain cysteine proteases. These two enzymes can enhance EGFR signaling by reducing its ubiquitylation and preventing its lysosomal degradation [39, 40]. USP22 was shown to be implicated in EGFR deubiquitylation to promote its recycling and prevent degradation in lysosomes [41].

DUBs can also act indirectly to affect EGFR fate through regulation of sorting proteins. USP8 regulates the turnover of ESCRT-0 complex proteins and EGFR de-ubiquitylation by this enzyme promotes receptor degradation [42], while USP18 plays a role in EGFR mRNA translation [43]. The extreme number of DUBs and E3s implicated in EGFR regulation suggests that cycles of ubiquitylation and deubiquitylation may modulate internalization and trafficking of the receptor.

6. Conclusion

Receptor signaling and internalization are intimately related as both are triggered simultaneously by ligand binding. Initiated at the plasma membrane, signaling continues until either the ligand binding is lost, or the receptor-ligand pair is targeted to the lysosome for degradation. Each of the steps of a receptor journey through the endosomal pathway is regulated by a plethora of protein-protein interactions, in which ubiquitin and ubiquitin-interaction motifs are heavily involved. Therefore, it is not surprising that ubiquitin ligases and ubiquitin proteases lie at the core of the regulation of each of these processes.

Recent progress in the field highlights the complexity of this process as a large number of previously unrecognized membrane-trafficking constituents linked to signal transduction are still being identified. More discoveries will come and pave the way to a better understanding and, eventually, means of controlling these complex pathways.

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

AREG	amphiregulin
ARP2/3	actin-related protein 2/3
BTC	betacellulin
CALM	clathrin assembly lymphoid myeloid leukaemia
CCP	clathrin-coated pits
CME	clathrin-mediated endocytosis
DUBs	deubiquitinating enzymes
EGF	epidermal growth factor
EGFR	epidermal growth factor receptor
EPGN	epithelial mitogen
ErbB2/HER2	human epidermal growth factor receptor 2
EREG	epiregulin
ESCRT	endosomal sorting complexes required for transport
Grb2	growth factor receptor bound protein 2
HBEGF	heparin-binding EGF-like growth factor
HECT	homologous to E6-AP C-Terminus
HIP1R	huntingtin interacting protein-1 related
HSC3	human tongue squamous carcinoma 3
MVBs	multivesicular bodies
Myb1	myeloblastosis viral oncogene homolog
N-WASP	neuronal Wiskott-Aldrich syndrome protein
RING	really interesting new gene
RNF126	ring finger protein 126
RTK	receptor tyrosine kinase
TGFA	transforming growth factor alpha
TOM1L1	target of Myb1 like 1 membrane-trafficking protein
ZNRF1	zinc and ring finger 1