EGF receptor

EVOLUTIONARY HOMOLOGS

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Interaction between EGFR receptor subtypes

The epidermal growth factor receptor (EGFR) and p185c-neu proteins associate as dimers to create an efficient signaling assembly. Overexpression of these receptors together enhances their intrinsic kinase activity and concomitantly results in oncogenic cellular transformation. The ectodomain is able to stabilize the dimer, whereas the kinase domain mediates biological activity. Potential interactions are examined of the cytoplasmic kinase domains of the EGFR and p185c-neu tyrosine kinases by homology molecular modeling. This analysis indicates that kinase domains can associate as dimers and, based on intermolecular interaction calculations, that heterodimer formation is favored over homodimers. The study also predicts that the self-autophosphorylation sites located within the kinase domains are not likely to interfere with tyrosine kinase activity, but may regulate the selection of substrates, thereby modulating signal transduction. In addition, the models suggest that the kinase domains of EGFR and p185c-neu can undergo higher order aggregation such as the formation of tetramers. Formation of tetrameric complexes may explain some of the experimentally observed features of their ligand affinity and hetero-receptor internalization (Murali, 1996).

EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF-sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor-receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF-induced sEGFR dimerization from the growth hormone and interferon-[gamma] receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF-induced sEGFR dimerization suggests possible mechanisms for both ligand-induced homo- and heterodimerization of the EGFR (or erbB) family of receptors (Lemmon, 1997).

Interaction of EGFs with EGF receptor

TGF-alpha and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a common receptor. Proteolytic cleavage of the transmembrane TGF-alpha precursor's external domain releases several TGF-alpha species. However, membrane-bound TGF-alpha forms remain on the surface of TGF-alpha-expressing cell lines. To evaluate the biological activity of these forms, two cleavage sites were modified in the TGF-alpha precursor coding sequence, making impossible the precursor's processing into the 50 amino acid TGF-alpha. Overexpression of this cDNA in a receptor-negative cell line, as well as partial purification, and N-terminal sequence analysis indicate the existence of two transmembrane TGF-alpha forms. These solubilized precursors induce tyrosine autophosphorylation of the EGF/TGF-alpha receptor in intact receptor-overexpressing cells, and anchorage-independent growth of NRK fibroblasts. Cell-cell contact between TGF-alpha precursor-overexpressing cells and cells expressing high numbers of receptors also result in receptor activation. These findings suggest a role for transmembrane TGF-alpha forms in intercellular interactions in proliferating tissues (Brachmann, 1989).

Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in the affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. The intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha has been compared in two different cell systems. TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha, in contrast to the mostly degraded EGF. In addition, TGF-alpha does not induce a complete down-regulation of cell surface receptors, as observed with EGF, which,in the case of TGF-alpha, is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF (Ebner, 1991).

The epidermal growth factor receptor plays crucial roles throughout the development of multicellular organisms, and inappropriate activation of the receptor is associated with neoplastic transformation of many cell types. The receptor is thought to be activated by ligand-induced homodimerization. However, in the absence of bound ligand the receptor has an ability to form a dimer and exists as a preformed dimer on the cell surface. The receptor dimerization has been analyzed by inserting cysteine residues at strategic positions about the putative alpha-helix axis of the extracellular juxtamembrane region. The mutant receptors spontaneously formed disulphide bridges and transformed NIH3T3 cells in the absence of ligand, depending upon the positions of the cysteine residue inserted. Kinetic analyses of the disulphide bonding indicate that EGF binding induces flexible rotation or twist of the juxtamembrane region of the receptor in the plane parallel with the lipid bilayer. The binding of an ATP competitor to the intracellular domain also induces similar flexible rotation of the juxtamembrane region. All the disulphide-bonded dimers have flexible ligand-binding domains with the same biphasic affinities for EGF as the wild-type. These results demonstrate that ligand binding to the flexible extracellular domains of the receptor dimer induce rotation or twist of the juxtamembrane regions (hence the transmembrane domains), and dissociate the dimeric, inactive form of the intracellular domains. The flexible rotation of the intracellular domains may be necessary for the intrinsic catalytic kinase to become accessible to the multiple tyrosine residues present in the regulatory domain and various substrates, and may be a common property of many cell-surface receptors, such as the insulin receptor (Moriki, 2001).

The responses of mouse blastocysts to TGF-alpha/EGF treatment are mediated by EGF receptors (EGFR) located on the apical surface of the trophectoderm (TE). Experiments using gold-labeled EGF confirm the presence of these apically located EGFRs. Immunoelectron microscopy (IEM) studies using anti-EGFR antibodies indicate that the receptor is preferentially distributed on the basolateral surface of the TE. The receptor is also present on the inner cell mass (ICM) and is likely to be functional, since treatment of isolated ICMs with TGF-alpha affects [35S]methionine uptake and incorporation into acid-insoluble material. IEM was also used to demonstrate that EGF, which is not synthesized by the mouse preimplantation embryo, is present in both the oviduct and the uterus. Maternally derived EGF is present in both ICM and TE cells in freshly isolated blastocysts, but is present in greatly reduced amounts following overnight culture of blastocysts in vitro. IEM was also used to demonstrate that TGF-alpha is preferentially localized to the ICM and polar TE. The co-localization of TGF-alpha and functional EGFRs to the ICM and polar TE suggests potential autocrine, juxtacrine, and paracrine roles for TGF-alpha in blastocyst development (Dardik, 1992).

The epidermal growth factor (EGF), the transforming growth factor alpha (TGFalpha) and the epidermal growth factor receptor (EGFr) have been immunolocalized at two developmental stages: (1) during the testicular postnatal development (i.e. at the perinatal, prepubertal and adult periods), and (2) during the seminiferous epithelium cycle in the different germ cell types. While TGFalpha is essentially observed in somatic cells, specifically in perinatal Leydig cells and in mature Sertoli cells, EGF is localized both in germ cells and in somatic cells with a preferential tubular expression. Identification of EGFr in different testicular cell types indicates that during postnatal development and spermatogenesis, testicular cells are potentially responsive to EGF because they express EGFr. Indeed, in the course of the gonadal development, the EGFr distribution is found both in somatic and germ cells with a specific germ cell pattern depending upon the seminiferous epithelium cycle. A predominant EGFr staining is found during the meiotic process and the spermiogenesis. This work suggests involvement of the TGFalpha/EGF system in the local control of testicular cells during development and particularly of the system's potential direct involvement in crucial steps of spermatogenesis such as meiosis and spermiogenesis (Caussanel, 1996).

Epidermal growth factor (EGF) and type alpha transforming growth factor (TGF-alpha) bind to a specific region in subdomain III of the extracellular portion of the EGF receptor (EGFR). Binding leads to receptor dimerization, auto-and transphosphorylation on intracellular tyrosine residues, and activation of signal transduction pathways. The binding and biological actions of EGF and TGF-alpha were compared in Chinese hamster ovary (CHO) cells expressing either wild-type human EGFR (HER497R) or a variant EGFR that has an arginine-to-lysine substitution in the extracellular domain at codon 497 (HER497K) within subdomain IV of EGFR. Both receptors exhibit two orders of binding sites with EGF. Similar results were obtained with TGF-alpha in cells expressing HER497R. In contrast, only one order of low-affinity binding sites was seen with TGF-alpha in the case of HER497K. Although EGF and TGF-alpha enhance tyrosine phosphorylation of both receptors, CHO cells expressing HER497K exhibit an attenuated growth response to EGF and TGF-alpha and a reduced induction of the protooncogenes FOS, JUN, and MYC. Moreover, high concentrations of TGF-alpha inhibit growth in these cells but not in cells expressing HER497R. These findings indicate that a region in subdomain IV of EGFR regulates signal transduction across the cell membrane and selectively modulates the binding characteristics of TGF-alpha (Moriai, 1994).

Epidermal growth factor (EGF) has been shown to stimulate mPL-I secretion and inhibit mPL-II secretion. Does transforming growth factor alpha (TGF-alpha) regulate the production of mouse placental lactogen I (mPL-I) and mPL-II in a manner similar to that of EGF? In contrast to the activity of EGF, TGF-alpha inhibits secretion of mPL-I by placental cells isolated from mice on day 7 of pregnancy. Maximum inhibition of mPL-I secretion occurs on the third day of a 5-day culture period and ranges between 37% and 56%. Incubation of cells with hTGF-alpha and EGF is not followed by a change in the mPL-I concentration of the medium, suggesting the peptides antagonize each other's effects. TGF-alpha inhibits secretion of mPL-II; maximum inhibition ranged between 62% and 84% in multiple trials. EGF and TGF-alpha bind to the same receptors on placental cells, and both peptides stimulate receptor phosphorylation. There are three types of mPL-containing cells in placental cultures: cells that contain only mPL-I, cells that contain only mPL-II, and cells that contain both mPLs. TGF-alpha affects the differentiation of the subpopulations of PL-containing cells in a manner that differs from that of EGF. The data suggest that TGF-alpha and EGF do not regulate the production of mPL-I and mPL-II in a similar manner (Yamaguchi, 1995).

The crystal structure, at 2.5 Å resolution, is reported of a truncated human EGFR ectodomain bound to TGFalpha. TGFalpha interacts with both L1 and L2 domains of EGFR, making many main chain contacts with L1 and interacting with L2 via key conserved residues. The results indicate how EGFR family members can bind a family of highly variable ligands. In the 2:2 TGFalpha:sEGFR501 complex, each ligand interacts with only one receptor molecule. There are two types of dimers in the asymmetric unit: a head-to-head dimer involving contacts between the L1 and L2 domains and a back-to-back dimer dominated by interactions between the CR1 domains of each receptor. Based on sequence conservation, buried surface area, and mutagenesis experiments, the back-to-back dimer is favored to be biologically relevant (Garrett, 2002).

Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. A computational model has been used to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and an experimental test was provided by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGFL47M. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling (DeWitt, 2002).

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 Å resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique 'receptor-mediated dimerization' was verified by EGFR mutagenesis (Ogiso, 2002).

Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. The 2.8 Å resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state is described. The structure reveals an autoinhibited configuration, where the dimerization interface present in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists (Ferguson, 2003).

Activation of EGF receptor by dimerization

Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. This study shows that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. These data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation (Jura, 2009).

Intercellular signaling in animals relies on receptor tyrosine kinases that consist of an extracellular ligand binding domain and a cytoplasmic kinase domain. A distinct subfamily of these receptors includes the epidermal growth factor receptor (EGFR, also known as ErbB1 or Her1) and its three homologs in humans: ErbB2/Her2 and ErbB4/Her4, which are kinase active, and ErbB3/Her3, which is not. The ligand-dependent dimerization of various combinations of EGFR family members results in phosphorylation of their C-terminal tail segments, which is crucial for cellular proliferation and survival (Jura, 2009).

Activation of the catalytic domain of EGFR family members is controlled primarily by an allosteric interaction between two protein kinase domains in an asymmetric dimer, rather than by phosphorylation (Gotoh, 1992; Stamos, 2002; Zhang, 2006). The kinase domain of one receptor molecule (the activator) plays a role analogous to that of a cyclin bound to a cyclin-dependent protein kinase and activates the kinase domain of a second receptor (the receiver) (Zhang, 2006). The formation of the asymmetric dimer appears to underlie the activation of all EGFR family members (Qiu, 2008; Zhang, 2006; Jura, 2009 and references therein)

Upon binding ligand, the extracellular domains of EGFR family members dimerize such that their C-terminal ends are brought close together at the junction with the transmembrane segments (Burgess, 2003). The transmembrane segments connect to the cytoplasmic juxtamembrane segments of the receptor. The role of the juxtamembrane segment of EGFR family members is distinct from that of typical receptor tyrosine kinases because it activates, rather than inhibits, the kinase domain (Thiel, 2007). The nature of this coupling between the juxtamembrane segment and the kinase domain is not understood (Jura, 2009).

The juxtamembrane segment of human EGFR spans residues 645 to 682, and the N-terminal half (residues 645 to 663) is referred to as JM-A and the C-terminal half (residues 664 to 682) as JM-B. An examination of crystal lattice contacts in a previously reported structure of the Her4 kinase domain (Wood, 2008) reveals that the JM-B portion of the juxtamembrane segment forms a clamp that reaches across from the N-terminal lobe of the receiver kinase domain in an asymmetric dimer to engage the C-terminal lobe of the activator kinase domain. The significance of this interaction has not been noted previously, but this study demonstrates that this interface involving JM-B, which is referred to as the 'juxtamembrane latch,' is crucial for receptor activation (Jura, 2009).

This study shows that JM-A segments on the receiver and the activator are both required for dimerization and activation, and it is proposed that the two JM-A segments in an asymmetric kinase domain dimer form short α helices that are likely to interact in an antiparallel manner and connect to the C-terminal ends of the dimeric form of the transmembrane helices. This allows a model for the entire activated receptor to be built, in which ligand engagement by the extracellular domains stabilizes the formation of the JM-A helical dimer, which in turn stabilizes the asymmetric kinase domain dimer, resulting in activation (Jura, 2009).

A structure of the EGFR kinase core has been determined in which formation of the juxtamembrane latch is blocked by the C-terminal tails of the receptor. This structure forms a symmetrical dimer of inactive kinase domains and suggests a potential mechanism whereby alternative dimers can prevent ligand-independent activation (Jura, 2009).

A single ligand is sufficient to activate EGFR dimers

Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. This study reports cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. Crystal structures are reported of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1β that resolve two types of ErbB dimers when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers (Alvarado, 2009; Alvarado, 2010). These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions (Liu, 2012).

EGFR dynamics change during activation in native membranes as revealed by NMR

The epidermal growth factor receptor (EGFR; see Drosophila Egfr) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP) was developed. In contrast to previous crystallographic results, the current experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. It is proposes that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase (Kaplan, 2016).

Localized and global activation of EGFR

When a cell is stimulated locally with ligands, is the activation of intracellular signaling localized or propagated over the entire cell? To address this intriguing and fundamental question, the spatiotemporal pattern of EGF signaling in single live COS cells. Lateral propagation was analyzed in single live COS cells following local stimulation, achieved by the use of laminar flows containing rhodamine-labeled EGF. The spatiotemporal pattern of EGF signaling was visualized by fluorescent indicators for Ras activation and tyrosine phosphorylation. Contrary to the findings in previous reports, both signals are localized to the stimulated regions in control COS cells expressing EGF receptor at the basal level. However, the signals spread over the entire cell when EGF receptors are overexpressed or when receptor/ligand endocytosis is blocked. Evidence is thus presented that ligand-independent propagation of EGF signaling occurs only when the receptor density on the plasma membrane is high, such as in carcinoma cells (Sawano, 2002).

There are many interesting biological consequences of the observed local and global activation of EGF signaling. One of the well-studied downstream signaling targets of EGFR is the Ras-Raf-MEK-ERK pathway. Since this pathway converges on the nucleus, the spatial information pertaining to the site of EGFR activation is likely to be lost. In contrast, other EGFR-dependent pathways, such as membrane trafficking, turnover of focal adhesions, and cytoskeleton organization, are spatially restricted, contributing to directional cell morphology and motility. In these experiments, the localized spatial pattern of EGF signaling is well correlated with local morphological changes, potentially involving not only the Ras pathway, but also other EGF-induced signaling molecules, such as Rho, focal adhesion kinase, and phosphatidylinositol-3 kinase. In organogenesis, local activation of EGF signaling appears to be important for coordinated control of region-specific detachment from the extracellular matrix. This process may allow for proliferation of cells with regulated EGFR expression while maintaining architectural order. A variety of carcinoma cells overexpress EGFR and show dysregulated cell motility upon EGF treatment, as do A431 cells. Ligand-independent propagation of EGF signaling may be one of the molecular mechanisms underlying invasion and metastasis, criteria by which malignant tumors are characterized (Sawano, 2002).

To investigate the function of c-Jun during skin development and skin tumor formation, c-jun was conditionally inactivated in the epidermis. Mice lacking c-jun in keratinocytes develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun deficient mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, it has been shown that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation (Zenz, 2003).

Genetic screens performed in worms identified major regulators of the epidermal growth factor receptor (EGFR) pathway, including the ubiquitin ligase Cbl/SLI-1. This study focused on the less-characterized Lst2 protein and confirmed suppression of MAPK signals. Unexpectedly, human Lst2, a monoubiquitinylated phosphoprotein, does not localize to endosomes, despite an intrinsic phosphoinositol-binding FYVE domain. By constructing an ubiquitinylation-defective mutant and an ubiquitin fusion, it is concluded that endosomal localization of Lst2, along with an ability to divert incoming EGFR molecules to degradation in lysosomes, is regulated by ubiquitinylation/deubiquitinylation cycles. Consistent with bifurcating roles, Lst2 physically binds Trim3/BERP, which interacts with Hrs and a complex that biases cargo recycling. These results establish an ubiquitin-based endosomal switch of receptor sorting, functionally equivalent to the mechanism inactivating Hrs via monoubiquitinylation (Mosesson, 2009).

To ensure fidelity of signaling outcomes, activated receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), are subjected to signal desensitizing mechanisms, primarily entailing accelerated endocytosis and degradation in lysosomes. Activated receptors concentrate over clathrin-coated pits at the plasma membrane, which invaginate to form coated vesicles. Vesicles then uncoat prior to fusing with tubulo-vesicular organelles, denoted early endosomes (EEs). Receptor cargos subsequently undergo sorting, either for recycling back to the plasma membrane or for lysosomal destruction at late endosomal compartments termed multivesicular bodies (MVBs). RTKs often undergo ubiquitinylation through recruitment of Cbl family ubiquitin ligases. Mono- and oligoubiquitins in the context of RTKs drive progression of cargos via endosomes, toward lysosomes. Active sorting in endosomes is attributed to various ubiquitin-binding domains (UBDs) carried by multiple adaptors (Mosesson, 2009 and references therein).

Small GTPases of the Rab subfamily are also attributed pivotal functions; enrichment of specific Rabs in different endosomal compartments permits local activation of a particular complement of effectors, which execute requisite endocytic tasks. Activated Rab5, which operates at the cell surface/EE interface, stimulates production of phosphatidylinositol 3-phosphate (PI3P) in EE membranes, thereby nucleating complexes of effector proteins like EEA1, which harbors a PI3P-binding domain called FYVE. In addition to EEA1, ~30 other proteins share the double zinc finger FYVE motif. Notably, PI3P binding by specific FYVE domain proteins is highly regulated both in cis and in trans. For example, a FYVE-containing amino-terminal fragment of Hrs failed to localize to EEs. This may be attributed to ancillary interactions involving the flanking coiled-coil domain, or dimerization of FYVE domains. Similarly, the FYVE domain of EEA1 may not suffice for endosomal localization. Instead, endosomal localization of EEA1 is thought to be complemented in living cells by p38-induced phosphorylation. Thus, FYVE-mediated localization of a variety of endocytic adaptors to EEs is a multifocal regulatory node that impacts on both endocytosis and signaling. This study extends the complexity to regulation by monoubiquitinylation (Mosesson, 2009).

This study describes a mammalian FYVE domain protein, termed hLst2, whose endosomal localization is regulated by monoubiquitinylation. Consistent with its primitive ortholog in C. elegans, which functions as a negative regulator of the worm's EGFR (Yoo et al., 2004), cellular depletion of human Lst2 augments EGF-induced signaling. Like many endosomal adaptors, hLst2 undergoes constitutive monoubiquitinylation, but despite the intrinsic FYVE domain, it displays primarily nonendosomal localization. By identifying a specific lysine acceptor, monoubiquitinylation was discovered as a means to prevent FYVE domain-dependent association of hLst2 with PI3P-enriched endosomes. In line with a unique ubiquitin/PI3P switch, a nonubiquitinylated hLst2 localizes to EEs, promotes degradative sorting of activated EGFRs, and reduces signaling.

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