A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.     

Utilization of a receptor reserve for effective amplification of mitogenic signaling by an epidermal growth factor mutant deficient in receptor activation

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study stems from our results showing that L15 of epidermal growth factor (EGF) is important in both EGF receptor (EGFR) binding and activation, and the L15A analog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activation (Nandagopal et al., [1996] Protein Engng 9:781-788). We address the cellular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerization, shown by chemical cross-linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, however, that L15A can induce EGFR autophosphorylation in intact murine keratinocytes by utilizing spare receptors, the ratio of total phosphotyrosine content per receptor being significantly lower than that elicited by wild-type. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A-activated receptors can stimulate mitogen-activated protein kinase (MAPK) that is important for mitogenesis. The lack of linear correlation between levels of receptor dimerization, autophosphorylation, and MAPK activation suggests that signal amplification is mediated by cooperative effects. Flow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S-phase are both decreased relative to 1 nM wild-type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild-type differ in the number of receptors each activates to induce the threshold response, which may be attained by cooperative activation of receptor dimers/oligomers by van der Waal's weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival.     

Transforming growth factor-alpha short-circuits downregulation of the epidermal growth factor receptor

Transforming growth factor-alpha (TGFalpha) is an epidermal growth factor receptor (EGFR) ligand which is distinguished from EGF by its acid-labile structure and potent transforming function. We recently reported that TGFalpha induces less efficient EGFR heterodimerization and downregulation than does EGF (Gulliford et al., 1997, Oncogene, 15:2219-2223). Here we use isoform-specific EGFR and ErbB2 antibodies to show that the duration of EGFR signalling induced by a single TGFalpha exposure is less than that induced by equimolar EGF. The protein trafficking inhibitor brefeldin A (BFA) reduces the duration of EGF signalling to an extent similar to that seen with TGFalpha alone; the effects of TGFalpha and BFA on EGFR degradation are opposite, however, with TGFalpha sparing EGFR from downregulation but BFA accelerating EGF-dependent receptor loss. This suggests that BFA blocks EGFR recycling and thus shortens EGF-dependent receptor signalling, whereas TGFalpha shortens receptor signalling and thus blocks EGFR downregulation. Consistent with this, repeated application of TGFalpha is accompanied by prolonged EGFR expression and signalling, whereas similar application of EGF causes receptor downregulation and signal termination. These findings indicate that constitutive secretion of pH-labile TGFalpha may perpetuate EGFR signalling by permitting early oligomer dissociation and dephosphorylation within acidic endosomes, thereby extinguishing a phosphotyrosine-based downregulation signal and creating an irreversible autocrine growth loop.     

Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.     

The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor

Hepatitis C virus (HCV) frequently establishes a persistent infection, leading to chronic liver disease. The NS5A protein has been implicated in this process as it modulates a variety of intracellular signalling pathways that control cell survival and proliferation. In particular, NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)-stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear. As EGFR signalling involves trafficking to late endosomes, we investigated whether NS5A perturbs EGFR signalling by altering receptor endocytosis. We demonstrate that NS5A partially localizes to early endosomes and, although it has no effect on EGF internalization, it colocalizes with the EGFR and alters its distribution. This redistribution correlates with a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment and also results in a concomitant increase in the total levels of EGFR. These observations suggest that NS5A controls EGFR signalling by diverting the receptor away from late endosomes. This represents a novel mechanism by which a viral protein attenuates cell signalling and suggests that NS5A may perturb trafficking pathways to maintain an optimal environment for HCV persistence.     

Targeted antireceptor therapy with monoclonal antibodies leads to the formation of inactivated tetrameric forms of ErbB receptors

mAbs capable of disabling heterodimeric kinase complexes of the epidermal growth factor receptor (EGFR) and human EGFR type 2/neu have therapeutic relevance to various human cancers. In this study, we demonstrate that in addition to the dimer, EGFR and human EGFR type 2 can associate as homo- and heterotetramers. EGF-induced phosphorylation of the tetramers was significantly lower than that of the dimers, indicating that the tetrameric receptor complexes have impaired signaling activity. Targeting v-erb-b2 erythroblastic leukemia viral oncogene homolog (erbB) receptors with mAbs promoted erbB tetrameric assembly, suggesting that a component of the antitumor activity may be mediated by the ability of Abs to shift the equilibrium from active dimeric to impaired tetrameric receptor complex states. This study suggests a novel therapeutic approach to disable signaling of erbB and potentially other receptors in tumors by biologic agents capable of inducing receptor tetramerization.     

Deletion-mutant epidermal growth factor receptor in human gliomas: effects of type II mutation on receptor function.

Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.     

Ligand-independent and EGF receptor-supported transactivation: lessons from beta2-adrenergic receptor signalling

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) is currently understood to be mediated by matrix metalloproteases (MMPs) and the release of EGF-like ligands. This ligand-mediated process also suggests that downstream of EGFR the signalling in response to GPCR ligands or EGF appears to be indistinguishable. Here we provide evidence that transactivation of EGFR by the beta2-adrenergic receptor (beta2-AR) is independent of MMPs and results in an incomplete downstream signalling involving extracellular signal-activated kinase (ERK) but not PLCgamma1 and Akt. In contrast, beta2-AR has the ability to activate PLCgamma1 when the EGFR is primed either by co-stimulation with EGF or by increased basal activity due to over-expression. In that way but not via the beta2-AR-mediated transactivation the EGFR docking sites pY992 and pY1173 may be generated which are critical for PLCgamma1. This EGFR-supported transactivation is strongly dependent on EGFR tyrosine kinase, c-Src, and the c-Src-specific EGFR tyrosine residue 845 and represents a novel paradigm of EGFR transactivation.     

A tale of two Cbls: interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation

The precise role of Cbl in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking remains to be fully uncovered. Here, we showed that mutant EGFR1044, which was truncated after residue 1044, did not associate with c-Cbl and was not ubiquitinated initially in response to EGF but was internalized with kinetics similar to those of wild-type EGFR. This finding indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. We also showed that the previously identified internalization-deficient mutant receptor EGFR1010LL/AA bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. We next investigated EGFR trafficking following EGFR internalization. We found that c-Cbl disassociation from EGFR occurred well in advance of EGFR degradation and that this event was concurrent with the selective dephosphorylation of EGFR at Y1045. This finding suggests that once EGFR is ubiquitinated, continual Cbl association is not required for EGFR degradation. Because EGFR1044 is ubiquitinated and degraded similarly to wild-type EGFR, we examined the role of another prominent Cbl homologue, Cbl-b, and found that Cbl-b was associated with both EGFR and EGFR1044. Further study showed that Cbl-b bound to EGFR at two regions: one in the C-terminal direction from residue 1044 and one in the N-terminal direction from residue 958. Moreover, Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. Using RNA interference to knock down both c-Cbl and Cbl-b, we were able to abolish EGFR downregulation. This knockdown had no affect on the rate of EGF-induced EGFR internalization. We found that the two Cbls accounted for total receptor ubiquitination and that while c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. Furthermore, these data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking of EGFR.     

Integrin alphavbeta3 is a coreceptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin alphavbeta3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and alphavbeta3, respectively, to initiate viral entry and signaling. Alphavbeta3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and alphavbeta3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and alphavbeta3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.     

Epidermal growth factor receptor (EGFR) signaling in cancer

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.     

Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor

Heterogeneous receptor-receptor interactions may play a role in intracellular signaling. Accordingly, the interaction of two dissimilar tyrosine kinase receptors, Ron and epidermal growth factor receptor (EGFR) was investigated. The functional interaction of Ron and EGFR in cell scatter and oncogenic transformation was investigated in vivo. Transfection of a dominant negative form of EGFR into human embryonic kidney cells stably expressing Ron (293-Ron) dramatically reduced the scatter response induced by the Ron ligand hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL). The scatter response of the 293-Ron cells was also attenuated by treatment of the cells with the specific EGFR inhibitor AG 1478. Co-transfection of Ron and dominant-negative EGFR, or co-transfection of EGFR and a dominant-negative form of Ron reduced focus formation in NIH/3T3 cells. Western analysis of NIH/3T3 cells overexpressing murine Ron and expressing endogenous levels of EGFR was used to demonstrate that Ron and EGFR co-immunoprecipitate. Stimulation of the cells in vitro with the Ron ligand HGFL or with the EGFR ligand epidermal growth factor (EGF) appeared to induce phosphorylation of both receptors. Co-immunoprecipitation and phosphorylation of phosphatidyl inositol 3-kinase (PI3-K) was also observed. This novel finding of a functional and biochemical interaction between Ron and EGFR suggests that heterologous tyrosine kinase receptor interactions may play a role in cellular processes such as scatter and transformation.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

GAPex-5 mediates ubiquitination, trafficking, and degradation of epidermal growth factor receptor

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.     

Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ΔEGFR

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal, we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.     

NOK/STYK1 has a strong tendency towards forming aggregates and colocalises with epidermal growth factor receptor in endosomes

Our previous studies showed that the overexpression of Novel Oncogene with Kinase-domain (NOK)/STYK1 led to cellular transformation, tumorigenesis and metastasis. This report characterises the subcellular distribution of NOK in HeLa cells and its localisation in early endosomes. Confocal immunolocalisation studies indicated that NOK had structural subtypes and was distributed into two distinct expression patterns: a dot pattern (DP) and an aggregation pattern (AP). The results of an immunohistochemistry (IHC) analysis of pathological tissues also showed that high expression level of endogenous NOK was expressed in an aggregate-like structure in vivo. Importantly, we found that NOK was localised in endosomes and colocalised with epidermal growth factor receptor (EGFR) in activated endosomal vesicles. However, as the stimulation time increased, NOK and EGFR began to progress through different pathways. EGFR was gradually degraded after treatment with EGF for approximately 20 min, whereas NOK levels were not reduced. This result suggests that NOK mainly plays a role in facilitating the trafficking of EGFR from early endosomes to later endosomes/lysosomes. Taken together, NOK has a strong tendency towards forming aggregates, which may have physiological implications and provide the first evidence that this novel receptor kinase is colocalised with EGFR in endosomes to participate in a post-internalisation step of EGFR.