Desensitization of the EGF receptor alters its ability to undergo EGF-induced dimerization

Treatment of A431 cells with EGF has been shown to induce the formation of EGF receptor dimers. Sucrose density gradient centrifugation as well as surface radio-iodination followed by crosslinking were used to study further the properties of EGF receptor monomers and dimers as well as the regulation of dimer formation. We have shown previously that treatment of A431 cells with high doses of EGF at 37 degrees C leads to the desensitization of the EGF receptor without a significant loss of cell surface 125I-EGF binding [Kuppuswamy and Pike (1989) J. biol. Chem. 264, 3357-3363; Cunningham et al. (1989) J. biol. Chem. 264, 15351-15356]. Desensitization of the EGF receptor led to a decrease in the ability of receptor monomers to be induced to form dimers by EGF both in vivo and in vitro. These data suggest that the sensitivity of a cell to EGF may be modulated by altering the capacity of the EGF receptor to form oligomers.     

Ligand-induced,receptor-mediated dimerization and activation of EGF receptor

The EGF receptor mediates many cellular responses in normal biological processes and in pathological states. Recent structural studies reveal the molecular basis for ligand binding specificity and how ligand binding induces receptor dimerization. Receptor dimerization is mediated by receptor-receptor interactions in which a loop protruding from neighboring receptors mediates receptor dimerization and activation.     

EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.     

Heparin activates PKR by inducing dimerization

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway. PKR is activated to undergo autophosphorylation upon binding to double-stranded RNAs or RNAs that contain duplex regions. Activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis. PKR is also activated by heparin, a highly sulfated glycosaminoglycan. We have used biophysical methods to define the mechanism of PKR activation by heparin. Heparins as short as hexasaccharide bind strongly to PKR and activate autophosphorylation. In contrast to double-stranded RNA, heparin activates PKR by binding to the kinase domain. Analytical ultracentrifugation measurements support a thermodynamic linkage model where heparin binding allosterically enhances PKR dimerization, thereby activating the kinase. These results indicate that PKR can be activated by small molecules and represents a viable target for the development of novel antiviral agents.     

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized cells, all antibodies were able to activate the EGF-R tyrosine kinase, as measured by EGF-R autophosphorylation and phosphorylation of other substrates on tyrosine residues. EGF-R tyrosine kinase activation correlated strongly with the induction of EGF-R dimerization. (i) Both processes specifically occurred in a narrow antibody concentration range; (ii) both processes required the presence of detergent; and (iii) both processes depended on antibody bivalence since monovalent Fab fragments were inactive yet regained full activity after cross-linking by a second bivalent antibody. These data demonstrate that antibody bivalence is essential and sufficient for EGF-R activation and that activation occurs regardless of the EGF-R epitope recognized. Finally, EGF-R dimerization was shown not to depend on receptor autophosphorylation since it still occurred in the absence of ATP. Also, partial inhibition of the tyrosine kinase activity by the specific EGF-R tyrosine kinase inhibitor tyrphostin AG 213 did not affect formation of EGF-R dimers. Taken together these results demonstrate that induction of EGF-R dimerization is sufficient and in case of antibody action, essential, for activation of the EGF-R tyrosine kinase and thus provide strong support for an intermolecular mechanism of EGF-R tyrosine kinase activation.     

The apoptosome activates caspase-9 by dimerization

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.     

Allosteric interactions prime androgen receptor dimerization and activation

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Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation,dimerization and EGF hormone binding

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N‐glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF–EGFR binding takes place through a large‐scale induced‐fitting mechanism. Proteins 2017; 85:561–570. © 2016 Wiley Periodicals, Inc.     

Hedgehog activates the EGF receptor pathway during Drosophila head development.

The Hedgehog (Hh) and Epidermal growth factor receptor (EGFR) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, we demonstrate a direct link between these two pathways in Drosophila melanogaster. Hh and EGFR signaling are each required for the formation of a specific region of the head of the adult fruitfly. We show that hh and vein (vn), which encodes a ligand of the Drosophila EGFR (Schnepp, B., Grumbling, G., Donaldson, T. and Simcox, A. (1996) Genes Dev. 10, 2302-13), are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, we demonstrate that Hh activates vn expression. We also show that Hh activation of vn is mediated through the gene cubitus interruptus (ci) and that this activation requires the C-terminal region of the Ci protein. Finally, we demonstrate that wingless (wg) represses vn expression, thereby limiting the domain of EGFR signaling.     

Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway

echinoid (ed) encodes an cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the EGFR signaling pathway during Drosophila eye development. Based on our previous genetic mosaic and epistatic analysis, we proposed that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes EGFR signaling. In this report, we demonstrate that Ed functions as a homophilic adhesion molecule and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting EGFR signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. In addition, Ed and Nrg colocalize in the Drosophila eye and are efficiently co-immunoprecipitated. Together, our results suggest a model in which Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes EGFR signaling.     

Activation of EGF receptor kinase by L1-mediated homophilic cell interactions

Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo.     

C-terminal dimerization activates the nociceptive transduction channel transient receptor potential vanilloid 1

Covalent modification of the specific cysteine residue(s) by oxidative stress robustly potentiates transient receptor potential vanilloid 1 (TRPV1) and sensitizes nociception. Here we provide biochemical evidence of dimerization of TRPV1 subunits upon exposure to phenylarsine oxide and hydrogen peroxide (H(2)O(2)), two chemical surrogates of oxidative stress. A disulfide bond formed between apposing cysteines ligates two C termini, serving as the structural basis of channel sensitization by oxidative covalent C-terminal modification. Systematic cysteine scanning of the C terminus of a cysteineless TRPV1 channel revealed a critical region within which any cysteine introduced phenylarsine oxide activation to mutant TRPV1. Oxidative sensitization persisted even when this region is substituted with a random peptide linker containing a single cysteine. So did insertion of this region to TRPV3, a homolog lacking the corresponding region and resistant to oxidative challenge. These results suggest that the non-conserved linker in the TRPV1 C terminus senses environmental oxidative stress and adjusts channel activity during cumulative oxidative damage by lowering the activation threshold of gating elements shared by TRPV channels.     

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.     

Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization

A cyclic decapeptide (1,

), which acts on the extracellular region of the EGF receptor, preventing it from dimerizing, has been developed. Peptide 2, which was labeled with fluorescein at the N-terminus of peptide 1, was synthesized based on structure–activity relationship studies. Peptide 2 essentially retained the inhibitory activity of peptide 1 against the receptor autophosphorylation. Confocal microscopy studies revealed that in carcinoma cells, the fluorescence of peptide 2 was localized inside some vesicles. Treatment of intact cells by peptide 1 in combination with peptide 2 decreased the fluorescence intensity significantly compared to treatment with only peptide 2. These results indicate that peptide 2 competes with peptide 1 for binding to the cellular surface. Six derivatives of peptide 2, in which constituent amino acids, with the exception of two cysteines and proline were randomized, were synthesized and used to treat the cells. Peptides 6 and 9 showed the highest fluorescence intensity in cells. From the results of the EGF receptor autophosphorylation assay, these two derivatives were proven to have higher inhibitory activity than peptide 2, which would therefore be a useful delivery peptide and fluorescent probe to find new inhibitors against the EGF receptor. Peptides 6 and 9 are promising leads for EGF receptor inhibitors.     

EGF induces increased ligand binding affinity and dimerization of soluble epidermal growth factor (EGF) receptor extracellular domain.

The binding of epidermal growth factor (EGF) to its cell surface receptor (EGF-R) results in a number of intracellular responses including the activation of the receptor intracellular tyrosine kinase. Receptor oligomerization induced by ligand binding has been suggested to play an important role in signal transduction. However, the mechanisms involved in oligomerization and signal transduction are poorly understood. We have produced and purified several milligrams of recombinant extracellular domain of the EGF receptor (EGF-Rx) using the baculovirus/insect cell expression system. The baculovirus-generated EGF-Rx is glycosylated, has had its signal peptide correctly cleaved, and exhibits a dissociation constant for EGF similar to that for solubilized full-length receptor, of about 100 nM. The binding of EGF to EGF-Rx leads to the formation of receptor dimers and higher oligomerization states which are irreversibly captured using the covalent cross-linking agent disuccinimidyl suberate. Interestingly, purified receptor monomers and dimers, stabilized by the cross-linker in the presence of EGF, exhibit increased binding affinity toward EGF as compared with receptor monomers which have not been exposed to EGF. It appears that the high affinity state of receptor can be maintained by the covalent cross-linking agent. These results indicate that in addition to ligand binding, the extracellular domain of EGF receptor possesses the inherent ability to undergo ligand-induced dimerization and that the low affinity state is converted to a high affinity state by EGF.     

How IGF-1 activates its receptor

This study presents a new model for IGF-I receptor activation in which the transmembrane domains are held apart until ligand binding brings them together in an activated state.     

Altering APP proteolysis: increasing sAPPalpha production by targeting dimerization of the APP ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers--which bind to dimers of the ectodomain--and Aβ fragments--which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP(695) 328-332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease.     

spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor

Signaling from the EGF receptor (EGFR) can trigger the differentiation of a wide variety of cell types in many animal species. We have explored the mechanisms that generate this diversity using the Drosophila peripheral nervous system. In this context, Spitz (SPI) ligand can induce two alternative cell fates from the dorsolateral ectoderm: chordotonal sensory organs and non-neural oenocytes. We show that the overall number of both cell types that are induced is controlled by the degree of EGFR signaling. In addition, the spalt (sal) gene is identified as a critical component of the oenocyte/chordotonal fate switch. Genetic and expression analyses indicate that the SAL zinc-finger protein promotes oenocyte formation and supresses chordotonal organ induction by acting both downstream and in parallel to the EGFR. To explain these findings, we propose a prime-and-respond model. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to raise the apparent threshold for induction by SPI. Subsequently, sal-dependent SAL upregulation is triggered as part of the oenocyte-specific EGFR response. Thus, a combination of SAL in the responding nucleus and increased SPI ligand production sets the binary cell-fate switch in favour of oenocytes. Together, these studies help to explain how one generic signaling pathway can trigger the differentiation of two distinct cell types.