Drosophila Epidermal Growth Factor Receptor Research Articles

Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye.

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.

Extracellular matrix induced by steroids and aging through a G-protein-coupled receptor in a Drosophila model of renal fibrosis.

ABSTRACTAldosterone is produced by the mammalian adrenal cortex to modulate blood pressure and fluid balance; however, excessive, prolonged aldosterone promotes fibrosis and kidney failure. How aldosterone triggers disease may involve actions independent of its canonical mineralocorticoid receptor. Here, we present a Drosophila model of renal pathology caused by excess extracellular matrix formation, stimulated by exogenous aldosterone and by insect ecdysone. Chronic administration of aldosterone or ecdysone induces expression and accumulation of collagen-like Pericardin in adult nephrocytes – podocyte-like cells that filter circulating hemolymph. Excess Pericardin deposition disrupts nephrocyte (glomerular) filtration and causes proteinuria in Drosophila, hallmarks of mammalian kidney failure. Steroid-induced Pericardin production arises from cardiomyocytes associated with nephrocytes, potentially reflecting an analogous role of mammalian myofibroblasts in fibrotic disease. Remarkably, the canonical ecdysteroid nuclear hormone receptor, Ecdysone receptor (EcR), is not required for aldosterone or ecdysone to stimulate Pericardin production or associated renal pathology. Instead, these hormones require a cardiomyocyte-associated G-protein-coupled receptor, Dopamine-EcR (DopEcR), a membrane-associated receptor previously characterized in the fly brain to affect behavior. DopEcR in the brain is known to affect behavior through interactions with the Drosophila Epidermal growth factor receptor (Egfr), referred to as dEGFR. Here, we find that the steroids ecdysone and aldosterone require dEGFR in cardiomyocytes to induce fibrosis of the cardiac-renal system. In addition, endogenous ecdysone that becomes elevated with age is found to foster age-associated fibrosis, and to require both cardiomyocyte DopEcR and dEGFR. This Drosophila renal disease model reveals a novel signaling pathway through which steroids may modulate mammalian fibrosis through potential orthologs of DopEcR.

The role of Sevenless in Drosophila R7 photoreceptor specification

Sevenless (Sev) is a Receptor Tyrosine Kinase (RTK) that is required for the specification of the Drosophila R7 photoreceptor. Other Drosophila photoreceptors are specified by the action of another RTK; the Drosophila EGF Receptor (DER). Why Sev is required specifically in the R7 precursor, and the exact role it plays in the cell's fate assignment have long remained unclear. Notch (N) signaling plays many roles in R7 specification, one of which is to prevent DER activity from establishing the photoreceptor fate. Our current model of Sev function is that it hyperactivates the RTK pathway in the R7 precursor to overcome the N-imposed block on photoreceptor specification. From this perspective DER and Sev are viewed as engaging the same transduction machinery, the only difference between them being the level of pathway activation that they induce. To test this model, we generated a Sev/DER chimera in which the intracellular domain of Sev is replaced with that of DER. This chimerical receptor acts indistinguishably from Sev itself; a result that is entirely consistent with the two RTKs sharing identical transduction abilities. A long-standing question in regard to Sev is the function of a hydrophobic domain some 60 amino acids from the initiating Methionine. If this represents a transmembrane domain, it would endow Sev with N-terminal intracellular sequences through which it could engage internal transduction pathways. However, we find that this domain acts as an internal signal peptide, and that there is no Sev N-terminal intracellular domain. phyllopod (phyl) is the target gene of the RTK pathway, and we show that R7 precursors are selectively lost when phyl gene function is mildly compromised, and that other photoreceptors are removed when the gene function is further reduced. This result adds a key piece of evidence for the hyperactivation of the RTK pathway in the R7 precursor. To facilitate the hyperactivation of the RTK pathway, Sev is expressed at high levels. However, when we express DER at the levels at which Sev is expressed, strong gain-of-function effects result, consistent with ligand-independent activation of the receptor. This highlights another key feature of Sev; that it is expressed at high levels yet remains strictly ligand dependent. Finally, we find that activated Sev can rescue R3/4 photoreceptors when their DER function is abrogated. These results are collectively consistent with Sev and DER activating the same transduction machinery, with Sev generating a pathway hyperactivation to overcome the N-imposed block to photoreceptor specification in R7 precursors.

The Drosophila Epidermal Growth Factor Receptor does not act in the nucleus

Mammalian members of the ErbB family, including the epidermal growth factor receptor (EGFR), can regulate transcription, DNA replication and repair through nuclear entry of either the full-length proteins or their cleaved cytoplasmic domains. In cancer cells, these nuclear functions contribute to tumor progression and drug resistance. Here, we examined whether the single Drosophila EGFR can also localize to the nucleus. A chimeric EGFR protein fused at its cytoplasmic C-terminus to DNA-binding and transcriptional activation domains strongly activated transcriptional reporters when overexpressed in cultured cells or in vivo However, this activity was independent of cleavage and endocytosis. Without an exogenous activation domain, EGFR fused to a DNA-binding domain did not activate or repress transcription. Addition of the same DNA-binding and transcriptional activation domains to the endogenous Egfr locus through genome editing led to no detectable reporter expression in wild-type or oncogenic contexts. These results show that, when expressed at physiological levels, the cytoplasmic domain of the Drosophila EGFR does not have access to the nucleus. Therefore, nuclear EGFR functions are likely to have evolved after vertebrates and invertebrates diverged.

Graf regulates hematopoiesis through GEEC endocytosis of EGFR.

GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. We show that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. We also find that loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on our findings, we propose a model in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation.

TGF-α ligands can substitute for the neuregulin Vein in Drosophila development.

ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-α-type and a single neuregulin-like called vein (vn). Here, we used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, we found that the essential role of vn in development could be largely replaced by expression of any TGF-α ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. Our results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity.

The rhomboid-like superfamily: molecular mechanisms and biological roles.

The rhomboid proteases were first discovered as regulators of Drosophila EGF receptor signaling; soon after, it was recognized that they represented the founder members of a widespread family of intramembrane serine proteases conserved in all kingdoms. More recently still, the family was promoted to a superfamily, encompassing a wide variety of distantly related proteins. One of the surprises has been that many members of the rhomboid-like superfamily are not active proteases. Given the size of this clan, and its relatively recent discovery, there is still much to learn. Nevertheless, we already understand much about how rhomboid proteases perform their surprising function of cleaving transmembrane domains. We also already know that members of the rhomboid-like superfamily participate in biological functions as diverse as growth factor signaling, mitochondrial dynamics, inflammation, parasite invasion, and the machinery of protein quality control. Their potential medical significance is now becoming apparent in several areas.

Maternal control of the Drosophila dorsal-ventral body axis.

The pathway that generates the dorsal-ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. For further resources related to this article, please visit the WIREs website. The authors have declared no conflicts of interest for this article.

The Drosophila Chmp1 protein determines wing cell fate through regulation of epidermal growth factor receptor signaling.

Receptor down-regulation by the multivesicular body (MVB) pathway is critical for many cellular signaling events. MVB generation is mediated by the highly conserved ESCRT (0, I, II, and III) protein complexes. Chmp1 is an ESCRT-III component and a putative tumor suppressor in humans. However, published data on Chmp1 activity are conflicting and its role during tissue development is not well defined. We investigated the function of Drosophila Chmp1 and found that it is an essential gene. In the wing, loss of Chmp1 activity causes a cell fate change from intervein to vein, and interactions between Chmp1 and Drosophila Epidermal Growth Factor Receptor (DER) regulators suggest that Chmp1 negatively regulates DER signaling. Chmp1 knockdown also decreases Blistered expression, which is repressed by DER signaling. We find that Chmp1 protein localizes to the late endosome in Drosophila embryos, which is consistent with its effects on DER signaling resulting from its function in the ESCRT-III complex. Drosophila Chmp1 negatively regulates DER signaling, likely through its role in MVB formation. Loss of Chmp1 activity in the Drosophila wing induces a cell fate change from intervein to vein that should provide a useful tool for future studies of ESCRT protein activity.

The Elongin Complex Antagonizes the Chromatin Factor Corto for Vein versus Intervein Cell Identity in Drosophila Wings

Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG) and Enhancers of Trithorax and Polycomb (ETP) families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor – MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. We show here that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C), increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. We propose that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid.

Trafficking of the EGFR ligand Spitz regulates its signaling activity in polarized tissues

Summary Epidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but that it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, whereas membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Taken together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.

Her2 activation mechanism reflects evolutionary preservation of asymmetric ectodomain dimers in the human EGFR family

The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human. DOI:http://dx.doi.org/10.7554/eLife.00708.001.

Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways

Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC50 values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood–brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects.

Molecular dynamics simulations of transitions for ECD epidermal growth factor receptors show key differences between human and drosophila forms of the receptors

Recent X-ray structural work on the Drosophila epidermal growth factor receptor (EFGR) has suggested an asymmetric dimer that rationalizes binding affinity measurements that go back decades (Alvarado et al., Cell 2010;142:568-579; Dawson et al., Structure 2007;15:942-954; Lemmon et al., Embo J 1997;16:281-294; Mattoon et al., Proc Natl Acad Sci USA 2004;101:923-928; Mayawala et al., Febs Lett 2005;579:3043-3047; Ozcan et al., Proc Natl Acad Sci USA 2006;103:5735-5740). This type of asymmetric structure has not been seen for the human EGF receptor family and it may or may not be important for function in that realm. We hypothesize that conformational changes in the Drosophila system have been optimized for the transition, whereas the barrier for the same transition is much higher in the human forms. To address our hypothesis we perform dynamic importance sampling (DIMS) (Perilla et al., J Comput Chem 2010;32:196-209) for barrier crossing transitions in both Drosophila and human EFGRs. For each set of transitions, we work from the hypothesis, based on results from the AdK system, that salt-bridge pairs making and breaking connections are central to the conformational change. To evaluate the effectiveness of the salt-bridges as drivers for the conformational change, we use the effective transfer entropy based on stable state MD calculations (Kamberaj and Der Vaart, Biophys J 2009;97:1747-1755) to define a reduced subset of degrees of freedom that seem to be important for driving the transition (Perilla and Woolf, J Chem Phys 2012;136:164101). Our results suggest that salt-bridge making and breaking is not the dominant factor in driving the symmetric to asymmetric transition, but that instead it is a result of more concerted and correlated functional motions within a subset of the dimer structures. Furthermore, the analysis suggests that the set of residues involved in the transitions from the Drosophila relative to the human forms differs and that this difference in substate distributions relates to why the asymmetric form may be more common to Drosophila than to the human forms. We close with a discussion about the residues that may be changed in the human and the Drosophila forms to potentially shift the kinetics of the symmetric to asymmetric transition.

Trafficking of the EGFR ligand Spitz regulates its signaling activity in polarized tissues

Epidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but that it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, whereas membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Taken together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.

The Cell Surface Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Contributes to Epidermal Growth Factor Receptor-mediated Cell Migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Human epidermal growth factor receptor (HER1) aligned on the plasma membrane adopts key features of Drosophila EGFR asymmetry

Current models suggest that ligand-binding heterogeneity in HER1 [human EGFR (epidermal growth factor receptor] arises from negative co-operativity in signalling HER1 dimers, for which the asymmetry of the extracellular region of the Drosophila EGFR has recently provided a structural basis. However, no asymmetry is apparent in the current crystal structure of the isolated extracellular region of HER1. This receptor also differs from the Drosophila EGFR in that negative co-operativity is found only in full-length receptors in cells. Structural insights into HER1 in epithelial cells, derived from FLIM (fluorescence lifetime imaging microscopy) and two-dimensional FRET (Förster resonance energy transfer) combined with Monte Carlo and molecular dynamics simulations, have demonstrated a high-affinity ligand-binding HER1 conformation consistent with the extracellular region aligned flat on the plasma membrane. This conformation shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative co-operativity is conserved from invertebrates to humans, but that, in HER1, the extracellular region asymmetry requires interactions with the plasma membrane.

Yan, an ETS‐domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye

We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan(-/-) cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways.

Human Epidermal Growth Factor Receptor (EGFR) Aligned on the Plasma Membrane Adopts Key Features of Drosophila EGFR Asymmetry

The ability of epidermal growth factor receptor (EGFR) to control cell fate is defined by its affinity for ligand. Current models suggest that ligand-binding heterogeneity arises from negative cooperativity in signaling receptor dimers, for which the asymmetry of the extracellular region of the Drosophila EGFR has recently provided a structural basis. However, no asymmetry is apparent in the isolated extracellular region of the human EGFR. Human EGFR also differs from the Drosophila EGFR in that negative cooperativity is found only in full-length receptors in cells. To gain structural insights into the human EGFR in situ, we developed an approach based on quantitative Förster resonance energy transfer (FRET) imaging, combined with Monte Carlo and molecular dynamics simulations, to probe receptor conformation in epithelial cells. We experimentally demonstrate a high-affinity ligand-binding human EGFR conformation consistent with the extracellular region aligned flat on the plasma membrane. We explored the relevance of this conformation to ligand-binding heterogeneity and found that the asymmetry of this structure shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative cooperativity is conserved from invertebrates to humans but that in human EGFR the extracellular region asymmetry requires interactions with the plasma membrane.

Dynamic Importance Sampling Calculations on ECD Epitelial Growth Factor Receptor Conformational Changes

The epidermal growth factor receptor (EGFR) is a major target for cancer drug development. Multiple x-ray structures provide some indications of the conformational changes that occur within the extracellular domain (ECD) upon activation by a growth factor ligand. These x-ray structures suggest a change at both the monomer level and at the dimer level. Yet the molecular details of how the conformations change with activation are not fully understood. We present calculations for the monomer of the transitions from a the tethered to an extended form of human EGFR, human ErbB2 and Drosophila EGFR. Our DIMS calculations for the dimer explore the recent asymmetric Drosophila dimer structure and create transitions from the symmetric to the asymmetric forms for the human EGFR and the Drosophila EGFR. The computations provide estimates of candidate reaction paths between the conformations as well as suggesting the effects of ligand binding and the transitions from the extracellular domains to the kinase activation.