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In the current study, a 2D similarity/docking-based study was used to predict the potential binding modes of icotinib, almonertinib, and olmutinib into EGFR. The similarity search of icotinib, almonertinib, and olmutinib against a database of 154 EGFR ligands revealed the highest similarity scores with erlotinib (0.9333), osimertinib (0.9487), and WZ4003 (0.8421), respectively. In addition, the results of the docking study of the three drugs into EGFR revealed high binding free energies (ΔGb = −6.32 to −8.42 kcal/mol) compared to the co-crystallized ligands (ΔGb = −7.03 to −8.07 kcal/mol). Analysis of the top-scoring poses of the three drugs was done to identify their potential binding modes. The distances between Cys797 in EGFR and the Michael acceptor sites in almonertinib and olmutinib were determined. In conclusion, the results could provide insights into the potential binding characteristics of the three drugs into EGFR which could help in the design of new more potent analogs.

The present investigation grounded on estimation of electron properties of the structures of EGFR proteins-ligand complexes using our laboratory-developed methodology AlteQ approach, which describes the molecular electron density of the complex in space for a certain point in three-dimensional coordinates. Briefly, the system embodies molecular electron density as a sum of Slater's type atomic increments of the molecular system. Further, using this methodology, we calculated different electron characteristics of selected EGFR protein-ligand complexes and established the relationship between different electron properties with their experimental pharmacological activity value (pIC

Background The epidermal growth factor receptor (EGFR) system is involved in cancer pathogenesis and serves as an important target for multiple cancer treatments. EGFR and its ligands epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), betacellulin (BTC), amphiregulin (AREG) and transforming growth factor α (TGF-α) have potential applications as prognostic or predictive serological biomarkers in cancer. The aim was to establish EGFR and EGFR ligand reference intervals in healthy women. Methods EGFR and EGFR ligands were measured in serum from 419 healthy women aged 26–78 years. The need for age partitioned reference intervals was evaluated using Lahti’s method. EGFR and EGF were analyzed using ELISA assays, whereas HB-EGF, BTC, AREG and TGF-α were analyzed using the highly sensitive automated single molecule array (Simoa) enabling detection below the lower reference limit for all six biomarkers. Results Reference intervals for EGFR and the EGFR ligands were determined as the 2.5th and 97.5th percentiles. All six biomarkers were detectable in all serum samples. For EGFR, EGF, HB-EGF and TGF-α, reference intervals were established for women 55 years, whilst common reference intervals were established for AREG and BTC including women aged 26–78 years. Conclusions Age specific reference intervals were determined for EGFR, EGF, HB-EGF, BTC, AREG and TGF-α.

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional ‘ring’ enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development., Author summary The EGFR signaling pathway plays a major role in innumerable developmental processes in all animals and its deregulation leads to different types of cancer, as well as many other developmental diseases in humans. Here we explored the integration of inputs from the Wnt- and TGF-beta signaling pathways and the leg-specifying transcription factors Distal-less and Sp1 at enhancer elements of EGFR ligands. These enhancers trigger a specific EGFR-dependent developmental output in the fly leg that is limited to specifying distal-most fates. Our findings suggest that activation of the EGFR pathway during fly leg development occurs through the activation of multiple EGFR ligand enhancers that are active at different positions along the proximo-distal axis. Similar enhancer elements are likely to control EGFR activation in humans as well. Such DNA elements might be ‘hot spots’ that cause formation of EGFR-dependent tumors if mutations in them occur. Thus, understanding the molecular characteristics of such DNA elements could facilitate the detection and treatment of cancer.

RNase 5 is a bona fide EGFR ligand that promotes oncogenic transformation in pancreatic cancer.

Background Prior to large studies in breast cancer patients and healthy individuals we established a sensitive three-plex immunoassay to measure the EGFR ligands amphiregulin (AR), betacellulin (BTC) and transforming growth factor α (TGF-α) simultaneously in human serum samples. Method The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 μL of serum. Results AR, BTC and TGF-α were first established as three single-plex assays. Multiplexing the three single-plex assays showed no significant cross reactivity between the reagents. The concentrations of the ligands in serum samples showed correlations r2 ≥ 0.84 between the single-plex and three-plex methods. The three-plex assay demonstrated limit of detection levels at 0.16 ng/L for AR, 0.23 ng/L for BTC and 0.22 ng/L for TGF-α. Total coefficients of variations were 8.5%–31% for AR, 11%–21.8% for BTC and 12.4%–16.2% for TGF-α. Spiking experiments showed a mean recovery of 97% for AR, 86% for BTC and 81% for TGF-α. The concentrations of the EGFR ligands did not change significantly after series of freeze thaw cycles or incubation at 22 °C for up to 24 h. Conclusion This robust three-plex assay with up to 40-fold increase in sensitivity relative to conventional ELISA is the first published method that has the required sensitivity to measure AR, BTC and TGF-α simultaneously in human blood samples.

This is the first of a comprehensive three-part review of the foundation for and therapeutic targeting of HER2/neu. No biological molecule in oncology has been more extensively or more successfully targeted than HER2/neu. This review will summarize the pertinent biology of HER2/neu and the EGF receptor family to which it belongs, with attention to the biological foundation for the design and clinical development of the entire range of HER2/neu-targeted therapies, including efforts to mitigate resistance mechanisms. In conjunction with the subsequent two parts (HER2/neu tissue expression and current HER2/neu-targeted therapeutics), this comprehensive survey will identify opportunities and promising areas for future evaluation of HER2/neu-targeted therapies, highlighting the importance of HER2/neu as an increasingly important therapeutic target.

Quantitative information about the distribution of morphogens is crucial for understanding their effects on cell-fate determination, yet it is difficult to obtain through direct measurements. We have developed a parameter estimation approach for quantifying the spatial distribution of Gurken, a TGFalpha-like EGFR ligand that acts as a morphogen in Drosophila oogenesis. Modeling of Gurken/EGFR system shows that the shape of the Gurken gradient is controlled by a single dimensionless parameter, the Thiele modulus, which reflects the relative importance of ligand diffusion and degradation. By combining the model with genetic alterations of EGFR levels, we have estimated the value of the Thiele modulus in the wild-type egg chamber. This provides a direct characterization of the shape of the Gurken gradient and demonstrates how parameter estimation techniques can be used to quantify morphogen gradients in development.

Background: Whilst RAS mutations predict which aCRC patients (pts) will not benefit from anti-EGFR agents, RAS-wt status does not reliably predict who will. Several studies report that high EGFR ligand expression (EREG/AREG) is predictive of anti-EGFR agent benefit but progression towards clinical utility is limited by lack of consensus on a clinical dichotomisation point and additional tumor material required. Here we explore EGFR copy number as a biomarker of prognosis and predictor of Pan benefit in a randomised trial in aCRC. Methods: EGFR CN, EREG/AREG RNA expression, RAS/RAF mutations were assessed in tumor from 275 pts randomised to 2nd-line irinotecan (Ir) or IrPan (PICCOLO, Lancet Onc 14:749-59). EGFR CN status was measured by the Affymetrix OncoScan array, analysed using Biodiscovery Nexus software and defined as normal (2 copies) or gain (>2 copies). Prognostic analysis was in Ir alone pts. Predictive analysis, in the 234 RAS-wt pts, compared baseline values with outcomes using Cox proportional hazards models. Results: 196 (71.3%) pts were classified as EGFR gain and 79 (28.7%) as normal. EGFR gain was significantly associated with high EREG and AREG RNA expression (both p

Although an exogenous EGF is dispensable for EGFRL858R-mediated NIH3T3 cell transformation in vitro (Greulich et al., PLoS Med 2005), an exogenous EGF promotes growth of H1650 and H1975 lung adenocarcinoma cells that carry EGFR mutations in vitro (Sordella et al., Science 2004). However, it is unknown whether an exogenous EGFR ligand plays a role in growth of EGFR-mutant lung tumors in vivo. Here, using conditional transgenic mice expressing EGFRL858R or KRASG12D along with an exogenous TGFalpha (an EGFR ligand) in lung epithelium, we sought to determine the role of an EGFR ligand in EGFRL858R-lung tumors or wild type EGFR-lung tumors (KRASG12D-lung tumors) in vivo. As previously reported, conditional expression of TGFalpha (an EGFR ligand) in lung epithelium caused pulmonary fibrosis but not lung tumors (Korfhagen et al., J Clin Invest 1994; Hardie et al., AJP-LCMP 2004), indicating that the expression of an exogenous EGFR ligand is not sufficient to induce lung tumors. Conditional expression of EGFRL858R or KRASG12D in lung epithelium caused lung tumors as previously reported (Fisher et al., Genes Dev 2001; Politi et al., Gene Dev 2006). In our study using a new rtTA driver, Scgb1a1-rtTA line 2 (Perl et al., AJRCMB 2006), the majority of EGFRL858R-lung tumors occurred in alveolar regions while KRASG12D-lung tumors occurred in both alveolar and airway regions. Importantly, co-expression of EGFRL858R along with TGFalpha in lung epithelium induced the growth of lung tumors in airway regions but not in alveolar regions while the expression of EGFRL858R alone did not induce lung tumors in airway regions. Co-expression of EGFRL858R along with TGFalpha also shortened survival of the mice compared to the expression of either EGFRL858R or TGFalpha alone (median 32 days for EGFRL858R/TGFalpha vs 118 days for EGFRL858R alone or 248 days for TGFalpha alone). Co-expression of KRASG12D with TGFalpha did not influence tumor location or survival of the mice compared to the expression of either KRASG12D or TGFalpha alone. Consistent with the mouse study, TGFalpha expression in human EGFR-mutant lung cancer but not wild-type EGFR lung cancer was associated with worse overall survival (median 44 months for TGFalpha high vs 75 months for TGFalpha low; p Citation Format: Koichi Tomoshige, Yutaka Maeda. TGFalpha (an EGFR ligand) promotes growth of EGFR-mutant lung tumors in airway regions but not in alveolar regions in a transgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1838. doi:10.1158/1538-7445.AM2017-1838

3590 Background: Arginine methylation of the epidermal growth factor receptor (meEGFR) increases binding affinity of EGF and other EGFR ligands, reduces the efficacy of anti-EGFR agents in vivo, and is reported to have a role in predicting response to anti-EGFR agents. This study aimed to investigate the predictive impact of meEGFR in metastatic colorectal cancer (mCRC) patients (pts) treated with anti-EGFR agents using blood-based testing. Methods: 15 mL of blood were collected from mCRC pts with documented disease progression following anti-EGFR treatment (Rx). Circulating tumor cells (CTCs) were isolated using antibody (ab)-independent micro-fluidic cassette-based technology (Parsortix system), which separates CTCs on the basis of size and deformability. CTCs were identified based on negative staining for CD45ab and positive staining for EpCAMab. meEGFR was identified based on positive staining for me-R198/200ab on CTCs. Associations between meEGFR-CTCs and total CTCs with progression free survival (PFS) were determined by Kaplan-Meier method and compared by the log-rank test. Results: A total of 47 mCRC pts were prospectively included in this study. CTCs were identified in 30 out of 47 cases (64%). Of those 30, meEGFR-CTCs were identified in 19 cases (63%). Mean total CTCs and meEGFR-CTCs counts were 3.6 (range 0-52) and 2.3 (range 0-30) cells per 7.5ml, respectively. There was no association between meEGFR-CTCs and clinic-pathological features (age, sex, tumor site & grade), line of anti-EGFR Rx, previous irinotecan used, or NRAS, BRAF, PIK3CA, and MSI status. However, in RASwt/BRAFwtmCRC pts, high levels of meEGFR ratio (defined as > 0.25 meEGFR-CTCs per total CTCs) was associated with significantly inferior PFS with anti-EGFR Rx (median PFS 5.4 mo vs. 8 mo, HR 3.4, 95% CI of 1.5-7.9, P = 0.004). By contrast, high levels of total CTCs ( > 3 cells/per 7.5 ml) had no impact on PFS with anti-EGFR Rx. Conclusions: We have successfully isolated CTCs from mCRC pts’ blood using Parsortix system. Elevated levels of arginine methylated EGFR is associated with a shorter PFS with anti-EGFR-based Rx. Assessment of meEGFR-CTCs may provide a “liquid biopsy” biomarker for reduced efficacy from anti-EGFR Rx.

Secreted signaling molecules typically float in the outer leaflet of the plasma membrane, or freely diffuse away from the signaling cell, suggesting that a signal should be sensed equally by all neighboring cells. However, we demonstrate that Spitz (Spi) mediated Epidermal Growth Factor Receptor (EGFR) signaling is spatially biased to selectively determine the induction of a single bract cell on the proximal side of each mechanosensory organ on the Drosophila leg. Dynamic and oriented cellular protrusions emanating from the socket cell, the source of Spi, robustly favor the Spi/EGFR signaling response in a particular cell among equally competent neighbors. We propose that these protrusive structures enhance signaling by increasing contact between the signaling and responding cells. The planar polarized direction of the protrusions determines the direction of the signaling outcome. This asymmetric cell signaling serves as a developmental mechanism to generate spatially patterned cell fates.

BACKGROUND: In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas. METHODS: Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx. RESULTS: Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis. CONCLUSION: Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.

Nicole K. Nickerson1, Jennifer L. Gilmore1, Kah Tan Allen1, David J. Riese II2, Kenneth P. Nephew1,3 and John Foley1,3,4 1Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 2Harrison School of Pharmacy, Auburn University, Auburn, AL 3Indiana University Cancer Center Indiana University School of Medicine, Indianapolis, IN 4Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN United States of America

Intracrine action is increasingly being appreciated as a physiologically relevant signaling mechanism. Growing out of the study of angiotensin biology, intracrine physiology is becoming better understood and general principles of intracrine action have been proposed. Here the field will be briefly reviewed and some predictions of intracrine theory discussed to illustrate these principles of intracrine action. The potential relevance of these ideas to the working of the local renin–angiotensin systems and to diverse other biological processes such as differentiation and neoplasia is discussed.

We aim to develop protein therapeutics that neutralize growth factors that activate EGF receptor family members in breast cancer. Rather than targeting receptors themselves (as do Herceptin, Iressa, etc), we propose to target the activating ligands. Our model is Argos from Drosophila, which we showed naturally inhibits EGF receptor signaling in fruit flies by inactivating the ligand. We hope to effectively humanize Argos - making it bind human EGFR ligands and/or to use human protein scaffolds for this. In the past year, we crystallized a complex between the minimal functional fragment of Argos and its target (Spitz), and are about to complete structure determination which will provide critical information for therapeutic design. We also established an experimental approach for screening libraries of Argos variants for those that bind human EGF-like ligands (our therapeutic aim). This approach employs yeast surface (rather than phage) display. We are now poised to combine our technical position and new structural information to identify Argos (and Dkk) variants that bind human EGFs and represent starting points for developing new therapeutics.

EGF receptor (EGFR) promotes intestinal epithelial restitution, an important early process in the reepithelialization of ulcers. During epithelial restitution, the mechanism of EGFR activation is not known. We evaluated the role of TNF-converting enzyme (TACE), a metalloprotease disintegrin that proteolytically processes plasma membrane-anchored EGFR ligand precursors into their mature active forms, in wound-induced EGFR activation and epithelial restitution. With the use of scrape-wounded rat intestinal epithelial-1 (RIE-1) cell monolayers to model epithelial ulceration and restitution, we observed the rapid wound-dependent release of EGFR ligands into culture medium. RIE-1 cells express TACE, and treatment with phorbol ester, an established TACE stimulus, triggered the extracellular release of an EGFR ligand, transforming growth factor-α. Blockade of TACE using TNF processing inhibitor (TAPI-1), a specific hydroxamate inhibitor of metalloprotease disintegrins, prevented release of EGFR ligands from wounded RIE-1 cell monolayers. The restitution of wounded RIE-1 cell monolayers was also dose-dependently inhibited by TAPI-1, establishing the role of metalloprotease disintegrins in this process. These results have established a mechanism of EGFR activation in wounded intestinal epithelium and show an important functional role for metalloprotease disintegrin-mediated ectodomain shedding during intestinal epithelial restitution. Therefore, activation of the TACE-EGFR system might promote the healing of intestinal tract ulcers in patients.

[Graphic][1] Cells surround the source of EGFR ligand (green) three at a time. GOULD/ELSEVIERA specialized cell type in flies leaves its specifying hub in groups of 3 cells, according to Veronique Brodu, Philip Elstob, and Alex (NIMR, Mill Hill, London, UK). Each group gets

EGFR requires ADAM17 activity to preserve skin barrier homeostasis., ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17ΔKC) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17ΔKC skin, and topical treatment of A17ΔKC mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (EgfrΔKC) closely resembled A17ΔKC mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17–EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects.

3519^ Background: We investigated the expression of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG) as well as the amplification of the EGFR-gene in tumor specimens of mCRC patients (pts) treated first-line with anti-EGFR targeted cetuximab together with CAPOX or CAPIRI. Expression levels were correlated with overall response rate (ORR), progression free survival (PFS) and overall survival (OS) to determine their relationship with effectiveness in this setting. Methods: A total of 185 mCRC pts were randomized to cetuximab (400mg/m² day 1, followed by 250mg/m² weekly) plus CAPIRI (irinotecan 200mg/m², day 1; capecitabine 800mg/m² twice daily days 1-14, every 3 weeks; 20% dose reduction of both agents for pts older than 65 years) or plus CAPOX (oxaliplatin 130mg/m² day 1; capecitabine 1000mg/m² twice daily days 1-14, every three weeks). The primary study endpoint was ORR. KRAS mutational status did not correlate with treatment outcome. The cut-offs for EGFR-amplification using FISH, AREG and EREG levels determined by RT-qPCR were calculated using ROC analysis for ORR. Results: Within the subgroup of KRAS wildtype tumors, analysis of EREG- and AREG-expression was possible in 99 pts and of EGFR-amplification in 63 pts. Higher AREG levels correlated significantly with higher ORR (83% vs 46%, p=0.006, OR 0.31), longer PFS (9.6mo vs 4.9, p

e16017 Background: Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancer, but no predictive biomarkers of cetuximab activity have yet been identified. Methods: We report on a patient with HNSCC who had a complete tumor regression following treatment with cetuximab given as a single agent after initial surgery and radiation therapy. The EGFR protein expression level, gene copy number and sequence were assessed from both normal and tumor tissues from this patient. Results: Besides protein overexpression and gene amplification in the tumor tissue, sequencing of the EGFR gene from the patient revealed the presence of two somatic mutations, one in the kinase domain (R705G) and the other in the ligand binding domain (P546S). Cells that stably express these EGFR mutants were treated with cetuximab and their sensitivity to the drug was compared to cells expressing the wildtype gene. While the P546S mutation sensitized NIH-3T3 cells to cetuximab, R705G had a marginal effect. The double mutant (P546S/R705G) behaved the same as the P546S mutant, indicating that the mutation in the kinase domain does not contribute to the increased sensitivity to cetuximab. No mutations were found in K-RAS or B-RAF genes and there was no indication that the tumor was HPV-positive. Conclusions: Our results support a role for the P546S mutation in cetuximab sensitivity. To our knowledge, this is the first report of a somatic mutation in the EGFR ligand binding domain that may contribute to increased sensitivity to cetuximab. Other factors including EGFR copy number, EGFR over-expression and the immune response, as indicated by a very adverse side effect that correlated with the antitumor activity, may have also contributed to the observed response. It remains to be determined how frequently this mutation occurs in patients with HNSCCs and other cancers. Prospective evaluation of cetuximab anti-tumor activity in patients harboring P546S mutation needs to be clinically evaluated.

Researchers at the Weizmann Institute of Science have shown that antibodies targeting tumor-specific EGFR ligands could boost the effectiveness of standard chemotherapies to treat pancreatic and other cancers.

U.S. researchers have identified an EGFR-mimicking peptide that blocks tumor growth by luring ligands away from the receptor, thus preventing its activation. The decoy could have advantages over marketed EGFR inhibitors, although its pharmacokinetic and toxicity profiles will have to be worked out first.

Argos, a secreted inhibitor of the Drosophila epidermal growth factor receptor, and the only known secreted receptor tyrosine kinase inhibitor, acts by sequestering the EGFR ligand Spitz. We use computational modeling to show that this biochemically-determined mechanism of Argos action can explain available genetic data for EGFR/Spitz/Argos interactions in vivo. We find that efficient Spitz sequestration by Argos is key for explaining the existing data and for providing a robust feedback loop that modulates the Spitz gradient in embryonic ventral ectoderm patterning. Computational analysis of the EGFR/Spitz/Argos module in the ventral ectoderm shows that Argos need not be long-ranged to account for genetic data, and can actually have very short range. In our models, Argos with long or short length scale functions to limit the range and action of secreted Spitz. Thus, the spatial range of Argos does not have to be tightly regulated or may act at different ranges in distinct developmental contexts.

The EGFR signalling cascade is responsible for coordinating a wide variety of events during Drosophila eye development. It remains something of a mystery how it is that cells are able to interpret the signal so as to choose the appropriate response from the battery of possibilities: division, differentiation, cell shape change and so on. Since the cascade is essentially linear below the receptor, different cellular responses cannot be regulated by alternative signal transduction pathways. The main diversity lies upstream, in the multiple activating ligands. Spitz, Gurken and Vein have been long studied, but little is known about the physiological functions of the fourth ligand, Keren, although various roles have been predicted based on the differences between mutants in the known ligands and those of the receptor. Here, we have isolated a mutant in the keren gene, and demonstrate that Keren does indeed participate in EGFR signalling in the eye, where it acts redundantly with Spitz to control R8 spacing, cell clustering and survival. Thus, specificity cannot be determined by ligand choice, and must instead be a consequence of cell-intrinsic factors, although we speculate that there may be some quantitative differences in signalling elicited by the two ligands.

All ligands of the epidermal growth factor receptor (EGFR) are made as membrane anchored precursors that can be proteolytically processed and released from the plasma membrane. This process, which is referred to as protein ectodomain shedding, is emerging as a key regulator of the function of EGFR ligands. In light of the important roles of EGFR signaling in development and disease, it will be important to understand more about the regulation of proteolytic processing of EGFR ligands. This chapter describes a sensitive and semiquantitative method to measure ectodomain shedding of EGFR ligands that was designed to facilitate studies of this process in cells.