EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis.

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

Regulation of Hippo signaling by EGFR-MAPK signaling through Ajuba family proteins.

EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. We find that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrate that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. Our observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways.

ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor.

The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

Negative regulation of Egfr/Ras pathway by Ultrabithorax during haltere development in Drosophila.

In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development (E.B. Lewis, 1978. A gene complex controlling segmentation in Drosophila. Nature 276, 565-570). Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Here, we report that down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways.

Argos inhibits epidermal growth factor receptor signalling by ligand sequestration.

The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells.

The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.

SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signalling in the imaginal wing disc.

We have cloned a novel SOCS gene from Drosophila, socs36E, which is most homologous to the mammalian socs-5 gene. Socs36E is expressed zygotically, predominantly during embryogenesis, in a highly dynamic pattern. In vivo expression of SOCS36E in transgenic flies results in several adult phenotypes. Engrailed-GAL4 directed expression causes loss of the wing anterior cross vein, humeral outgrowths, absence of halteres and eye pigmentation defects. Expression of SOCS36E under apterous-GAL4 control resulted in outstretched wings. Full penetrance of these phenotypes required the presence of the SH2 and SOCS-box domains of SOCS36E. The observed phenotypes were consistent with defects in JAK/STAT or EGF-R signalling and were exacerbated in flies heterozygous for either the d-jak (hopscotch), d-stat (stat92E) or d-egf-r (der) genes. Conversely, inactivating one copy of the d-cbl gene, a negative regulator of the d-EGF-R, partially rescued the wing phenotypes. These genetic interactions imply that SOCS36E can suppress activities of the JAK/STAT and EGF-R signalling pathways in the wing disc and suggest that SOCS36E interacts with multiple pathways in vivo.

Ras promotes cell survival in Drosophila by downregulating hid expression.

Activation of Ras inhibits apoptosis during Drosophila development. Genetic evidence indicates that Ras antiapoptotic activity in the developing eye is regulated by the Drosophila EGF receptor and operates through the Raf/MAPK pathway. Decreased activity of this pathway enhances, and increased activity suppresses, apoptosis induced by ectopic expression of the cell death regulators reaper (rpr) and head involution defective (hid). In addition, ectopic activation of the Ras/MAPK pathway in the developing embryo and in the developing eye suppresses naturally occurring apoptosis and regulates the transcription of the proapoptotic gene hid. Null alleles of hid recapitulate the antiapoptotic activities of Ras/MAPK, providing genetic evidence that downregulation of hid is an important mechanism by which Ras promotes survival.

The effect of FMRFamide analogs on [35S]GTP-gamma-S stimulation in squid optic lobes.

Pharmacological study of Phe-Met-Leu-Phe-amide (FMRFa) receptors is hindered by the lack of selective ligands. The classification of these selective ligands is further hampered by the limited availability of functional assays. In this study, we evaluated several synthetic FMRFa analogs for agonist and antagonist activity by measuring their abilities to produce [35-S]-GTP-gamma-S stimulation or to inhibit FMRFa-induced [35S]-GTP-gamma-S binding in squid optic lobes. Analogs included acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa), desamino-Tyr-Phe-Leu-Arg-amide (daYFLRa), desamino Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa), desamino Tyr-Phe-norLeu-Arg-[TIC]-amide (daYFnLR[TIC]a), desamino Tyr-Trp-norLeu-Arg-amide (daYWnLRa), (D)-Tyr-Phe-norLeu-Arg-Phe-amide (D)-YFnLRFa), Phe-Leu-Arg-Phe-amide (FLRFa), and the D-amino acid analogs of FMRFa (D-FMRFa, F-(D)-MRFa and FM-(D)-RFa). For agonist studies, full dose-response curves were generated and analyzed for potency and efficacy (maximal percent effect). FMRFamide as well as analogs ac-FnLRFa, daYFnLRFa, daYFnLR[TIC]a, D-YFnLRFa, FLRFa, and (D)-FMRFa stimulated [35S]-GTP-gamma-S binding. Analogs daYWnLRa, daYFLRa, F-(D)-MRFa, and FM-(D)-RFa failed to stimulate either [35S]-GTP-gamma-S binding or to inhibit FMRFa-induced [35S]-GTP-gamma-S binding. The rank order of potency was daYFnLRFa > or = daYFnLRF[TIC]a > acFnLRFa > (D)YFnLRFa > FLRFa > or = FMRFa >> (D)-FMRFa. The order of efficacy was daYFnLRFa = acFnLRFa = (D)-YFnLRFa > FLRFa = FMRFa > or = (D)-FMRFa > or = daYFnLRF[TIC]a. Peptide analog daYFnLR[TIC]a was less efficacious (59% maximal stimulation) than analogs daYFnLRFa, acFnLRFa, and (D)-YFnLRFa (113-146% maximal stimulation). A maximal concentration of daYFnLR[TIC]a (10 microM) reduced daYFnLRFa, acFnLRFa, and (D)-YFnLRFa induced [35S]-GTP-gamma-S stimulation, indicating that daYFnLR[TIC]a is a partial agonist at the receptor stimulated by the FMRFamide analogs. Analysis of the structural requirements needed for promoting [35S]-GTP-gamma-S binding show that elongation (i.e., daYFnLRFa, D-YFnLRFa) or modification of Phe1 (ac-FnLRFa) leads to increased efficacy and potency. Moreover, elimination of the C-terminal Phe (daYWnLRa, daYFLRa,) leads to a loss of biological activity. However, substitution with L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid, a rigid analog of the C-terminal Phe (daYFnLR[TIC]a), leads to decreased efficacy but not loss of potency. The data suggest that immobilization or modification of the C-terminal Phe may produce highly selective and potent FMRFamide antagonists. These results agree with published receptor radioligand studies and indicate that the [35S]GTP-gamma-S assay may be useful in classifying novel FMRFamide-selective ligands.

Receptor inactivation by dye-neuropeptide conjugates: 1. The synthesis of Cys-containing dye-neuropeptide conjugates.

In an attempt to attenuate specifically identified receptors through photolysis, a four-step synthesis is of a useful tethered derivative of Azure-B (Az) was developed After characterization, this derivative was covalently attached to CFMRFamide, CFMRF, and CLRFamide (i.e., three different neuropeptide analogues of the putative neurotransmitter FMRFamide. This resulted in the formation of three dye-neuropeptide conjugates: Az-CFMRFamide, Az-CFMRF, and Az-CLRFamide.