Argos是EGFR（外皮生长因子受体）的一种被研究得非常明确的内分泌抑制因子，在果蝇体内起信号作用，在发育中的胚胎中有若干特定作用。除了在形态形成中起一个关键作用外，EGFR家族及其配体还与若干种人类癌症有关。这一事实使人们对与EGFR相互作用的分子更加感兴趣。现在，一项关于Argos在果蝇中如何发挥作用的新的研究表明，它不是抑制EGFR本身，而是与Spitz等本会激发EGFR的分子结合。这一结果为设计行为像Argos的分子指出了新的策略，例如，当EGFR配体的过度表达有助于人类癌症形成时对其予以抑制。
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 cancers1-3. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors3, 4. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR5, with clearly identified roles in multiple stages of development3. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels6, 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.
Figure 1 Quantitative analysis of Spitz/Argos interactions. a, SPR sensorgrams show that Spitz (at 1 µM, solid line), but not sDER2 (at 1 µM, dotted line) binds immobilized Argos. b, Representative binding curves for interaction of soluble Argos with immobilized Spitz (filled triangles) and immobilized sDER2 (open triangles). c, sDER2 binds immobilized Spitz (filled squares) but not immobilized Argos (open squares). d, In analytical ultracentrifugation, Argos (open triangles) sediments at 48.8 4.3 kDa (47.6 kDa expected without glycosylation), whereas Spitz (diamonds) sediments as a 16.2 1.7 kDa species (11.8 kDa expected without glycosylation). Equimolar Argos/Spitz mixtures fit best to a single 59 kDa species (filled triangles). The grey straight line represents expected results for a 1:1 Argos:Spitz complex. e, sDER2 (open circles) sediments as a 101.8 0.6 kDa species, which approximately doubles upon adding a 1.2-fold excess of Spitz (filled circles); Spitz alone (diamonds) sediments as in d. The grey line represents expected results for an sDER2 dimer.
Figure 2 Argos sequesters Spitz away from DER. a, Binding to immobilized sDER2 was measured for samples containing 250 nM Spitz and the indicated mole fraction of Argos. Binding is abolished when Argos is in excess. Mean s.d. (n 3) is shown. b, Western blot showing robust Spitz-induced tyrosine phosphorylation of DER2 expressed in S2 cells and the effect of adding different concentrations of Argos, using an anti-phosphotyrosine antibody (anti-pY, left panels). Western blot showing DER2 protein levels using an anti-DER2 antibody (right panels). c, Spitz–488 binds to the 40% of living D2f cells that express DER, but not to parental S2 cells. Argos–633 gives an identical punctate staining pattern on both parental and DER-expressing S2 cells, which is largely abolished by 10 µg ml-1 low-molecular-weight heparin. d, A fivefold excess of Argos prevents cell-surface labelling by Spitz–488. Mean fluorescence intensity is defined in the Methods.
Figure 3 Argos does not displace Spitz pre-bound to cell surface DER. a, Western blot showing DER phosphorylation in serum-starved D2f cells left untreated (- ), treated with 30 nM Spitz alone on ice (Spi) or treated with 30 nM Spitz plus 300 nM Argos in the order indicated. b, Parallel experiments analysing the effect of adding 1 µM unlabelled Argos on Spitz–488 binding (at 100 nM) to living D2f cells. Histograms describe cellular distribution of fluorescence intensity for each experiment (see Methods), plus a control experiment in which Spitz-488 was added to parental cells ('S2 Spi only'). The percentage of cells with fluorescence intensity >500 (filled bars) is reported above each graph. Spitz–488 alone, Spi; pre-mixed Spitz and Argos, Spi + Aos; Aos Spi and Spi Aos are defined in the text; addition of premixed Argos, Spitz and heparin, Spi + Aos + heparin (30 µg ml-1 in a, 20 µg ml-1 in b). c, Coomassie-stained SDS–PAGE gel shows Argos (50 µg ml-1 in input) but not Spitz (4 µg ml-1 in input) sedimenting with excess heparin–sepharose beads (washed with 150 mM NaCl buffer). Both Argos and Spitz sediment from an Argos/Spitz mixture, indicating that heparin-bound Argos retains Spitz-binding capacity.
