Cytokines and growth factors signal by modulating the interactions between multiple receptor components to form an activated receptor complex. The quantitative details of the activation mechanisms of this important class of receptors are not well understood. Using receptor phosphorylation measurements in live cells, as well as mathematical modeling and data fitting, we have characterized the multistep mechanism by which the GDNF-family neurotrophin artemin (ART), together with its co-receptor GDNF-family receptor α3 (GFRα3), brings about activation of the Ret receptor tyrosine kinase through formation of a pentameric signaling complex: ART–(GFRα3)2–(Ret)2. By systematically varying the concentrations of ART and cell-surface GFRα3, we establish both the sequence of steps by which the signaling complex forms and the affinities of all the steps, including the two-dimensional affinities of the steps involving protein-protein interactions between membrane-bound species. Our results reveal the ways in which the individual binary interactions involved in the activation of a multicomponent receptor govern the receptor's functional properties.
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We thank A. Rossomando and B.J. Gong for providing rat ART, C. Graf for providing the anti-GFRα3 antibodies GFRA3B and GFRA3N, J. Campos-Rivera for assistance with FACS measurements and E. Day for helpful discussions.
The authors declare no competing financial interests.
Quantification of Ret and GFRα3 expression on NB41A3-mGFRα3 cells by FACS. (PDF 50 kb)
Contribution of ligand-dependent trafficking to the shape of the ART-dependent dose-response curves measured by KIRA ELISA. (PDF 48 kb)
Binding of blocking antibody GFRA3B to GFRα3 as measured by flow cytometry. (PDF 28 kb)
Plots of experimental receptor activation data with best fits to equilibrium models 1–4. (PDF 78 kb)
Comparison of Ret phosphorylation quantified by western blot and KIRA ELISA. (PDF 64 kb)
Description of methods and representative input file for the DynaFit modeling. (PDF 39 kb)
Estimated parameters with standard errors for models 1–4 in the global fitting analysis. (PDF 45 kb)
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Schlee, S., Carmillo, P. & Whitty, A. Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret. Nat Chem Biol 2, 636–644 (2006). https://doi.org/10.1038/nchembio823
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