Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret

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 alpha3 (GFRalpha3), brings about activation of the Ret receptor tyrosine kinase through formation of a pentameric signaling complex: ART-(GFRalpha3)(2)-(Ret)(2). By systematically varying the concentrations of ART and cell-surface GFRalpha3, 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.