Quantitative investigation of the activation mechanism of the RET receptor tyrosine kinase
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Cells process a wide range of signals by means of multi-component receptors that span the plasma membrane. Our knowledge about the individual proteins involved in these signaling cascades has grown considerably over recent years. However, critical information about the detailed mechanisms of receptor activation, and the quantitative relationships between stimulus and biological response, is still missing. Here, I used the RET receptor tyrosine kinase (RTK), together with its glycosylphosphatidylinositol-coupled co-receptor GFRα3 and their activating growth factor artemin (ART), as a model system to investigate the quantitative and mechanistic features of receptor activation and signaling. I used a set of anti-RET agonist antibodies to induce different extents of receptor clustering on the cell surface, and studied how this factor affects the amplitude and kinetics of membrane-proximal and downstream signaling events, as well as the biological response of neurite outgrowth. Using simulations of the RET-GFRα3-ART system, I studied the effect of co-receptor involvement in the activation mechanism, as well as the importance of the specific activation pathway for the RET system’s response to variations in the expression levels of different components. The principal findings of my work include the following: 1) Higher order receptor clustering is required for full RET activation, as well as for the biological response of neurite outgrowth. 2) The activated forms of the receptor brought about by the agonist antibodies and by ART plus GFRα3 are identical with respect to the ability to activate the transient extracellular signal-regulated kinase (ERK) and Akt responses, but the antibodies show a reduced ability to induce sustained activation of ERK, Akt or c-Jun N-terminal kinase (JNK). 3) The involvement of GFRα3 co-receptor in the activation mechanism of RET provides cells with the ability to regulate their sensitivity to ligand without affecting the maximum amplitude of the pRET response. 4) This effect is limited if the co-receptor GFRα3 is pre-dimerized. Overall, my work aims to elucidate broad principles that underlie the quantitative relationships between RET activation, signaling, and the resulting cellular functional response, that can be applied to other receptor systems.