Proteomic and structural profiling of PXXP-SH3 interactions as key regulators of membrane receptor activity (360G-Wellcome-201750_Z_16_Z)

Membrane receptor signalling occurs upon growth factor stimulation. Interestingly, I have recently shown that fibroblast growth factor receptor 2 signalling can also be triggered under non-stimulated conditions. This occurs when proteins with Src homology 3 (SH3) domain bind its C-terminal proline-rich-motif (PXXP) and modulate its activity to induce a homeostatic or oncogenic response. Whether this occurs in other receptors remains unclear. .Notably, the genome encodes for > 50 PXXP-containing receptors and > 300 SH3 domain-containing proteins. Thus, I will test the hypothesis that SH3-PXXP interactions are abundant and critical for functional regulation of receptors which dictates cellular response. I will use 2 model receptors to: Generate short biotinylated PXXP-peptides immobilized on streptavidin-coated-beads to "fish-out" bound proteins from cell lysates. I will then employ mass-spectrometry to determine the identity of the SH3 domain-containing hits (to be validated by CRISPR/Cas9 pull-downs and fluorescent-lifetime-imaging). Run pilot biophysical experiments like microscale thermophoresis to reduce confounding elements such us complex formation rather than direct SH3-PXXP binding events. Evaluate functional modulation of receptors by running Reverse-Phase-Protein-Array and functional assays. This project will set the foundation for characterizing novel interactions to enhance our understanding of signalling mechanisms; with impact on structural modelling of receptors and cancer therapeutics.