FPU2023-Development of ligand-directed labelling technology to unravel the complexity of β-adrenoceptors in native environments

The current technologies to study the organization, function and signalling of GPCRs are based in artificial systems, involving genetically modified proteins overexpressed in heterologous cells. These include GPCRs fused to bioluminescent domains, fluorescent proteins or suicide enzymes which can be covalently labelled with a fluorophore. These approaches tend to simplify the natural cellular environment and these modifications may lead to wrong conclusions, since native GPCRs likely have different properties from those recombinantly expressed in heterologous cells. Ligand-directed (LD) labelling approaches represent a powerful technology to extend the techniques above commented to the study native proteins. They are based on molecular probes consisting in an affinity unit (i.e. a ligand), a fluorescent label and a reactive moiety with a relative geometry and distance that, upon the affinity unit binding, directs the labelling reaction to a proximal amino acid residue, which can chemically react to form a covalent bond and leave the binding site unoccupied. The PhD project we are proposing involves designing, synthesising and characterising new ligand-directed labelling probes for β-adrenoceptors with the following two main objectives: (A) Developing a technology that will offer a high degree of control on native GPCR labelling and (B) Employing this technology to develop new “user-friendly” assays for endogenous GPCRs to be applied in native environments.