Abstract
G protein-coupled receptors (GPCRs) are central actors of cell signaling. They are the target of one third of the current cellular drugs. Thus, it is crucial to understand the molecular mechanism behind G-protein dependent and independent signaling pathways. G-protein independent cell signaling is mediated by the interaction of the cytoplasmic C-termini of GPCRs with arrestin. These C-termini are intrinsically disordered regions (IDRs) and are variable in length and sequence, making their study more elusive. This interaction is regulated by the phosphorylation of GPCR C-terminal domains by specific GPCR kinases (GRKs). My thesis objectives aimed at understanding the impact of GRK phosphorylation on the conformation and the interaction of GPCR C-termini with their arrestin partner. The strategy implemented was to produce excised recombinant C-terminal domain of three well-studied GPCRs: vasopressin V2 (V2R), growth hormone secretagogue 1a (GHSR) and β2-adrenergic (β2AR) receptors. Phosphorylation by GRK2, as well as, the use of phosphomimetic variants of these GPCR C-terminal domains allowed us to study the impact of phosphorylation on their conformation and interaction with arrestin-2. Structural and functional analyses of these disordered regions were performed using complementary biophysical methods such as solution-state nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS) and fluorescence spectroscopy (FRET). My thesis work postulates for a new picture of GPCR signaling dependent of arrestine. It shows that phosphorylation by GRKs changes the conformation of the C-terminal domain of GPCRs and modulates the binding region of these domains with arrestin.