G protein-coupled receptors (GPCRs) represent the largest family of cell surface proteins. Agonist binding to GPCRs activates G proteins regulating many cellular effectors. Classically, G protein activation occurs at the plasma membrane and is rapidly terminated by β-arrestin recruitment to the activated GPCRs, promoting G protein uncoupling from receptor, GPCR internalisation and signalling arrest. However, recent studies revealed that upon internalisation, some GPCRs continue to activate G proteins from internal compartments leading to sustained production of second messengers far from the plasma membrane. This different spatiotemporal signalling profile allows distinct cellular functions from the ones occurring at the plasma membrane that can be exploited in the near future to design new pharmacological approaches. My collaborators and I recently observed that for some GPCRs such as the vasopressin type 2 receptor, formation of a GPCR-G protein-β-arrestin complex (baptised megaplex) in internal compartments is required for non-canonical Gs protein signalling. The initial research programme I propose aims to tackle the following important questions underlying this novel signalling mode: 1. Is G protein selectivity different at intracellular compartments vs at plasma membrane? 2. Is megaplex formation restricted to Gs isoform? 3. What is the specific role of β-arrestin within the megaplex? Environmental stimulants (hormones, neurotransmitters) can be compared to keys able to bind to and activate (open) cell surface receptors (locks). When a key opens a lock, proteins detecting this opened lock (called G proteins) are activated at the cell surface leading to specific outcomes. Some proteins (ß-arrestins) are then recruited to remove G proteins from the receptor and bring it inside the cell leading to signalling arrest. Recently, we observed that active G proteins can also be found inside the cell mediating different outcomes than when located at cell surface and that ß-arrestins was promoting receptor signalling inside the cell instead of stopping it. My objective is to explore this new G protein signalling, to learn how ß-arrestins can promote internalised receptor signalling, and determine if they activate other G proteins than the ones at the cell surface. This could lead to development of new medications with less side effects.