G protein-coupled receptors (GPCRs) at the cell surface regulate most physiological processes and are important drug targets with ~34% of all prescribed drugs targeting them. Classically, upon agonist stimulation, GPCRs activate heterotrimeric G proteins, causing downstream signaling throughout the cell. In order to terminate G protein signaling, cells have devised a specialized desensitization mechanism that includes receptor phosphorylation by GPCR kinases and subsequent recruitment of β-arrestins (βarrs) to the phosphorylated receptors. The GPCR–βarrs interaction both blocks the G protein-binding site at the receptor core and promotes receptor endocytosis. Recently, however, we discovered that some GPCRs interact with βarrs exclusively through their phosphorylated C-terminal tails. Since βarrs do not block the G protein-binding site in this `tail' conformation, the receptor can associate with βarrs and G proteins simultaneously to form GPCR–G protein– βarr `megaplexes.' The assembly of these megaplexes allows the receptor to continue to stimulate G protein signaling while being internalized into endosomes by βarrs. Thus, the existence of the core and tail GPCR–βarr complex conformations suggests that βarrs act as spatiotemporal master regulators of G protein signaling: When bound to the receptor core, βarrs regulate the duration of G protein signaling whereas βarrs control the cellular location from where G proteins are activated from when associated with the receptor C-terminal tail. As the underlying properties that promote these two complex conformations remain elusive, my research objectives over the next 5 years involve determining these molecular driving forces on a general scale. Our preliminary data suggest that phosphorylation site clusters located within the receptor C-terminal tail are required for the association with βarrs in the tail conformation. Therefore, we plan to establish whether the presence of these phosphorylation site clusters correlates with the capacity of GPCRs to engage in mechanisms that lead to sustained endosomal G protein signaling. In regards to the GPCR–βarr core conformation, the fingerloop domain (FLD) of βarrs inserts itself into the transmembrane core of most GPCRs via its hydrophobic tip and receptor- specific residues. To characterize this interaction on a general scale, we will examine whether these receptor- specific βarr-FLD residues correlate with G protein subtype coupling of different GPCRs. Finally, βarrs modulate the activity of phosphodiesterases (PDEs), which terminate Gs-cAMP signaling. However, our preliminary data raise the possibility that this modulation occurs specifically by βarrs in the core conformation. Therefore, we will apply a combination of cell biological, biochemical, and proteomics approaches to examine whether modulation of PDEs and other desensitization mechanism is mediated specifically by distinct βarr conformations. My vision with this research program is to elucidate how...