ABSTRACT: Seven transmembrane receptors (7TMRs), alternatively known as G protein-coupled receptors (GPCRs), are involved in the signaling and regulation of many physiological processes. Currently, over one third of all clinically approved drugs target GPCRs, including the β-adrenoceptors (β-ARs) and the arginine vasopressin type 2 receptor (V2R), which are GPCRs of great cardiovascular significance. Thus, a better understanding of different modes of GPCR signaling stands to greatly inform the drug discovery process for this important class of receptor targets. Classically, agonist-bound active GPCRs binds to and activate the heterotrimeric G protein (Gαβγ), leading to dissociation between the Gα subunit and the remaining Gβγ subunits. This dissociation initiates the generation of second messenger molecules such as cyclic AMP (cAMP), which propagate a wave of signaling that eventually leads to a physiological response. To attenuate G protein signaling, GPCR kinases (GRKs) phosphorylate the C-terminal tail of GPCRs, allowing for the binding and activation of β- arrestins (βarrs), which mediates receptor desensitization and internalization. However, recent works have shown that some GPCRs engage in sustained G protein signaling from within internalized cellular compartments (e.g. endosomes) after receptor internalization rather than staying desensitized. With data directly implicating βarrs in mediating this phenomenon, we and others further show that the aforementioned mode of sustained G protein signaling is potentially mediated by two distinct novel GPCR-transducer complexes: a GPCR–G protein– βarr mega-complex (‘megaplex’), whereby a single GPCR directly engages and activates both G protein and βarr, and a GPCR–Gβγ–βarr complex that subsequently forms after G protein dissociation. This observation is inconsistent with the classical model of GPCR signaling described above, which states that βarr and G protein binding to a GPCR is mutually exclusive. While the discovery of these complexes is well established, and provides a potential explanation for sustained G protein signaling, the structural basis governing the interactions of each complex component, and by extension how sustained signaling is mediated, remains to be elucidated. Accordingly, this proposal aims to determine the structure of both the megaplex and the GPCR–Gβγ–βarr complex, with the following aims: (1) To optimize megaplex purification and complex formation for cryo-EM structural studies, (2) to investigate the effects of the GPCR–Gβγ–βarr complex on sustained G protein signaling, and to obtain its structure by cryo-EM. Structural elucidation of these complexes translates directly into a better understanding of this newly appreciated mode of sustained signaling, and will serve as the foundation for the design of therapeutics that confers spatiotemporal control of signaling by GPCRs, such as those of cardiovascular importance. In particular, our structural approach will generate a...