Project Summary G protein-coupled receptors (GPCRs) can be activated by partial agonists, resulting in submaximal signaling with reduced side effects, compared to the full agonist. Increased studies have indirectly demonstrated that partial agonists stabilize intermediate conformational states between the inactive and fully activated states with a reduced heterotrimeric Gabg protein coupling activity from the fully activated state. Due to the technical hurdles in delineating GPCR conformational states and populating individual intermediate states to study them individually, a mechanistical understanding of partial agonism signaling has been challenging. By creating conformation-biased mutants, we identified five adenosine A2A receptor (A2AR) conformational states, including two inactive states (S1 and S2), two intermediate states (S3 and S4), and a fully active state (S5), using 19F nuclear magnetic resonance (NMR) spectroscopy. This result is a significant advancement to previous research. The R291A mutant predominantly accumulates the intermediate S4 state while the R291AR293A mutant populates both intermediate S3 state and the full activated state S5. This finding enables us to study the roles of these intermediates and their complexes. We will use these two mutants to examine the roles of intermediate states S3 and S4 and their interactions with G proteins and consequent signaling effects. In Aim 1, we will characterize whether and how the intermediate states S3 and S4 interact with Gasbg. These characterizations will include the study of conformational transitions and dynamics of intermediate states and the effects of Gasbg and ligands on their transitions and dynamics. In Aim 2, we will map the conformational states of the Gas and determine its intermediate states that interact with the S3 and S4 states of the A2AR. In Aim 3, we will determine if the intermediate states S3 and S4 of the A2AR induce Gasbg states that are competent for GTP hydrolysis, G protein dissociation, and contribution to submaximal signaling without the S5 state being involved. We expect to correlate conformational and dynamic characteristics of the intermediate states of the A2AR and Gas protein created from Aims 1 and 2 to the signaling efficacies of conformation-biased mutants with ligand stoichiometries, measured in Aim 3. The completion of this proposed project will advance our understanding of the roles that intermediate conformations play in GPCR signaling, lead to a conceptual innovation in understanding receptor activation beyond a simple two-state model, and potentially guide drug design based on GPCR and G protein conformational responses to ligands. Moreover, the conformation-biased mutants will guide an approach development in resolving the structures of intermediate complexes.