The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), and GPCRs represent the largest group of targets for drug development. While GPCRs are known to signal through G protein independent pathways, G protein activation represents the primary mode of cellular signaling. The goal of this proposal is to determine the structural basis by which GPCRs activate specific G proteins. The proposal builds on the foundation provided by the high-resolution structure of the ?2AR-Gs complex determined during the first funding cycle, and insights into the dynamic behavior of this complex obtained during the most recent funding cycle. The ?2AR-Gs structure provided a single snap-shot of the nucleotide-free complex, a very transient step in a series of interactions between GPCRs and G proteins that make up the G protein cycle. The studies outlined in this proposal will provide more mechanistic details about steps that precede and follow the nucleotide-free complex, and will shed light on the mechanism of G protein coupling specificity as well as the similarities and differences in the mechanism of activation of different G protein isoforms. The ?2AR-Gs complex reflects activation of only one alpha subunit isoform by a Family A GPCR. In Aim 1 we propose to obtain structures of GPCRs in complex with Gq and with a pertussis toxin sensitive Gi/o family member. In addition, we propose to obtain a structure of the glucagon receptor in complex with Gs, to identify similarities and differences in activation of this G protein by Family A and Family B GPCRs. The dynamic process of GPCR-G protein complex formation is not amenable to study by protein crystallography; however, the interactions that precede the formation of the nucleotide-free complex may be a major determinant of G protein coupling specificity. In Aim 2 we propose to characterize the process of complex formation and dissociation using double electron-electron resonance spectroscopy. These studies will provide high-resolution distance constraints that will allow us to model the interactions involved in the formation of GPCR complexes with Gs and Gi proteins. The ?2AR-Gs complex, together with other structural studies during the first funding cycle revealed that the alpha helical domain of Gs is highly dynamic. Molecular dynamics simulations conducted during the recent funding period suggest that alpha helical domain dynamics may play an essential role in G protein function. In Aim 3 we will use double electron-electron resonance spectroscopy and single molecule spectroscopy to directly monitor alpha helical domain dynamics during different stages of the G protein cycle. The studies outlined here build upon the experience, reagents and methods accumulated during the past eight years, and will provide unprecedented insights into G protein activation by GPCRs.