PROJECT SUMMARY/ABSTRACT Heterotrimeric G proteins are involved in the regulation of many physiological processes and are essential to multiple cell signaling pathways. G protein heterotrimers are formed through association of α, β, and γ subunits with guanidine diphosphate (GDP) bound to the Gα subunit. The canonical signaling mechanism involves displacement of GDP from the Gα subunit by guanidine triphosphate (GTP) and subsequent disassociation of the Gβγ dimeric complex from Gα-GTP; the Gβγ and Gα-GTP complexes then act to regulate downstream effectors in the cell. G protein coupled receptors (GPCRs) represent the most well-studied class of proteins responsible for catalyzing the exchange of GDP for GTP on the Gα subunit. However, other nonreceptor guanine exchange factor (GEF) proteins also exist and have been shown to be crucial to regulation of G protein signaling. Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) is a protein that has been shown to act as a GEF and molecular chaperone for Gα subunits of Gi, Gq and G12/13 families. Although Ric-8A acts as a GEF to activate both Gαq/Gαi, previous research has demonstrated that the affinity of Ric-8A is different for Gαq in comparison to Gαi, and that the kinetics of the Ric-8A-catalyzed guanine nucleotide reaction are unique in relation to these two subtypes. Further, Ric-8A contains several phosphorylation sites and the phosphorylated form of Ric-8A has enhanced GEF activity toward Gα subunits. The effect of phosphorylation of Ric-8A has been shown to be different for Gαq relative to Gαi. Aim 1 of this work is to systematically measure the kinetics of GEF activity of Ric-8A for Gαq/Gαi using stopped-flow fluorescence spectroscopy to allow for a direct comparison, and to determine the effect of Ric-8A phosphorylation. Aim 2 is to elucidate the structure of Ric-8A:Gαq complexes in the GDP-free and GDP-bound forms by X-ray crystallography and cryo-electron microscopy. These studies will provide new insight into the distinct mechanism of Ric-8A GEF activity for Gαq/Gαi, and provide, for the first time, an atomic-resolution model of the Ric-8A:Gαq complex. This work will enhance our understanding of the role of Ric-8A in G protein signaling and have broad implications in human health and disease by laying the foundation for development of future therapeutics that target cytoplasmic G protein activation.