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 ?, ?, and ? subunits with guanidine diphosphate (GDP) bound to the G? subunit. The canonical signaling mechanism involves displacement of GDP from the G? subunit by guanidine triphosphate (GTP) and subsequent disassociation of the G?? dimeric complex from G?-GTP; the G?? and G?-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 G? 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 G? subunits of Gi, Gq and G12/13 families. Although Ric-8A acts as a GEF to activate both G?q/G?i, previous research has demonstrated that the affinity of Ric-8A is different for G?q in comparison to G?i, 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 G? subunits. The effect of phosphorylation of Ric-8A has been shown to be different for G?q relative to G?i.
Aim 1 of this work is to systematically measure the kinetics of GEF activity of Ric-8A for G?q/G?i 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:G?q 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 G?q/G?i, and provide, for the first time, an atomic-resolution model of the Ric-8A:G?q 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.
The goal of this project is to understand the guanine nucleotide exchange mechanism catalyzed by Ric-8A on G? subunits that contributes to its role in G protein signaling with wide-ranging implications in understanding human development and many disease pathologies. The importance of G protein signaling is highlighted by the fact that approximately 35% of drugs currently approved by the FDA target these pathways. The products of this research will include a systematic description of the reaction kinetics of Ric-8A catalyzed guanine nucleotide exchange for G?q/G?i, and an atomic model of the Ric-8A:G?q complex structure in the GDP-free and GDP- bound states.
