Investigation of Ric8-Gαi nucleotide exchange activity

Abstract: Ric8 is a chaperone protein for the Gα subunit of the G protein coupled receptor (GPCR), the most common and diverse membrane receptor group in eukaryotic cells. The GPCR initiates cellular response upon recognition of an extracellular ligand, by transducing the signal across the cellular membrane and activating the Gα subunit of the G protein heterotrimer on the interior of the membrane. When in the inactive state, GDP is bound to Gα in the heterotrimer. Upon ligand binding, the GPCR facilitates exchange of GDP for GTP, resulting in dissociation of Gα:GTP from the complex and continuation of the signaling cascade. The Gα subunit has an intrinsic ability to exchange GDP for GTP at a very slow rate, but the Ric8 chaperone protein greatly increases the rate of nucleotide exchange by over tenfold. The purpose of this project was to identify potential Gα binding sites on the Ric8 protein and assess the nucleotide exchange behavior of different Ric8 mutants. The mutants created for these assays targeted specific potentially catalytic amino acids based off the structure of Ric8 bound to Gα, and catalytic activity was measured for each using fluorescence spectroscopy. By comparing the reaction rate for each Ric8 mutant to the reaction rate of wild type Ric 8 and the intrinsic reaction rate of Gα, the effect of mutating each amino acid residue can be compared to normal function for both proteins.