Effect of ?-helical domain of Gi/o ? subunit on GDP/GTP turnover

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of alpha, beta, and gamma subunits, and G alpha has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with G alpha, GDP is released, and GTP interacts to G alpha. The GTP-bound activated G alpha dissociates from GEF and G beta gamma, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, G alpha subunits are subcategorized into four subfamilies: G alpha i/o, G alpha s, G alpha q/11, and G alpha 12/13. Although the G alpha i/o subtype family proteins, G alpha i3 and G alpha oA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with G alpha oA possessing faster rates than G alpha i3. The structural factors responsible for these differences remain unknown. In this study, we employed hydrogen/deuterium exchange mass spectrometry and mutational studies to investigate the factors responsible for these functional differences. The G alpha subunit consists of a Ras-like domain (RD) and an alpha-helical domain (AHD). The RD has GTPase activity and receptor-binding and effector-binding regions; however, the function of the AHD has not yet been extensively studied. In this study, the chimeric construct containing the RD of G alpha i3 and the AHD of G alpha oA showed a GDP/GTP turnover profile similar to that of G alpha oA, suggesting that the AHD is the major regulator of the GDP/GTP turnover profile. Additionally, site-directed mutagenesis revealed the importance of the N-terminal part of alpha A and alpha A/alpha B loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the G alpha subfamily.