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

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits, and Ga has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with Ga, GDP is released, and GTP interacts to Ga. The GTP-bound activated Ga dissociates from GEF and Gbg, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, Ga subunits are subcategorized into four subfamilies: Gai/o, Gas, Gaq/11, and Ga12/13. Although the Gai/o subtype family proteins, Gai3 and GaoA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with GaoA possessing faster rates than Gai3. 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 Ga subunit consists of a Ras-like domain (RD) and an α-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 Gai3 and the AHD of GaoA showed a GDP/GTP turnover profile similar to that of GaoA, 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 αA and αA/αB loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the Ga subfamily.