Measurement of GTP-binding and GTPase activity of heterotrimeric Gα proteins.

Heterotrimeric G-proteins are important signaling intermediates in all eukaryotes. These proteins link signal perception by a cell surface localized receptor to the downstream effectors of a given signaling pathways. The minimal core of the heterotrimeric G-protein complex consists of Gα, Gβ, and Gγ subunits, the G protein coupled receptor (GPCR) and the regulator of G-protein signaling (RGS) proteins. Signal transduction by heterotrimeric G-proteins is controlled by the distinct biochemical activities of Gα protein, which binds and hydrolyses GTP. Evaluation of the rate of GTP binding, the rate of GTP hydrolysis, and the rate of GTP/GDP exchange on Gα protein are required to better understand the mechanistic aspects of heterotrimeric G-protein signaling, which remains significantly limited for the plant G-proteins. Here we describe the optimized methods for measurement of the distinct biochemical activities of the Arabidopsis Gα protein.