In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.