Guilt by dissociation

J. Biol. Chem. 289, 1271–1281 (2014)

Signals downstream of G protein–coupled receptors (GPCRs) are transduced and amplified by G protein heterotrimers, consisting of subunits α, β and γ. In the canonical G protein cycle, activation of GPCRs causes exchange of Gα-bound GDP for GTP and Gα dissociation from Gβγ dimers. This part of the cycle is less clear for the G_i/o family of G proteins because of conflicting in vitro and in vivo results, but three models have been debated that differ mainly by whether the Gα_i/o subunit dissociates from Gβγ and to what extent. Therefore, it has been unclear whether a rearranged heterotrimer (Gα_GTPGβγ) or a dissociated trimer (Gα_GTP, Gβγ) is the active GTP-bound form capable of transmitting the GPCR signal to effectors. To reconcile these discrepancies, Bondar and Lazar used two-photon polarization microscopy, which can report on changes in protein-protein interactions and protein conformational changes of fluorescently labeled proteins (FPs) or native proteins. They also looked at localization of GRK3, a kinase associated with GPCRs whose binding to Gβγ is favored upon dissociation of activated G_i/o, and looked at regulation of GIRK channels downstream of it. They found that at endogenous concentrations, 85–95% of nonmodified G_i/o molecules dissociate upon activation, suggesting that dissociated Gα and Gβγ are the major activated form that mediates downstream signaling in a natural setting. However, some FP-tagged G_i/o constructs were able to activate downstream signaling without dissociation, suggesting that G_i/o dissociation is not strictly required for activity.