Biochemical Purification and Functional Analysis of Complexes between the G-Protein Subunit Gβ5 and RGS Proteins

Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Gγ-like (GGL) domain that enables their association with the G-protein β subunit Gβ5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse–chase analysis, Gβ5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gβ5–RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein–protein interactions involving the Gβ5–RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gβ5, RGS, and Gα subunits fused to the cyan and yellow versions of green fluorescent protein

Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K+ channel and control regulation of RGS7 by Gβ5

AbstractRegulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K+ channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit Gβ5, is regulated by Gβ5 by two distinct mechanisms. First, Gβ5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase-activating protein activity