G protein activation without subunit dissociation depends on a Gαi-specific region

G proteins transmit a variety of extracellular signals into intracellular responses. The G alpha and G beta gamma subunits are both known to regulate effectors. Interestingly, the G alpha subunit also determines subtype specificity of G beta gamma effector interactions. However, in light of the common paradigm that G alpha and G beta gamma subunits dissociate during activation, a plausible mechanism of how this subtype specificity is generated was lacking. Using a fluorescence resonance energy transfer ( FRET)-based assay developed to directly measure mammalian G protein activation in intact cells, we demonstrate that fluorescent G alpha(i1,2,3), G alpha(z), and G beta(1)gamma(2) subunits do not dissociate during activation but rather undergo subunit rearrangement as indicated by an activation-induced increase in FRET. In contrast, fluorescent G alpha(0) subunits exhibited an activation-induced decrease in FRET, reflecting subunit dissociation or, alternatively, a distinct subunit rearrangement. The alpha(B/C)-region within the alpha-helical domain, which is much more conserved within G alpha(i1,2,3) and G alpha(z) as compared with that in G alpha(0), was found to be required for exhibition of an activation-induced increase in FRET between fluorescent G alpha and G beta gamma subunits. However, the alpha(B/C)-region of G alpha(i1) alone was not sufficient to transfer the activation pattern of G alpha(i) to the G alpha(o) subunit. Either residues in the first 91 amino acids or in the C-terminal remainder (amino acids 93-354) of G alpha(i1) together with the alpha(B/C)-helical region of G alpha(i1) were needed to transform the G alpha(o)-activation pattern into a G alpha(i1)-type of activation. The discovery of subtype-selective mechanisms of G protein activation illustrates that G protein subfamilies have specific mechanisms of activation that may provide a previously unknown basis for G protein signaling specificity.