G Protein Activation without Subunit Dissociation Depends on a Gα_i-specific Region*

G proteins transmit a variety of extracellular signals into intracellular responses. The Gα and Gβγ subunits are both known to regulate effectors. Interestingly, the Gα subunit also determines subtype specificity of Gβγ effector interactions. However, in light of the common paradigm that Gα and Gβγ 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 Gα_i1,2,3, Gα_z, and Gβ₁γ₂ subunits do not dissociate during activation but rather undergo subunit rearrangement as indicated by an activation-induced increase in FRET. In contrast, fluorescent Gα_o subunits exhibited an activation-induced decrease in FRET, reflecting subunit dissociation or, alternatively, a distinct subunit rearrangement. The α_B/C-region within the α-helical domain, which is much more conserved within Gα_i1,2,3 and Gα_z as compared with that in Gα_o, was found to be required for exhibition of an activation-induced increase in FRET between fluorescent Gα and Gβγ subunits. However, the α_B/C-region of Gα_il alone was not sufficient to transfer the activation pattern of Gα_i to the Gα_o subunit. Either residues in the first 91 amino acids or in the C-terminal remainder (amino acids 93–354) of Gα_il together with the α_B/C-helical region of Gα_i1 were needed to transform the Gα_o-activation pattern into a Gα_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.