The heterotrimeric G proteins act as key molecular switches in eukaryotic cells to transmit signals from hundreds of receptors to multiple effectors. The conventional theory of G protein activation entails the physical dissociation of Gβγ from the GTP-bound Gα subunit, thereby producing two separate signals for effector regulation. The concept of subunit dissociation is primarily based on the Gβγ-binding characteristics of Gα subunits in solution, but there is little or no in vivo evidence in support of this mechanism. Here, a difference in subunit dissociation was observed among G protein families by co-immunoprecipitation assays. Gα subunits of G_q and G_s family remained bound to Gβγ even when they were activated by AlF₄^- or GTPγS, whereas Gα subunits of G_i family became sepa...[ Read more ]

The heterotrimeric G proteins act as key molecular switches in eukaryotic cells to transmit signals from hundreds of receptors to multiple effectors. The conventional theory of G protein activation entails the physical dissociation of Gβγ from the GTP-bound Gα subunit, thereby producing two separate signals for effector regulation. The concept of subunit dissociation is primarily based on the Gβγ-binding characteristics of Gα subunits in solution, but there is little or no in vivo evidence in support of this mechanism. Here, a difference in subunit dissociation was observed among G protein families by co-immunoprecipitation assays. Gα subunits of G_q and G_s family remained bound to Gβγ even when they were activated by AlF₄^- or GTPγS, whereas Gα subunits of G_i family became separated from the Gβγ dimers under the same condition. To determine whether subunit dissociations of various G protein families are required for the activation of their effectors, a series of Gβ-Gα fusion proteins were created. Fusion proteins as well as their constitutively active mutants were examined for effector activations. Fusion proteins of G_s and G_q families remain functional even when the Gα and Gβ subunits were tightly fused with a single residue linker, while the G_i fusion proteins with the same linker region failed regulate their effector. In addition to the major effector, the Gβ₁-Gα_q fusion proteins remained capable of activating other G_q effectors including GRK2, p63RhoGEF and TPR1. Moreover, the G_q fusion proteins could also cooperate with “free” Gβγ dimers released from G_i activation to synergistically activate PLCβ3. These results suggest that only the activation of G_i family heterotrimer requires subunit dissociation but not for G_q and G_s families. The in cellulo crosslinking of the G protein subunits also confirmed this family-wise difference. Such difference in subunit dissociation explains some long-lasting confusions such as Gβγ signal specificity.