Activation of heterotrimeric G proteins is commonly believed to trigger the dissociation of Gβγ dimers from GTP-bound Gα subunits. Such a model is mainly based on the observation of purified G proteins in non-physiological environments, with little supportive evidence in living cells. For several G proteins, unequivocal subunit dissociation upon activation could not be detected in vivo. Thus the traditional idea of complete subunit dissociation may need further validation or refinement. To examine if subunit dissociation is universally required for the functional activation of G proteins, fusion proteins of Flag-tagged Gβ₁ linked to various Gα subunits (G_q, G₁₁, G₁₆, G_s, G_i3, and G_z) via a single amino acid or 16 residues were generated. Fusion proteins as well as their constitu...[ Read more ]

Activation of heterotrimeric G proteins is commonly believed to trigger the dissociation of Gβγ dimers from GTP-bound Gα subunits. Such a model is mainly based on the observation of purified G proteins in non-physiological environments, with little supportive evidence in living cells. For several G proteins, unequivocal subunit dissociation upon activation could not be detected in vivo. Thus the traditional idea of complete subunit dissociation may need further validation or refinement. To examine if subunit dissociation is universally required for the functional activation of G proteins, fusion proteins of Flag-tagged Gβ₁ linked to various Gα subunits (G_q, G₁₁, G₁₆, G_s, G_i3, and G_z) via a single amino acid or 16 residues were generated. Fusion proteins as well as their constitutively active (QL) mutants were examined for adenylyl cyclase or PLCβ activities in transfected cells. Fusion proteins with a single amino acid linking Gβ to constitutively active Gα_q (β-1-αqQL) or Gα₁₁ (β-1-α11QL) significantly stimulated PLCβ in COS-7 cells. Similarly, β-1-αsQL efficiently stimulated adenylyl cyclase. However, β-1-αi3QL and β-1-αzQL did not inhibit adenylyl cyclase but those with a long linker (β-16-αi3QL and β-16-αzQL) remained fully functional. In addition to PLCβ, the fusion proteins of Gα_q remained capable of interacting with p63RhoGEF and GRK2, two other important effectors for G_q signaling. The β-1-αqQL induced activation of p63RhoGEF was further confirmed with significant downstream SRF activation. The demonstrated functionality of the one-linker fusion proteins of Gα_q, Gα₁₁ and Gα_s supports an activation mechanism without subunit dissociation. In contrast, fusion proteins of Gα_i3 and Gα_z suggest that these G proteins may require physical dissociation from Gβγ for effector regulation. The different activation mechanisms of various G proteins may provide explanations for some long-standing arguments in G protein signal crosstalk.