Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα s mutant proteins (from a screen of 66 Gα s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα s . This extended Linker 2 is the target site of a natural product inhibitor of G q . Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β 2 /β 3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.G protein-coupled receptors mainly signal through heterotrimeric G-proteins. Results We have demonstrated that mutations in the extended Linker 2 impaired the activation of Gα s by β-adrenergic receptors. Conclusion We have proposed a potential novel conduit from β-adrenergic receptors to the helical domain of Gα s subunit via the extended Linker 2. Significance Our systematic mutagenesis studies provide insights into the activation mechanism of G-proteins by receptors.