Catalytic site mutations confer multiple states of G protein activation

Heterotrimeric guanine nucleotide-binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gαq or Gα11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein-coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance-based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands

Identifying the G protein, G z α

The signalling pathway associated with pertussis and cholera toxin sensitive G proteins have been extensively investigated. In contrast, the function and associated signal transduction cascade for the pertussis toxin insensitive G protein, G(Zα), have remained elusive. Therefore, the aim of this study was to identify the signal transduction pathway associated with G(Zα) by using the protein identification techniques of matrix assisted laser desorption ionization-time of flight mass spectroscopy and N-terminal Edman sequencing. We have chosen this technique to identify proteins that G(Zα) associates with and to gain insights into the potential role this G protein plays in cells. As G(Zα) is predominantly localized in neuronal tissues, homogenates of whole brain tissue were used. G(Zα) and its associated proteins were immunoprecipitated from brain tissue and identified. The immunoprecipitation of four proteins (140, 46, 41 and 36 kDa) was shown to be inhibited in the presence of the G (Zα) peptide. These proteins were subsequently identified as phospholipase C (PLC)-γ, β or γ-actin, G(Zα) and G(β), the β subunit of heterotrimeric G proteins, respectively. These results suggest that G(Zα) exists in a protein complex with the actin cytoskeleton and an important intracellular signalling enzyme, PLC-γ. These methods are powerful techniques for determining protein-protein interactions, and provide the first step to the identification of signalling proteins that G(Zα) associates with. However further experimentation will be required to determine the biological relevance of these protein interactions.</p