Molecular mechanisms of G protein activation

G protein-coupled signal transduction pathways mediate many vital biological processes such as cardiovascular regulation, inflammation, sensory detection, and synaptic transmission. The mechanism whereby G protein-coupled receptors (GPCRs) selectively activate their cognate G proteins is critical to these processes, but the mode of transmission of the signal from receptor to G protein has been poorly defined. A GPCR is spatially separated from the guanine nucleotide-binding pocket of its G protein and therefore cannot directly interact with this domain. GPCR activation must cause a conformational change in the G protein that begins at the receptor-contact regions and is transferred to the nucleotide-binding pocket to cause GTP exchange for GDP. The main objective of this thesis is to understand how the activation signal is transferred from the transmembrane domains of GPCRs to the G protein surface, activating the G protein and leading to downstream signaling pathway activation.; This dissertation addresses the molecular mechanisms mediating GPCR-G protein coupling using the pheromone response pathway of the yeast Saccharomyces cerevisiae as a model system. Identifying the contact regions between the receptor and G protein is critical to understanding the mechanisms of G protein activation. Therefore, three aspects of G protein signaling were examined for functions in receptor-G protein coupling: the C-terminal domain of the Ggamma subunit, the first and second intracellular loop domains of the alpha-factor receptor, and the dimeric nature of the receptor. The C-terminal domain of G protein gamma subunits appear to interact with GPCRs, but, surprisingly, the data indicate that this domain of yeast Ggamma is dispensable in vivo. Second, the first and second intracellular loop domains of the receptor were studied by mutagenesis analysis, and it was discovered that the first intracellular loop and the juxtacytoplasmic ends of transmembrane domains 2 and 3 of the yeast pheromone receptor are involved in G protein coupling. Finally, GPCRs exist as dimeric complexes, and it is unknown whether both receptors interact with the G protein. This work demonstrates that receptor dimerization is critical for G protein coupling and activation. Taken together, this thesis presents work that significantly furthers our understanding of receptor-G protein coupling.