A universal allosteric mechanism for G protein activation.

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

Molecular and Functional Profiling of Gαi as an Intracellular pH Sensor.

Heterotrimeric G proteins (Gα, Gß and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. Previously, human Gαi and its yeast homolog Gpa1 have been reported to function as intracellular pH sensors, yet the pH sensing capabilities of Gαi and the underlying mechanism remain to be established. Herein, we identify a pH sensing network within Gαi, and evaluate the consequences of pH modulation on the structure and stability of the G-protein. We find that changes over the physiological pH range significantly alter the structure and stability of Gαi-GDP, with the protein undergoing a disorder-to-order transition as the pH is raised from 6.8 to 7.5. Further, we find that modulation of intracellular pH in HEK293 cells regulates Gαi-Gßγ release. Identification of key residues in the pH-sensing network allowed the generation of low pH mimetics that attenuate Gαi-Gßγ release. Our findings, taken together, indicate that pH-dependent structural changes in Gαi alter the agonist-mediated Gßγ dissociation necessary for proper signaling.