Phospho- and natural-variant regulation of heterotrimeric G protein signaling

Project Summary/Abstract Numerous cellular signaling pathways of critical importance to organism health rely on heterotrimeric G protein signaling. Heterotrimeric G proteins are composed of Gα, Gβ, and Gγ subunits. Classically, ligand binding to transmembrane G-protein-coupled-receptors (GPCRs) elicits conformational changes that lead to exchange

of GDP for GTP on the Gα subunit, and resultant separation of Gα from the Gβγ dimer, either or both of which then signal to downstream effector proteins until intrinsic GTPase activity of Gα restores the heterotrimeric state. G proteins participate in numerous signaling cascades in both metazoan and plant systems and many aspects

of G protein signaling are remarkably conserved across this evolutionary divide. The overarching goal of my research is to harness the simpler G protein complement of the model plant, Arabidopsis thaliana, to reveal how heterotrimeric G protein genotypes and molecular phenotypes control cellular signaling cascades, and thus

whole organism consequences. Our laboratory has been a leader in plant heterotrimeric G protein signaling for >25-years; we have elucidated G protein signaling via methods ranging from single cell electrophysiology to imaging to -omics approaches, to sophisticated whole organism phenotyping and genome-phenome analyses.

This proposal focuses on two types of molecular phenotypes – phosphorylation and natural variation -- and their attendant consequences on G protein biochemistry, cellular signaling, and whole organism vitality. We have ascertained that Gα phosphorylation at sites conserved across mammalian and plant Gαs can dramatically

impact the G protein cycle. In Theme 1, we plan to utilize >60 Gα phosphomimic and phosphonull lines we have created to determine the phenotypic consequences of Gα phosphostatus. We will assess how phosphorylation changes Gα molecular partners (i.e. how phosphorylation biases signaling), and phenotypic consequences. We

propose that phosphorylation “diversifies” the number of functionally distinct Gα proteins, resolving an apparent mismatch between the number of G protein subunits vs. the much greater numbers of GPCRs and phenotypes. Some G protein mutations are known to cause disease. Many more are statistically “associated’ with disease

outcomes, but whether they actually cause disease is difficult to ascertain due to the high heterozygosity of humans, low phenotypic penetrance, and inaccessibility of humans to experimentation. Arabidopsis is naturally inbred and can be genetically manipulated with ease. In Theme 2 research, we plan to determine the impact of

natural missense single nucleotide variants (SNVs) on Gα biochemistry. We will next experimentally reveal in Arabidopsis the phenotypic consequences of these mutations. Then we will ask whether, in nature, deleterious SNVs are accompanied by other SNVs in Gα that forestall the negative consequences of the missense

mutations, in positive “intrageneic epistasis”. Next, we will assess intergenic epistasis between Gα variants and regulatory proteins. Demonstration of such phenomena is both mechanistically revealing and will allow medical science to better ascertain whether or not an individual with a given Gα SNV is actually predisposed to disease.