Hormones and neurotransmitters modulate a variety of physiological processes in cell growth and behavior. Their cognate cell surface receptors, which have seven transmembrane domains, act by coupling to G proteins, promoting the dissociation of GDP and the subsequent loading of GTP. Signaling abates when GTP is hydrolyzed and GTPase activity is accelerated by Regulators of G Signaling proteins having GTPase accelerating protein (GAP) activity. Recently, we discovered a naturally-occurring 7TM-RGS protein in Arabidopsis (AtRGS1) that is a glucose or nucleotide-sugar receptor. It is the prototype of a receptor-GAP. We also showed that the Arabidopsis Gα subunit (AtGPA1) has rapid nucleotide exchange making nucleotide hydrolysis the rate limiting step. This property is in marked contrast to the slow nucleotide exchange property of all tested Gα subunits where GDP release is the rate limiting step of the G protein cycle. In contrast to animals, regulation of the G protein cycle is at the GTP hydrolysis step and is modulated by AtRGS1. This level of modulation is controlled by reversible phosphorylation of AtRGS1 and AtGPA1 at a mostly undeciphered set of phosphorylated amino acids designated here as the collective phospho-bar code. We do know that one phosphorylation pattern on AtRGS1 is necessary and sufficient to initiate AtRGS1 endocytosis and another on AtGPA1 changes the rate of AtRGS1-dependent G cycling. There are different clusters of complexes of the AtRGS1/G protein/kinase/phosphatase on the plasma membrane and these different clusters are activated by different agonists. Furthermore, we hypothesize that this initial clustering, the assortment of proteins in the cluster, and the subsequent trafficking of the cluster components after activation is encrypted by the phospho-bar code. Using the power of a genetic system will enable us to determine the physiological role of the bar code in a multicellular context. Both hypothesis- and discovery-driven approaches will be taken to determine precisely what structure imparts regulatory control. Our studies of the Arabidopsis G protein cycle have to date illustrated how the G-protein cycle can be regulated by mechanisms distinct from the classical GEF. Consequently, a greater degree of plasticity of the cycle is now appreciated and new entry points for regulation are revealed. Understanding the structure underlying these new mechanisms will provide a new means to regulate other G protein cycles. Understanding how AtRGS1 modulates the G protein cycle in a ligand-dependent manner opens up new possibilities to regulate G protein cycles through drug therapies. The core elements of heterotrimeric G protein coupled signaling are conserved in eukaryotes but the mechanism to regulate the active state of the G protein is not. This variation, genetically encoded in organisms divergent by as much as 1.6 billion years of evolution, represents the full range of plasticity of the G protein signaling system. Understanding this plasticity will reveal novel ways to regulate G signaling in humans.

The rationale for the proposed work is that an understanding of molecular mechanisms used in divergent signaling pathways will yield new drug targets, new ideas for manipulating human signaling pathways, and new tools to engineer human pathways.