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 plasticity of the G protein signaling system. Understanding this plasticity will reveal novel ways to regulate G signaling in humans. Whereas, in animal cells, the G protein is activated by agonist stimulation of a G-protein Coupled Receptor (GPCR) to promote guanine nucleotide exchange, in Arabidopsis, the G protein spontaneously exchanges guanine nucleotide without a GPCR; rather, Arabidopsis utilizes agonist inhibition of a 7 transmembrane (receptor like) Regulator of G Signaling protein (7TM-RGS) to control the activation state. Animal cells have ~800 GPCRs to discriminate among a broad spectrum of signals (mostly hormone agonists), in contrast to Arabidopsis which has essentially one G protein complex comprised of the heterotrimeric G protein and the 7TM-RGS protein. Nonetheless, despite the single G protein core, genetic evidence indicates that Arabidopsis G signaling discriminates a broad spectrum of agonists just as animal cells do. This project explores the possibility that signal discrimination is achieved by receptor-like kinases (RLK). Plant cells encode ~400 RLKs and preliminary evidence shows that some RLKs are also physical components of the G protein core. The project hypotheses are: a) ligand-dependent, phosphorylation of the 7TM-RGS at its C-terminal tail is the key step for G protein activation, b) an unknown arrestin-fold protein recognizes the phosphorylated 7TM-RGS and recruits clathrin to complete endocytosis/G protein uncoupling/activation, and c) the recycling of the phosphorylated, and endocytosed 7TM-RGS is regulated by an unknown phosphatase. To test these hypotheses, we will use the genetic model organism, Arabidopsis thaliana. Arabidopsis is the ideal system to elucidate this mechanism because it has a simple heterotrimeric G protein repertoire, it provides a multicellular context for G signaling, it is easily genetically manipulatd, and it has myriad physiologies that utilize G signaling, e.g. pathogen resistance, stress responses, cell division, light and hormone-dependent development and programmed cell death. Specifically, we will: 1) determine the physical relationship between an informative set of receptor kinases and the heterotrimeric G protein complex and determine how cognate ligands alter the physical composition and/or the protein conformations. 2) determine if the selected set of kinases phosphorylate the 7TM-RGS protein (and other G protein components) in vivo and in vitro and the cellular consequences of this phosphorylation (e.g. 7TM-RGS endocytosis). 3) Determine the mechanism to recognize phosphorylated AtRGS1 and to control its phosphorylation state. Successful completion of these three aims will introduce a newly-recognized mechanism to regulate G protein activation.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Melillo, Amanda A
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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