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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065989-12
Application #
9402078
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Melillo, Amanda A
Project Start
2002-09-01
Project End
2018-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
12
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Escudero, Viviana; Jordá, Lucía; Sopeña-Torres, Sara et al. (2017) Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the ?-subunit of the heterotrimeric G-protein. Plant J 92:386-399
Liao, Kang-Ling; Jones, Roger D; McCarter, Patrick et al. (2017) A shadow detector for photosynthesis efficiency. J Theor Biol 414:231-244
Mudgil, Yashwanti; Karve, Abhijit; Teixeira, Paulo J P L et al. (2016) Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis. Front Plant Sci 7:1255
Li, Bo; Makino, Shin-Ichi; Beebe, Emily T et al. (2016) Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein. Protein Expr Purif 126:33-41
Tunc-Ozdemir, Meral; Fu, Yan; Jones, Alan M (2016) Cautions in Measuring In Vivo Interactions Using FRET and BiFC in Nicotiana benthamiana. Methods Mol Biol 1363:155-74
Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar et al. (2016) Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex. J Biol Chem 291:13918-25
Urano, Daisuke; Miura, Kotaro; Wu, Qingyu et al. (2016) Plant Morphology of Heterotrimeric G Protein Mutants. Plant Cell Physiol 57:437-45
Urano, Daisuke; Jackson, David; Jones, Alan M (2015) A G protein alpha null mutation confers prolificacy potential in maize. J Exp Bot 66:4511-5
Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan et al. (2015) Evidence for an unusual transmembrane configuration of AGG3, a class C G? subunit of Arabidopsis. Plant J 81:388-98

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