2010/AFGN Collaborative Project: The Heterotrimeric G-Protein Interactome

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Arabidopsis 2010/AFGN Collaborative Project: The Interaction Network for the Heterotrimeric G-Protein Signaling PathwayPI Alan M. Jones, University of North CarolinaPI Joachim Ulhrig, University of KolnCo-PI Ralph Panstruga, Max Planck Institute for Plant Breeding- KolnBackground and Scientific Goals/Merit: A variety of signaling networks in Arabidopsis utilize one or both of the two heterotrimeric G-protein complexes to couple signals that are perceived at the cell surface by G-protein coupled receptors (GPCRs), that have a seven-transmembrane spanning (7TM) domain, to enzymes called effectors. The plant heterotrimeric G protein complex is a central node in an exemplary signaling network. To date, neither the downstream effectors nor the upstream GPCRs have been unequivocally identified. This paucity of information stands in contrast to humans and C. elegans G protein-coupled pathways, where 950 GPCRs and more than a dozen known effectors have been identified; thus, representing the most extensive signaling network in metazoans. None of the metazoan GPCRs and almost none of the canonical effectors are encoded in the Arabidopsis genome; thus, non-biased approaches are needed to fill this knowledge gap. To address this problem, a high through-put interaction screen to identify proteins that interact with the heterotrimeric G protein complex is descirbed. URL: Additional information and progress is located at http://www.bio.unc.edu/Faculty/Jones/lab/2010Project/Gene Set: The central core is defined by the following genes: GPA1 (At2g26300), AGB1 (At4g34460), AGG1 (At3g63420), AGG2 (At3g22945), and AtRGS1 (At3g26090). Selected interactors with this core that meet the defined criteria for priority are being pursued more thoroughly. For those selected, reverse and molecular genetic approaches are applied to determine on what branches of the G-protein signaling network these elements operate. Up to 100 genes encoding confirmed interactors will be knocked out either by insertional mutagenesis or a RNAi technique in order to determine the loss of function phenotype with regard to known phenotypes of G-protein mutants. A smaller subset will be pursued in great depth by examining in vivo interactions. Interactors are tested for in vivo interaction and those confirmed interactors are used as bait to initiate additional rounds of screening. Note that the gene set expands as progress is made. Gene Function: The physiological processes involving Arabidopsis G protein have been characterized. G protein interactors will be placed genetically in one or more of these pathways. Data dissemination: Data will be made public through a project website, TAIR, and interaction data repositories such as IntAct, DIP and bioGRID. Public data will include frequency of identification in the screen, screen conditions, gene expression patterns, protein type, homologs, interlogs, and links to the individual genes and other gene-specific resources. The data will be subjected to bioinformatic and graph-theoretical meta-analyses to identify the main signal transduction pathways and protein complexes (signalosomes) in the interaction network. Key functional signal mediators (hubs) as well as abundant regulatory patterns will be identified.Relevance to the NSF 2010 and AFGN programs: This research activity substantially aids NSF's goal for "Benchmarking Gene Function." The research has as its aim to identify the complete interacting set of elements in the G-protein signaling network; thus, it meets the need for "exploring exemplary networks and systems." This project coordinates efforts with two other 2010 projects that are focusing on the global interactome of Arabidopsis proteins. Broader Impact: A broader impact of this work will be realized through the training of undergraduate, pre-, and post-doctoral students. In addition, UNC and Shaw University will jointly mentor 3-6 minority students in a 10-week summer research program.