The identification and characterization of interactions between the heterotrimeric G protein beta subunit and two downstream effectors

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  • March 22, 2019
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  • Friedman, Erin Julie
    • Affiliation: College of Arts and Sciences, Department of Biology
Abstract
  • Cells have evolved mechanisms to sense and respond to signals from the external environment and from other cells. One such mechanism is the heterotrimeric guanine-nucleotide binding protein (G protein) signaling pathway. This suite of signaling components allows cells to perceive extracellular signals using a seven-pass transmembrane receptor and to effect change within the cell via the dissociation of the G protein Galpha subunit and Gbeta&gamma heterodimer, which both regulate downstream interacting proteins. In my dissertation work, I identified and characterized several interactions between the Gbeta subunit and its downstream effectors. First, I utilized an evolutionary approach to predict non-overlapping binding interfaces on the Gbeta protein surface. Gbeta proteins bind to a diverse group of interacting proteins, but structural data for many protein complexes are not available. I mapped the conservation of the Gbeta surface residues over time to identify eight regions of interest (ROIs) that I hypothesized composed novel binding interfaces. By analyzing the evolutionary history of known Gbeta effectors, I determined that one such ROI is required for the activation of the effector phospholipase Cbeta2. I next utilized a screen in the model plant Arabidopsis thaliana to identify genes whose overexpression compensated for the loss of Arabidopsis Gbeta (AGB1) and therefore were in the same genetic pathway as AGB1. Aci-reductone dioxygenase 1 (ARD1) interacts both genetically and physically with AGB1. I showed that ARD1 operates as an enzyme in the methionine salvage pathway, and AGB1 stimulates the enzymatic activity of ARD1. I used an evolutionary comparison to identify three ARD1 interaction sites on AGB1, and showed that mutation of these sites abolished AGB1 stimulation of ARD1. Finally, I predicted and tested the structural mechanism of ARD1 basal and stimulated activities, showing that a conserved tryptophan residue near its active site is critical for both functions, and a regulatory helix is critical for the stimulation of ARD1 by AGB1. Together, my results identified ARD1 as a novel Gbeta effector, characterized its function in Arabidopsis and its regulation by AGB1, and provided structural insight into the mechanism of the regulation of downstream effectors by Gbeta.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology."
Advisor
  • Jones, Alan M.
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Chapel Hill, NC
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  • Open access
Date uploaded
  • March 18, 2013

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