The transduction of biological signals such as light, hormones and neurotransmitters starts by a specific interaction of the ligand or stimulus with a receptor protein. The ultimate cell-specific responses to most biological signals are produced via G protein coupled receptor (GPCR) activation of specific GTP-binding proteins. Our lab has elucidated mechanisms of G protein interaction with cognate receptors and effectors as well as the structure-function relationships of G proteins. In the last grant period, detailed hypotheses of G protein function based upon crystallographic three-dimensional structural data were tested in biochemical and cellular assays, using a variety of model systems with chimeric constructs and site-directed mutagenesis of G protein alpha and beta/gamma subunits expression in heterologous systems. In this renewal, we will dissect out the interactions of Gbetagamma subunits with their effectors, and determine the mechanisms underlying the cellular specificity of Gbetagamma signaling. Although activation of G proteins by GPCRs is always thought to lead to G protein subunit dissociation, Gbetagamma signaling is known to play an important role in cellular responses especially when the Gi/o family of G proteins is activated. We will systematically determine whether Gbetagamma signaling is not an important part of the response when Gq, Gs, G12 and 13 are activated, and the basis for this specificity. There are a number of well-characterized Gbetagamma effectors, however, the number of Gbetagamma interacting proteins continues to grow. We have recently shown another function: the ability of Gbetagamma released from Gi-coupled receptors to interact with syntaxin and SNAP25A, components of the vesicular fusion or SNARE complex, and inhibit vesicular fusion. We will determine the molecular basis for this effect. In addition, we have discovered yet another Gbetagamma binding protein in a yeast-two-hybrid screen: the receptor for activated C kinase or RACK 1. The fact that Gbetagamma has so many different effectors (Hamm review, 1999) poses a problem for cells, which normally express a number of different Gbetagamma effectors. When a G protein-coupled receptor is activated, both Galpha and Gbetagamma-dependent signaling will take place, and if multiple Gbetagamma effectors are activated, they will have multiple effects in the cell. The implications for cellular physiology have never been systematically examined.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY010291-09
Application #
6485727
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Mariani, Andrew P
Project Start
1997-07-01
Project End
2006-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
9
Fiscal Year
2002
Total Cost
$377,500
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Yim, Yun Young; Zurawski, Zack; Hamm, Heidi (2018) GPCR regulation of secretion. Pharmacol Ther 192:124-140
Zurawski, Zack; Page, Brian; Chicka, Michael C et al. (2017) G?? directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes. J Biol Chem 292:12165-12177
Van Hook, Matthew J; Babai, Norbert; Zurawski, Zack et al. (2017) A Presynaptic Group III mGluR Recruits G??/SNARE Interactions to Inhibit Synaptic Transmission by Cone Photoreceptors in the Vertebrate Retina. J Neurosci 37:4618-4634
Yim, Yun Young; McDonald, W Hayes; Hyde, Karren et al. (2017) Quantitative Multiple-Reaction Monitoring Proteomic Analysis of G? and G? Subunits in C57Bl6/J Brain Synaptosomes. Biochemistry 56:5405-5416
Zurawski, Zack; Rodriguez, Shelagh; Hyde, Karren et al. (2016) G?? Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein-Coupled Receptors. Mol Pharmacol 89:75-83
Hamid, Edaeni; Church, Emily; Wells, Christopher A et al. (2014) Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex. J Neurosci 34:260-74
Betke, Katherine M; Rose, Kristie L; Friedman, David B et al. (2014) Differential localization of G protein ?? subunits. Biochemistry 53:2329-43
Betke, Katherine M; Wells, Christopher A; Hamm, Heidi E (2012) GPCR mediated regulation of synaptic transmission. Prog Neurobiol 96:304-21
Wells, Christopher A; Betke, Katherine M; Lindsley, Craig W et al. (2012) Label-free detection of G protein-SNARE interactions and screening for small molecule modulators. ACS Chem Neurosci 3:69-78
Wells, Christopher A; Zurawski, Zack; Betke, Katherine M et al. (2012) G?? inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25. Mol Pharmacol 82:1136-49

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