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Abstract

Grant Number:	1R03NS053754-01
Project Title:	Real-Time Fluorescence Assay:RGS Domain GAP Activit(RMI)

PI Information:	Name	Email	Title
	SIDEROVSKI, DAVID P.	dsiderov@med.unc.edu	ASSOCIATE PROFESSOR

Abstract: DESCRIPTION (provided by applicant): Members of the "regulator of G-protein signaling" (RGS)-protein superfamily have emerged as critical modulators of specific G-protein-coupled receptor (GPCR) signal transduction pathways. Via their "GTPase accelerating protein" (GAP) activity, RGS proteins deactivate heterotrimeric G-protein alpha subunits and thereby reduce GPCR signal transduction. Combining existing GPCR agonists with specific RGS domain inhibitors should potentiate cellular responses to these drugs. The diversity of RGS proteins with highly localized and dynamically regulated distributions in the human brain, makes them attractive targets for pharmacotherapy of central nervous system disorders such as Parkinson's disease. Unfortunately, no small molecule inhibitor (or activator) of RGS protein GAP activity is publicly available for study. Therefore, to identify small molecule tools for further advancing knowledge of RGS protein function in specific GPCR signaling pathways, and also to facilitate identification of lead compounds for developing RGS protein directed therapeutics, we will modify and validate novel, real-time, fluorescence-based assays of RGS protein function for automated high throughput molecular screening: a fluorescence resonance energy transfer (FRET)-based binding assay that employs cyan fluorescent protein-labeled G-alpha subunits and yellow fluorescent protein-labeled RGS proteins, a single-turnover GTP hydrolysis assay using a fluorescent sensor for inorganic phosphate production, and an assay of G-alpha nucleotide binding and hydrolysis that employs the fluor-modified nucleotide BODIPY(r) FL 2'-(or-3')-O-(N-(2-aminoethyl)urethane)guanosine 5'-triphosphate. Many useful drugs act by binding a particular type of protein receptor on the cell's surface: a G-protein coupled receptor. Our group has discovered a new family of proteins-the RGS proteins-that interfere with these receptors. We wish to create ways to screen for new drug compounds that can stop RGS proteins from interfering and thereby allow existing drugs to act more potently.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
G protein coupled receptor kinase, cell surface receptor, chemical structure function, drug discovery /isolation, drug screening /evaluation, fluorescence resonance energy transfer, small molecule
analgesia, antiparkinson drug, biomedical automation, lead, protein structure function, receptor binding
biotechnology, high throughput technology, hydrolysis

Institution:	UNIVERSITY OF NORTH CAROLINA CHAPEL HILL
	Office of Sponsored Research
	CHAPEL HILL, NC 27599
Fiscal Year:	2005
Department:	PHARMACOLOGY
Project Start:	30-SEP-2005
Project End:	31-AUG-2009
ICD:	NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
IRG:	ZNS1

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