David P Siderovski, Associate Professor
University Of North Carolina Chapel Hill Office Of Sponsored Research Chapel Hill, Nc 27599
Grant 1P01GM065533-019001 from National Institute Of General Medical Sciences IRG: ZGM1
Abstract: Recombinant protein expression, purification, and initial biochemical characterization is rapidly becoming the rate-limiting step in testing biological hypotheses related to complex signaling networks. Extensive sequence databases, sophisticated algorithms for domain prediction, and various high-throughput technologies such as gene array analysis provide a wealth of initial data easily converted into testable hypotheses. Conventional biochemical techniques carried out in individual laboratories are becoming increasingly incapable of providing the numerous biochemical reagents and associated analyses required to test this rapidly expanding universe of hypotheses. This proposal seeks to address this dilemma for the Projects of the PPG by providing a Protein Core capable of rapidly producing, purifying, and functionally characterizing the numerous and diverse proteins and domains required for the proposed studies. The Protein Core will rely heavily on automation of reagent handling and miniaturization of biological assays amenable to high-throughput processing. In particular, the Protein Core will house robotics for the rapid generation of arrayed clones and the automated purification of analytical amounts of arrayed proteins. The Protein Core also will have limited capacity for large scale protein purification as well as instrumentation to measure surface plasmon resonance and fluorescence for protein characterization. The four Projects of the PPG propose a wide variety of experimental techniques encompassing numerous protein components linked to diverse G protein signaling systems. Many of these avenues of research ultimately will rely on the purification and characterization of recombinantly-expressed proteins. This Protein Core is therefore designed to satisfy these common requirements in an efficient manner using a combination of automation and sophisticated biophysical techniques. By centralizing instrumentation and expertise, the PPG anticipates turning common research needs into a unique opportunity to advance standard biochemical techniques past the era of the individual researcher and into a future of highly-efficient data collection and analysis.
Keywords: biomedical facility, high throughput technology, protein purification, recombinant protein, fluorescent dye /probe, microarray technology, surface plasmon resonance
Project start date: 2002-04-01
Project end date: 2007-03-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to David P Siderovski
G PROTEIN COORDINATION BY RGS12 AND RGS14
David P Siderovski, Associate Professor
University Of North Carolina Chapel Hill Office Of Sponsored Research Chapel Hill, Nc 27599
Grant 5R01GM062338-05 from National Institute Of General Medical Sciences IRG: PHRA
Abstract: Verbatim from s ) Hormones and neurotransmitters can activate intracellular signal transduction by binding receptors linked to heterotrimeric guanine nucleotide-binding or "G" proteins. G protein a subunits cycle between active, GTP-bound and inactive, GDP-bound states, and thus signal duration is controlled by their intrinsic GTPase activity. "Regulator of G-protein signaling" (RGS) proteins are considered key desensitizers of G protein-coupled signaling given the ability of their hallmark "RGS-box" domains to accelerate Ga GTPase activity. However, as RGS proteins have only recently been identified, their physiological functions in the overall dynamics of signal onset, integration, and termination are poorly defined. Recent identification of Ras-family GTPase- and Ga-interaction domains ("RFL" and "GoLoco" domains) within RGS 12 and RGS 14 presents the opportunity to define the molecular mechanisms these two RGS proteins use to transact higher-order functions in G protein signaling modulation. This proposal is focused on determining the binding specificities and structural determinants of RGS 12/14 GoLoco and RFL domains, as well as the effects of domain interactions both on the nucleotide cycle of the bound G protein and on RGS-box GTPase-accelerating activity. Studies in Aim 1 test the hypothesis that the GoLoco region binds to GDP-bound Gi-class Ga subunits and acts as a receptor-independent guanine-nucleotide exchange factor. Studies in Aim 2 test the hypothesis that the RFL domains bind GTP-bound Ras-family G proteins and inhibit nucleotide dissociation. Studies in Aim 3 will define the Ga selectivites of RGS12/14 ROS-boxes as well as test the hypothesis that GoLoco/Gax and/or RFL/Ras-family protein interactions modify RGS box function. In all three Aims, binding specificity and affinity will be determined by a combination of yeast two-hybrid analyses, coprecipitation studies, biosensor measurements, and cell co-immunoprecipitation assays; effects of these interactions on nucleotide binding/hydrolysis will be analysed by in vitro nucleotide binding assays, single-turnover and steady state GTP hydrolysis measurements, and cellular readouts of receptor signaling outcomes. As perturbation of G protein-coupled signal transduction can cause human disease, yet forms the basis of many drug actions, defining the mechanisms by which RGS12 and RGS14 assemble and regulate specific heterotrimeric and Ras-family G proteins should ultimately lead to novel drug discovery targets with exquisite specificity.
Keywords: G protein, protein structure function, receptor coupling, chimeric protein, enzyme activity, genetic translation, guanosine diphosphate, guanosine triphosphate, guanosinetriphosphatase, SDS polyacrylamide gel electrophoresis, autoradiography, immunoprecipitation, surface plasmon resonance, yeast two hybrid system
Project start date: 2001-02-01
Project end date: 2006-01-31
5R01GM062338-05 (2005): $245504
5R01GM062338-04 (2004): $245504
5R01GM062338-03 (2003): $245504
5R01GM062338-02 (2002): $245504
1R01GM062338-01 (2001): $235634
G-protein Signal Coordination By RGS12
David P Siderovski, Associate Professor
University Of North Carolina Chapel Hill Office Of Sponsored Research Chapel Hill, Nc 27599
Grant 5R01GM062338-07 from National Institute Of General Medical Sciences IRG: MNPS
Abstract: Timely initiation and cessation of intercellular communication via heterotrimeric G protein-coupled receptors (GPCRs) is crucial for normal functioning of brain and sensory neurons. Aberration of this process can lead to pathophysiological deficits in peripheral or central nervous system activity; conversely, stimulation, prolongation, or antagonism of GPCR signaling underlies the actions of many psychoactive and neurotropic agents. In 1996, a new class of proteins was discovered that directly modulate the timing of GPCR signaling - the "regulators of G-protein signaling" (RGS) proteins. The overall goal of the Siderovski laboratory is to identify specific roles for RGS proteins in (patho)physiological process and thus establish select members of the RGS family as viable drug discovery targets. RGS12 is specifically involved in determining desensitization from GABA(B) receptor-mediated inhibition of presynaptic calcium current in dorsal root ganglia. Our long-term objective is to define the molecular determinants that constitute the desensitization function of RGS12. Beyond the hallmark GTPase-accelerating activity specified by the RGS12 RGS domain, we have now identified phospholipid- and phosphotyrosine-binding activity within RGS12, as well as its ability to interact with multiple components of the mitogen-activated protein kinase (MAPK) cascade. This proposal therefore describes biochemical/biophysical and cell-biological analyses of RGS12-mediated protein/protein and protein/lipid interactions, coupled with cellular studies of agonist-promoted MAPK signal transduction and electrophysiological assessment of neurotransmitter modulation of Cav2.2 channel activity in the presence of selective RGS12 mutants. Results from this work will help define the specific roles RGS12 partakes in signal transduction and ion channel modulation, as well as illuminate the potential for RGS12 as a drug discovery target for novel therapies for neuropathic pain and spasticity. Many drugs act by binding a particular protein receptor on the cell s surface a G-protein coupled receptor. Our group discovered a new protein family - the RGS proteins - that interfere with these receptors. We wish to study one RGS protein, RGS12, that interferes with the actions of the neurotransmitter GABA in neurons that control pain processing. This work should lead to the discovery of new pain-controlling drugs.
Keywords: G protein, protein, binding site, brain, calcium, cell, cell cell interaction, cell surface receptor, central nervous system, dorsal root, drug control, drug discovery /isolation, electrical potential, family, ion, lead, lipid, mitogen activated protein kinase, mutant, neuron, neurotransmitter, pain, phospholipid, phosphopeptide, play, receptor, role, small molecule, spinal ganglion, therapy
Project start date: 2001-02-01
Project end date: 2010-01-31
5R01GM062338-07 (2007): $258751
2R01GM062338-06 (2006): $266479
ENZYMATIC SCREEN FOR RGS PROTEIN MODULATORS
David P Siderovski, Associate Professor
University Of North Carolina Chapel Hill, Office Of Sponsored Research, Chapel Hill, Nc 27599
Grant 1R03DA030555-01 from Office Of The Director, National Institutes Of Health
Abstract: The superfamily of "regulator of G-protein signaling" (RGS) proteins share a defining RGS domain that accelerates the intrinsic GTP hydrolysis rate of heterotrimeric G-protein alpha subunits - an enzymatic activity that terminates signaling by activated G-protein coupled receptors (GPCRs). As GPCRs constitute the largest single class of protein target for existing drugs, small molecule modulators of RGS protein action should hold great promise for the development of novel therapeutics, yet no bona fide proof-of-principle small molecule has yet been developed. We recently created a novel, robust, and facile method of measuring RGS domain GTPase-accelerating function using fluorescence polarization. In lieu of the traditional, cumbersome, radioactive "single-turnover" assay, we have developed a high- throughput screening (HTS) method based on detecting RGS-accelerated GDP production by a G subunit with altered nucleotide binding and hydrolysis rates. This enzymatic assay differs considerably from the RGS/G protein-protein interaction assays that have previously been screened by the MLPCN. We request funding to deliver this HTS assay (including required protein reagents) to an MLPCN, as well as to facilitate our lab´s post-screening analysis of hits using various medium-throughput secondary and counterscreen assays. A particular class of cell-surface proteins, the G protein-coupled receptor superfamily, has for many decades provided valuable targets for drug discovery across a variety of clinical needs and diseases. A large family of negative regulators of these receptors (the "RGS proteins") was discovered over ten years ago, but their potential as additional drug discovery targets has yet to be tested owing to the present dearth of chemical modulators of their action. Here we describe a novel, enzymatic reaction for the high-throughput screening of small molecule libraries for modulators of RGS protein action; our intent is to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) with the assistance of one of the nodes within the NIH Molecular Libraries Probe Production Centers Network (MLPCN). Our long-term goal is to develop novel chemical probes and (ultimately) drugs that act at the level of the RGS protein to improve therapy of multiple pathological conditions caused by aberrant GPCR signal transduction
Keywords: Agonist; Anxiety; Assay; Attenuated; Binding; Binding (Molecular Function); Bioassay; Biochemical Reaction; Biologic Assays; Biological Assay; Brain; CNS Diseases; CNS disorder; Cell Communication and Signaling; Cell Signaling; Cell Surface Proteins; Cells; Central Nervous System Diseases; Central Nervous System Disorders; Chemicals; Clinical; Collaborations; Development; Disease; Disorder; Drug Therapy; Drugs; Encephalon; Encephalons; Enzymatic Reaction; Family; Fluorescence Polarization; Funding; G Protein-Complex Receptor; G Protein-Coupled Receptor Signaling; G(s), alpha Subunit; G(s)alpha; G-Protein Regulating Factors; G-Protein alpha Subunit; G-Protein, Gs alpha Family; G-Protein, Stimulatory Gs; G-Protein-Coupled Receptors; G-Proteins; G-substrate; GPCR Signaling; GTP; GTP Phosphohydrolases; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; GTP-Regulatory Proteins; GTPases; Goals; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Guanosine 5`-(tetrahydrogen triphosphate); Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Guanosinetriphosphatases; Heterotrimeric G-Proteins; Heterotrimeric GTP-Binding Proteins; High Throughput Assay; Human; Human, General; Hydrolysis; Idiopathic Parkinson Disease; In Vitro; Intracellular Communication and Signaling; Knowledge; Lead; Lewy Body Parkinson Disease; Man (Taxonomy); Man, Modern; Measures; Medication; Methods; Molecular Bank; Molecular Interaction; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Ns Protein, Regulatory; Nucleotides; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson`s; Parkinson`s disease; Parkinsons disease; Pb element; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacotherapy; Primary Parkinsonism; Production; Proteins; Proteins, Cell Surface; RGS Domain; RGS Family Protein; RGS Protein (G-Protein Signaling); RGS Proteins; RGS-Signaling Domain; Radioactive; Reagent; Receptor Protein; Regulating Factors, GTP-Binding Protein; Regulators of G Protein Signaling Domain; Regulators of G-Protein Signaling Proteins; Regulators, G-Protein Signaling; Schizophrenia; Schizophrenic Disorders; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Solubility; Specificity; Stimulatory GTP-Binding Protein, alpha Subunit; Synthesis Chemistry; Synthetic Chemistry; Testing; Therapeutic; United States National Institutes of Health; alpha-Gs; analog; aqueous; base; biological signal transduction; cerebellum protein substrate for cGMP dependent protein kinase; clinical relevance; clinically relevant; counterscreen; dementia praecox; disease/disorder; drug discovery; drug/agent; gene product; guanosinetriphosphatase; heavy metal Pb; heavy metal lead; high throughput screening; improved; inhibitor; inhibitor/antagonist; member; new therapeutics; next generation therapeutics; novel; novel therapeutics; protein G; protein function; protein protein interaction; public health relevance; receptor; repository; response; schizophrenic; small molecule; small molecule libraries; tool
Relevance: A particular class of cell-surface proteins, the G protein-coupled receptor superfamily, has for many decades provided valuable targets for drug discovery across a variety of clinical needs and diseases. A large family of negative regulators of these receptors (the "RGS proteins") was discovered over ten years ago, but their potential as additional drug discovery targets has yet to be tested owing to the present dearth of chemical modulators of their action. Here we describe a novel, enzymatic reaction for the high-throughput screening of small molecule libraries for modulators of RGS protein action; our intent is to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) with the assistance of one of the nodes within the NIH Molecular Libraries Probe Production Centers Network (MLPCN). Our long-term goal is to develop novel chemical probes and (ultimately) drugs that act at the level of the RGS protein to improve therapy of multiple pathological conditions caused by aberrant GPCR signal transduction
Project start date: 2010-05-01
Project end date: 2012-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PAR-09-129
1R03DA030555-01 (2010): $36979
MECHANISTIC STUDIES OF A NOVEL G-ALPHA NUCLEOTIDE CYCLE
David P Siderovski, Associate Professor
University Of North Carolina Chapel Hill, Office Of Sponsored Research, Chapel Hill, Nc 27599
Grant 5R01GM074268-04 from National Institute Of General Medical Sciences
Abstract: Proper chromosomal segregation by microtubule (MT) dynamics at the mitotic spindle is an essential component of cell division in all organisms; aberration of this process can lead to severe developmental abnormalities. Recent disparate genetic and biochemical evidence suggests the existence of a nucleotide cycle for G-protein alpha subunits required for proper mitotic spindle function during cell division. This cycle employs G-alpha regulatory proteins conserved across metazoa, including GoLoco motif nucleotide dissociation inhibitors, RGS-domain-containing GTPase-accelerating proteins, and the guanine nucleotide exchange factor RIC-8. Our long-term objective is to define the molecular determinants and spatiotemporal dynamics that underlie this novel G-alpha regulatory cycle and its effects on MT dynamics. Aim 1 is to delineate the functional interplay between the novel G-alpha regulatory factors that impinge on G-alpha subunit activity in cell division via a series of protein biochemical studies. Aim 2 details development and use of novel peptide biosensors for G-alpha nucleotide state to determine the active species that modulates MT dynamics and the spatial and temporal dynamics of this process in live cells. A novel GoLoco-insensitivity mutation to G-alpha will be employed to address the necessity of the G-alpha/GoLoco interaction to these processes. Aim 3 is to map the functional determinants of RGS14 and G-alpha-i1 proteins that directly modulate MT dynamics using in vitro biochemistry and cell viability, MT network, and cell cycle transit studies, thereby testing our initial hypothesis that the coordinated action of G-alpha and G-alpha regulators directly on tubulin and MTs might represent the force generator in mitotic spindle function during mitosis. This research program will lead to a new understanding of the diversity of G-protein action and its impact on microtubule dynamics, and further the ultimate goal of defining the precise molecular mechanisms of microtubule force generation at mitosis. This should facilitate new drug discovery for anticancer therapeutics, as many current anti-proliferative agents target microtubule dynamics. These studies will also afford insight into the regulation of asymmetric cell division and, consequently, mechanisms of cell polarity and cell-fate determination that have specific relevance to neural specification, developmental defects, and the potential future uses of neuroprogenitor stem cells as treatment for neurodegenerative disorders
Keywords: Address; Affect; Anti-Cancer Agents; Anti-Tumor Agents; Anti-Tumor Drugs; Antineoplastic Agents; Antineoplastic Drugs; Antineoplastics; Antiproliferative Agents; Antiproliferative Drugs; Binding; Binding (Molecular Function); Biochemical; Biochemistry; Biosensor; CDK; Cancer Drug; Cell Cycle; Cell Death; Cell Division Cycle; Cell Polarity; Cell Survival; Cell Viability; Cell division; Cells; Chemistry, Biological; Chemotherapeutic Agents, Neoplastic Disease; Cyclin-Dependent Kinases; Cyclin-Dependent Protein Kinases; DNA; Defect; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Deoxyribonucleic Acid; Development; Dissociation; Dysfunction; Functional disorder; Future; G(s), alpha Subunit; G(s)alpha; G-Protein alpha Subunit; G-Protein, Gs alpha Family; G-Protein, Stimulatory Gs; G-Proteins; GDP Dissociation Factor; GDP Dissociation Stimulators; GDP Exchange Factors; GDP-GTP Exchange Protein; GDP-GTP Reversing Factors; GEF; GTP GDP exchange factor; GTP Phosphohydrolases; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; GTP-Regulatory Proteins; GTPases; Generations; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Goals; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Exchange Factors; Guanine Nucleotide Exchange Protein; Guanine Nucleotide Regulatory Proteins; Guanine Nucleotide Releasing Factors; Guanosine Triphosphate Phosphohydrolases; Guanosinetriphosphatases; Guanyl-Nucleotide Exchange Factor; Guanyl-Nucleotide Releasing Factor; Health; Heterotrimeric G Protein Subunit; Heterotrimeric G-Proteins; Heterotrimeric GTP-Binding Proteins; Human; Human, General; In Vitro; Lead; Life; M Phase; M phase (cell cycle); Man (Taxonomy); Man, Modern; Maps; Micro-tubule; Microtubules; Mitosis; Mitosis Stage; Mitotic spindle; Molecular; Molecular Interaction; Mother Cells; Mutation; Nature; Nervous; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Ns Protein, Regulatory; Nucleotides; Organism; Outcome; Pb element; Peptides; Phenotype; Physiopathology; Process; Progenitor Cells; Programs (PT); Programs [Publication Type]; Proteins; RGS Domain; RGS-Signaling Domain; Racial Segregation; Receptor Protein; Regulation; Regulators of G Protein Signaling Domain; Regulatory Protein; Research; Series; Specificity; Stem cells; Stimulatory GTP-Binding Protein, alpha Subunit; Testing; Tubulin; Tubulin Interaction; Tumor-Specific Treatment Agents; alpha-Gs; anti-cancer therapeutic; anticancer agent; anticancer drug; anticancer therapeutic; cdk Proteins; cellular polarity; drug discovery; exchange factor; gene product; genetic regulatory protein; genome mutation; guanosinetriphosphatase; heavy metal Pb; heavy metal lead; inhibitor; inhibitor/antagonist; insight; living system; mutant; necrocytosis; neural; neurodegenerative illness; novel; pathophysiology; programs; receptor; regulatory gene product; relating to nervous system; segregation; sensor (biological); spatiotemporal; stem cell fate
Project start date: 2006-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2011
5R01GM074268-04 (2009): $226178
5R01GM074268-03 (2008): $226178
Sponsored Links Excellgen http://Excellgen.com
5R01GM074268-02 (2007): $222605
1R01GM074268-01A1 (2006): $227413
Real-Time Fluorescence Assay:RGS Domain GAP Activit(RMI)
David P Siderovski, Associate Professor
Pharmacologyuniversity Of North Carolina Chapel Hill
office Of Sponsored Research
chapel Hill, Nc 27599
Grant 1R03NS053754-01 from National Institute Of Neurological Disorders And Stroke IRG: ZNS1
Abstract: 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
Keywords: 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
Project start date: 2005-09-30
Project end date: 2008-08-31
1R03NS053754-01 (2005): $73000
STRUCTURAL DETERMINANTS OF HETEROTRIMERIC G-PROTEIN NUCLEOTIDE CYCLING
David P Siderovski, Professor And Director
University Of North Carolina Chapel Hill, Office Of Sponsored Research, Chapel Hill, Nc 27599
Grant 5R01GM082892-03 from National Institute Of General Medical Sciences
Abstract: G protein-coupled receptors (GPCRs) serve as catalytic activators of heterotrimeric G-proteins by exchanging GTP for the bound GDP on the G? subunit. This guanine nucleotide exchange factor (GEF) activity of GPCRs is the initial step in the G-protein cycle and determines the onset of various intracellular signaling pathways that govern critical physiological responses to extracellular cues. The structural basis for several steps in the G-protein nucleotide cycle have been made clear over the past decade, including intrinsic GTP hydrolysis by G? and acceleration of this hydrolysis (
Relevance: GAP activity´) by RGS domains; however, the precise structural determinants underlying receptor-mediated G-protein activation, and facilitation of signal onset by RGS proteins, remain incompletely defined. As GPCRs represent a rich set of drug targets, more thorough understanding of their mechanism of activating intracellular signaling should provide valuable further avenues for drug discovery. Currently, several distinct (and somewhat conflicting) models have been proposed to explain the communication between activated GPCRs and G-protein heterotrimers that leads to the structural changes required for guanine nucleotide exchange. This research effort is focused on a high-resolution elucidation of the structural details underlying heterotrimeric G-protein activation via nucleotide exchange. Aim 1 is to resolve the structural determinants of nucleotide exchange within G? via protein crystallography of fast-exchanging G? subunits we recently identified from the genomes of A. thaliana and C. elegans, as well as additional G? mutants with enhanced GDP release or propensity to exist in a stable, nucleotide-free state. In Aim 2, three complementary cellular systems (yeast pheromone signaling, mammalian cell GIRK currents, Dictyostelium cAMP responses) will be used to ascertain the structural determinants underlying non-GAP actions of RGS proteins that facilitate GPCR/heterotrimer signal onset kinetics. This second aim relies on our recent crystallographic evidence that the fast-hydrolyzing phenotype of the G?i1 mutant G202A arises from mimicry of the transition state for GTP hydrolysis normally stabilized by RGS domains. Aim 3 is to resolve the structural determinants of receptor-catalyzed nucleotide exchange via protein crystallography of functional receptor loop peptides bound to heterotrimeric G-protein subunits. This latter aim will be facilitated by our discovery of a G?i subfamily GEF peptide, KB-752, which acts as a surrogate for G??-mediated switch region changes; we have recently used KB-752 to establish the first crystal structure of a receptor loop bound to its G- protein target - the dopamine D2-receptor ic3 loop peptide D2N bound to G?i1. High-resolution structural models derived from these pursuits will be validated in biochemical and cellular studies of point mutants predicted from structural details to abrogate or enhance nucleotide exchange or switch receptor/G-protein coupling specificity. Success of this research program will lead to a new understanding of the precise structural determinants of GPCR/G-protein coupling, agonist-induced activation, and RGS protein facilitation. The The family of proteins known as G protein-coupled receptors represent the largest single fraction of targets for current drug therapies, including key medicines that control schizophrenia, bipolar disorder, and depression. While critically important for these drugs´ actions, the precise molecular details by which these receptor proteins activate biochemical processes inside cells is poorly understood. This research is thus directed towards building and validating structural models that describe the details of how receptors activate their coupled G-proteins, with such new knowledge providing valuable further avenues for drug discovery and design
Project start date: 2008-08-01
Project end date: 2012-07-31
Budget start date: 1-AUG-2010
Budget end date: 31-JUL-2011
PFA/PA: Real-Time Fluorescence Assay:RGS Domain GAP Activit(RMI)
David P Siderovski, Associate Professor
Pharmacologyuniversity Of North Carolina Chapel Hill
office Of Sponsored Research
chapel Hill, Nc 27599
Grant 3R03NS053754-01S2 from National Institute Of Neurological Disorders And Stroke IRG: ZNS1
Abstract: EXCEED THE SPACE PROVIDED. 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. 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. Ourgroup hasdiscovered 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. 7PERFORMANCE SITE ========================================Section End===========================================
Project start date: 2005-09-30
Project end date: 2009-08-31
3R03NS053754-01S2 (2008): $36875
G-PROTEIN SIGNAL COORDINATION BY RGS12
David P Siderovski, Associate Professor
University Of North Carolina Chapel Hill, Office Of Sponsored Research, Chapel Hill, Nc 27599
Grant 5R01GM062338-09 from National Institute Of General Medical Sciences
Keywords: 4-Aminobutanoic Acid; 4-Aminobutyric Acid; Address; Afferent Neurons; Agonist; Aminalon; Aminalone; Antigenic Determinants; Baclofen Receptors; Binding; Binding (Molecular Function); Binding Determinants; Binding Sites; Biochemical; Biological; Blood Coagulation Factor IV; Brain; Butanoic acid, 4-amino-; Ca++ element; Calcium; Cell Communication and Signaling; Cell Signaling; Cell surface; Cell to Cell Communication and Signaling; Cell-Cell Signaling; Cells; Central Nervous System; Coagulation Factor IV; Combining Site; Coupled; Dorsal Root Ganglia; Drugs; EC 2.7.2-; Encephalon; Encephalons; Epitopes; Extracellular Signal-Regulated Kinases; Factor IV; Family; G Protein-Complex Receptor; G Protein-Coupled Receptor Signaling; G-Protein Regulating Factors; G-Protein-Coupled Receptors; G-Proteins; GABA; GABA-B Receptor; GPCR Signaling; GTP Phosphohydrolases; GTP-Binding Protein Regulators; GTP-Binding Proteins; GTP-Regulatory Proteins; GTPases; Ganglia, Spinal; Goals; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Guanosine Triphosphate Phosphohydrolases; Guanosinetriphosphatases; Heterotrimeric G-Proteins; Heterotrimeric GTP-Binding Proteins; Intracellular Communication and Signaling; Ion Channel; Ionic Channels; L-tyrosine, dihydrogen phosphate (ester); Laboratories; Lead; Lipids; MAP Kinase Kinases; MAP kinase; MAPK; MAPK Kinases; MAPKKs; Mediating; Medication; Membrane Channels; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular; Molecular Interaction; Nature; Nerve Cells; Nerve Transmitter Substances; Nerve Unit; Nervous System, Brain; Nervous System, CNS; Neural Cell; Neuraxis; Neurocyte; Neurons; Neurons, Afferent; Neurons, Sensory; Neurotransmitters; Organism-Level Process; Organismal Process; Outcome; PTB Domain; Pain Control; Pain Therapy; Pain management; Pb element; Peripheral; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphatides; Phospholipids; Phosphopeptides; Phosphotyrosine; Phosphotyrosine Binding Domain; Physiologic Processes; Physiological Processes; Play; Process; Programs (PT); Programs [Publication Type]; Protein Family; Proteins; RGS Domain; RGS Family Protein; RGS Protein (G-Protein Signaling); RGS Proteins; RGS-Signaling Domain; Reactive Site; Receptor Protein; Regulating Factors, GTP-Binding Protein; Regulation; Regulators of G Protein Signaling Domain; Regulators of G-Protein Signaling Proteins; Regulators, G-Protein Signaling; Research; Role; Sensory Cell Afferent Neuron; Signal Transduction; Signal Transduction Systems; Signaling; Specific qualifier value; Specificity; Specified; Spinal Ganglia; Therapeutic Agents; Time; Tyrosine-O-phosphate; V (voltage); Work; biological signal transduction; desensitization; dorsal root ganglion; drug discovery; drug/agent; gamma-Aminobutyric Acid; gene product; guanosinetriphosphatase; heavy metal Pb; heavy metal lead; intercellular communication; member; mutant; neuronal; neuropathic pain; neurotropic; new therapeutics; next generation therapeutics; novel; novel therapeutics; painful neuropathy; presynaptic; programs; receptor; small molecule; social role; voltage
Project start date: 2001-02-01
Project end date: 2011-01-31
Budget start date: 1-FEB-2009
Budget end date: 31-JAN-2011
5R01GM062338-09 (2009): $258751
Real-Time Fluorescence Assay:RGS Domain GAP Activit(RMI)
David P Siderovski, Associate Professor
Pharmacologyuniversity Of North Carolina Chapel Hill
office Of Sponsored Research
chapel Hill, Nc 27599
Grant 3R03NS053754-01S1 from National Institute Of Neurological Disorders And Stroke IRG: ZNS1
Project start date: 2005-09-30
Project end date: 2009-08-31
3R03NS053754-01S1 (2007): $36500
Sponsored Links Excellgen http://Excellgen.com