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BioAssay: AID 485274

Summary of probe development for potentiators of the regulator of G-protein signaling 4 (RGS4)

HTS execution: Joseph Babcock, Zhihong Lin Ph.D., Shunyou Long M.S., Xiaofang Huang M.S., Owen McManus Ph.D., Meng Wu Ph.D. ..more
 Related BioAssays
 Related BioAssays
AID: 485274
Data Source: Johns Hopkins Ion Channel Center (JHICC_RGS4_pot_summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-09-27
Related Experiments
463111Primary cell-based high-throughput screening assay for identification of compounds that potentiate/activate regulator of G-protein signaling 4 (RGS4)Screeningdepositor-specified cross reference: Primary cell-based high-throughput screening assay for identification of compounds that potentiate/a
602282Validation assay for identification of compounds that activate the regulator of G-protein signaling 4 (RGS4)Screeningdepositor-specified cross reference
602283Counter screen for identification of compounds that activate the regulator of G-protein signaling 4 (RGS4): Non-induced cells with the primary screen assayScreeningdepositor-specified cross reference
624166Counter screen dose-response assay for SAR compounds that potentiate the regulator of G-protein signaling 4 (RGS4) in non-induced RGS4 cellsConfirmatorydepositor-specified cross reference
624179Dose response-assay for SAR compounds that potentiate the regulator of G-protein signaling 4 (RGS4)Otherdepositor-specified cross reference
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Summary

Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Richard Neubig, M.D., Ph.D. University of Michigan
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH087441-01A1
Grant Proposal PI: Richard Neubig, M.D., Ph.D.
Assay Implementation: Zhihong Lin Ph.D., Joseph Babcock, Shunyou Long M.S., Meng Wu Ph.D.
HTS execution: Joseph Babcock, Zhihong Lin Ph.D., Shunyou Long M.S., Xiaofang Huang M.S., Owen McManus Ph.D., Meng Wu Ph.D.


Signal transduction pathways mediated by G protein-coupled receptors (GPCRs) are major therapeutic targets with many known pharmacologic modulators. The regulators of G protein signaling (RGS proteins) are a key family of modulators and scaffolds for GPCRs which have been identified by many authors as appealing drug targets [1-5]. RGS proteins bind to activated G proteins and strongly inhibit their signals by catalyzing the hydrolysis of bound GTP [6-7]. Consistent with this mode of action, genetic deletion of RGS greatly enhances receptor signaling [8-10]. Furthermore, RGS proteins display isoform-specific tissue distribution [11-14] and their expression is altered in pathophysiological states including Parkinson's disease [15], neuropathic pain [16-17], schizophrenia, hypertension, and congestive heart failure [18]. An activator of RGS action would be expected to suppress the function of GPCR agonists such as vasoactivators, inflammatory mediators, endogenous neurotransmitters, or catecholamines such as L-DOPA. It has recently been established that loss of function of RGS2 causes hypertensive symptoms in mice [19] and may contribute to vasoactive disorders in humans as well [20-22]. Also, reduced expression of RGS4 has been correlated with schizophrenia [23-24]. Consequently, compounds that could either enhance the function of RGS proteins or could increase their expression might be of therapeutic benefit. The identification of selective modulators of RGS proteins would also greatly facilitate experimental studies of this sizeable protein family (with 20-30 members) by permitting rapid and specific chemical potentiation. Such RGS modulators would be particularly useful to determine which RGS proteins modulate particular receptor signaling pathways.

The goal of this project is to identify potentiators /activators of the regulator of G protein signaling 4 (RGS4), using a receptor-stimulated cell-based Ca++ assay.

[1]. Zhong, H., and Neubig, R.R. 2001. Regulator of G protein signaling proteins: novel multifunctional drug targets. J Pharmacol Exp Ther 297:837-845.
[2]. Neubig, R.R., and Siderovski, D.P. 2002. Regulators of G-protein signalling as new central nervous system drug targets. Nat Rev Drug Discov 1:187-197.
[3]. Druey, K.M. 2003. Regulators of G protein signalling: potential targets for treatment of allergic inflammatory diseases such as asthma. Expert Opin Ther Targets 7:475-484.
[4]. Cho, H., Harrison, K., and Kehrl, J.H. 2004. Regulators of G protein signaling: potential drug targets for controlling cardiovascular and immune function. Curr Drug Targets Immune Endocr Metabol Disord 4:107-118.
[5]. Riddle, E.L., Schwartzman, R.A., Bond, M., and Insel, P.A. 2005. Multi-tasking RGS proteins in the heart: the next therapeutic target? Circ Res 96:401-411.
[6]. Druey, K.M., Blumer, K.J., Kang, V.H., and Kehrl, J.H. 1996. Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family. Nature 379:742-746.
[7]. Yan, Y., Chi, P.P., and Bourne, H.R. 1997. RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis. J Biol Chem 272:11924-11927.
[8]. Jeong, S.W., and Ikeda, S.R. 2000. Endogenous regulator of G-protein signaling proteins modify N-type calcium channel modulation in rat sympathetic neurons. J Neurosci 20:4489-4496.
[9]. Clark, M.J., Neubig, R.R., and Traynor, J.R. 2004. Endogenous regulator of G protein signaling proteins suppress Galphao-dependent, mu-opioid agonist-mediated adenylyl cyclase supersensitization. J Pharmacol Exp Ther 310:215-222.
[10]. Fu, Y., Zhong, H., Nanamori, M., Mortensen, R.M., Huang, X., Lan, K., and Neubig, R.R. 2004. RGS-insensitive G-protein mutations to study the role of endogenous RGS proteins. Methods Enzymol 389:229-243.
[11]. Gold, S.J., Ni, Y.G., Dohlman, H.G., and Nestler, E.J. 1997. Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain. J Neurosci 17:8024-8037.
[12]. Nomoto, S., Adachi, K., Yang, L.X., Hirata, Y., Muraguchi, S., and Kiuchi, K. 1997. Distribution of RGS4 mRNA in mouse brain shown by in situ hybridization. Biochem Biophys Res Commun 241:281-287.
[13]. Kurrasch, D.M., Huang, J., Wilkie, T.M., and Repa, J.J. 2004. Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues. Methods Enzymol 389:3-15.
[14]. Doupnik, C.A., Xu, T., and Shinaman, J.M. 2001. Profile of RGS expression in single rat atrial myocytes. Biochim Biophys Acta 1522:97-107.
[15]. Tekumalla, P.K., Calon, F., Rahman, Z., Birdi, S., Rajput, A.H., Hornykiewicz, O., Di Paolo, T., Bedard, P.J., and Nestler, E.J. 2001. Elevated levels of DeltaFosB and RGS9 in striatum in Parkinson's disease. Biol Psychiatry 50:813-816.
[16]. Garnier, M., Zaratin, P.F., Ficalora, G., Valente, M., Fontanella, L., Rhee, M.H., Blumer, K.J., and Scheideler, M.A. 2003. Up-regulation of regulator of G protein signaling 4 expression in a model of neuropathic pain and insensitivity to morphine. J Pharmacol Exp Ther 304:1299-1306.
[17]. Xie, G.X., and Palmer, P.P. 2005. RGS proteins: new players in the field of opioid signaling and tolerance mechanisms. Anesth Analg 100:1034-1042.
[18]. Mittmann, C., Chung, C.H., Hoppner, G., Michalek, C., Nose, M., Schuler, C., Schuh, A., Eschenhagen, T., Weil, J., Pieske, B., et al. 2002. Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure. Cardiovasc Res 55:778-786.
[19].Heximer, S.P., Knutsen, R.H., Sun, X., Kaltenbronn, K.M., Rhee, M.H., Peng, N., Oliveira-dos-Santos, A., Penninger, J.M., Muslin, A.J., Steinberg, T.H., et al. 2003. Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. J Clin Invest 111:445-452.
[20]. Bodenstein, J., Sunahara, R.K., and Neubig, R.R. 2007. N-terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol Pharmacol 71:1040-1050.
[21]. Gu, S., Tirgari, S., and Heximer, S.P. 2008. The RGS2 gene product from a candidate hypertension allele shows decreased plasma membrane association and inhibition of Gq. Mol Pharmacol 73:1037-1043.
[22]. Yang, J., Kamide, K., Kokubo, Y., Takiuchi, S., Tanaka, C., Banno, M., Miwa, Y., Yoshii, M., Horio,T., Okayama, A., et al. 2005. Genetic variations of regulator of G-protein signaling 2 in hypertensive patients and in the general population. J Hypertens 23:1497-1505.
[23]. Chowdari, K.V., Mirnics, K., Semwal, P., Wood, J., Lawrence, E., Bhatia, T., Deshpande, S.N., B, K.T., Ferrell, R.E., Middleton, F.A., et al. 2002. Association and linkage analyses of RGS4 polymorphisms in schizophrenia. Hum Mol Genet 11:1373-1380.
[24]. Mirnics, K., Middleton, F.A., Stanwood, G.D., Lewis, D.A., and Levitt, P. 2001. Disease-specific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Mol Psychiatry 6:293-301.
[25]. Miret, Juan J., et al., Multiplexed G-Protein-Coupled Receptor Ca2+ Flux Assays for High-Throughput Screening. J Biomol Screen 10: 780-787 (2005) PMID: 16234348.
[26]. Zhang, J.-H., T.D.Y. Chung, and K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 4(2),67-73 (1999) PMID: 10838414.
[27]. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 24(2), 167-175 (2006). PMID: 16465162.


RGS4, M3 receptor, HTS assay, 384, primary, agonist, activator, potentiator, allosteric, FDSS, calcium, fluorescence, Kinetic, Fluo4-AM, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.
Please see the related bioassays (e.g., AID 463111) for details of assay protocols.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Additional Information
Grant Number: 1 R03 MH087441-01A1