Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango™ and PathHunter ™, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gαi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and Gαi-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. (Journal of Biomolecular Screening 2009:811-823)
Overington JP , Al-Lazikani B., Hopkins AL: How many drug targets are there? Nat Rev Drug Discov2006;5:993-996.
2.
Foreman JC, Johansen T.: Textbook of Receptor Pharmacology. 2nd ed. Boca Raton, FL: CRC Press; 2003.
3.
Leifert WR, Aloia AL, Bucco O., McMurchie EJ: GPCR-induced dissociation of G-protein subunits in early stage signal transduction . Mol Membr Biol2005;22:507-517.
4.
Siehler S.: Cell-based assays in GPCR drug discovery. Biotechnol J2008;3:471-483.
5.
McLoughlin DJ , Bertelli F., Williams C.: The A, B, Cs of G-protein-coupled receptor pharmacology in assay development for HTS. Expert Opinion on Drug Discovery2007;2:603-619.
Verkaar F., van Rosmalen JW, Blomenrohr M., van Koppen CJ, Blankesteijn WM, Smits JF, Zaman GJ: G protein-independent cell-based assays for drug discovery on seven-transmembrane receptors. Biotechnol Annu Rev2008;14:253-274.
8.
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI: Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature1990;346:561-564.
9.
Howlett AC: Pharmacology of cannabinoid receptors. Annu Rev Pharmacol Toxicol1995;35:607-634.
10.
Devane WA, Hanus L., Breuer A., Pertwee RG, Stevenson LA, Griffin G., et al: Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science1992;258:1946-1949.
11.
Stella N., Schweitzer P., Piomelli D.: A second endogenous cannabinoid that modulates long-term potentiation . Nature1997;388:773-778.
12.
Rodriguez de Fonseca F., Del Arco I., Bermudez-Silva FJ, Bilbao A., Cippitelli A., Navarro M.: The endocannabinoid system: physiology and pharmacology. Alcohol Alcohol2005;40:2-14.
13.
Garcia DE, Brown S., Hille B., Mackie K.: Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor. J Neurosci1998;18:2834-2841.
14.
Daigle TL, Kearn CS, Mackie K.: Rapid CB1 cannabinoid receptor desensitization defines the time course of ERK1/2 MAP kinase signaling. Neuropharmacology2008;54:36-44.
15.
Hausdorff WP , Caron MG, Lefkowitz RJ: Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J1990;4:2881-2889.
16.
Oakley RH, Hudson CC, Cruickshank RD, Meyers DM, Payne RE Jr, Rhem SM, et al: The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors. Assay Drug Dev Technol2002;1:21-30.
17.
Barnea G., Strapps W., Herrada G., Berman Y., Ong J., Kloss B., et al: The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci U S A2008;105:64-69.
18.
Olson KR, Eglen RM: Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol2007;5:137-144.
19.
Wehrman TS, Casipit CL, Gewertz NM, Blau HM: Enzymatic detection of protein translocation. Nat Methods2005;2:521-527.
20.
Melvin LS, Johnson MR, Harbert CA, Milne GM, Weissman A.A cannabinoid derived prototypical analgesic. J. Med. Chem.1984;27:67-71.
21.
D’Ambra TE, Estep KG, Bell MR, Eissenstat MA, Josef KA, Ward SJ, et al: Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor. J Med Chem1992 ;35:124-135.
22.
Rinaldi-Carmona M., Barth F., Héaulme M., Shire D., Calandra B., Congy C., et al: SR141716A, a potent and selective antagonist of the brain cannabinoid receptor. FEBS Lett1994;350:240-244.
23.
Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen1999;4:67-73.
24.
Felder CC, Joyce KE, Briley EM, Mansouri J., Mackie K., Blond O., et al: Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors. Mol Pharmacol1995;48:443-450.
25.
Bouaboula M. , Poinot-Chazel C., Bourrié B., Canat X., Calandra B., Rinaldi-Carmona M., et al: Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem J1995 ;312 (Pt 2):637-641.
26.
Hill SJ, Baker JG, Rees S.: Reporter-gene systems for the study of G-protein-coupled receptors . Curr Opin Pharmacol2001;1:526-532.
27.
Granas C., Lundholt B., Heydorn A., Linde V., Pedersen H., Krog-Jensen C., et al: High content screening for G protein-coupled receptors using cell-based protein translocation assays. Comb Chem High Throughput Screen2005;8:301-309.
28.
Hsieh C., Brown S., Derleth C., Mackie K.: Internalization and recycling of the CB1 cannabinoid receptor. J Neurochem1999;73:493-501.
29.
Coutts AA, Anavi-Goffer S., Ross RA, MacEwan DJ, Mackie K., Pertwee RG, et al: Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. J Neurosci2001;21:2425-2433.
30.
Lohse MJ, Benovic JL, Codina J., Caron MG, Lefkowitz RJ: beta-Arrestin: a protein that regulates beta-adrenergic receptor function . Science1990;248: 1547-1550.
31.
Rubino T., Viganò D., Premoli F., Castiglion IC, Bianchessi S., Zippel R., et al: Changes in the expression of G protein-coupled receptor kinases and beta-arrestins in mouse brain during cannabinoid tolerance: a role for RAS-ERK cascade. Mol Neurobiol2006;33:199-213.
32.
Jin W., Brown S., Roche JP, Hsieh C., Celver JP, Kovoor A., et al: Distinct domains of the CB1 cannabinoid receptor mediate desensitization and internalization. J Neurosci1999;19:3773-3780.
33.
Kouznetsova M., Kelley B., Shen M., Thayer SA: Desensitization of cannabinoid-mediated presynaptic inhibition of neurotransmission between rat hippocampal neurons in culture . Mol Pharmacol2002;61:477-485.
34.
Leterrier C. , Laine J., Darmon M., Boudin H., Rossier J., Lenkei Z.: Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors. J Neurosci2006;26:3141-3153.
35.
Bouaboula M. , Perrachon S., Milligan L., Canat X., Rinaldi-Carmona M., Portier M., et al: A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions. J Biol Chem1997;272:22330-22339.
36.
Pan X., Ikeda SR, Lewis DL: SR 141716A acts as an inverse agonist to increase neuronal voltage-dependent Ca2+ currents by reversal of tonic CB1 cannabinoid receptor activity. Mol Pharmacol1998;54:1064-1072.
37.
Anavi-Goffer S., Fleischer D., Hurst DP, Lynch DL, Barnett-Norris J., Shi S., et al: Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms. J Biol Chem2007;282: 25100-25113.
38.
van der Lee MM, Bras M., van Koppen CJ, Zaman GJ: {beta}-Arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1. J Biomol Screen2008;13:986-998.
39.
Breivogel CS , Scates SM, Beletskaya IO, Lowery OB, Aceto MD, Martin BR: The effects of delta9-tetrahydrocannabinol physical dependence on brain cannabinoid receptors . Eur J Pharmacol2003;459:139-150.
40.
Martin BR, Sim-Selley LJ, Selley DE: Signaling pathways involved in the development of cannabinoid tolerance . Trends Pharmacol Sci2004;25: 325-330.
41.
Bass CE, Martin BR: Time course for the induction and maintenance of tolerance to Delta(9)-tetrahydrocannabinol in mice. Drug Alcohol Depend2000;60:113-119.
42.
Appleyard SM , Patterson TA, Jin W., Chavkin C.: Agonist-induced phosphorylation of the kappa-opioid receptor. J Neurochem1997;69: 2405-2412.
43.
Kovoor A., Nappey V., Kieffer BL, Chavkin C.: Mu and delta opioid receptors are differentially desensitized by the coexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 in xenopus oocytes . J Biol Chem1997;272:27605-27611.
44.
Muccioli GG : Blocking the cannabinoid receptors: drug candidates and therapeutic promises. Chem Biodivers2007 ;4:1805-1827.
