CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a β-arrestin 2—based screening assay for the CB1R using BRET2 technology. When the existing BRET2 screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with β-arrestin 2, resulting in unsatisfactory assay performance. To enhance the β-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with β-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC50 and EC50 values ranging from 0.3 nM to 7.5 µM. Both primary and secondary screening were performed with Z' > 0.5, suggesting that the assay is a robust and cost-effective alternative to existing cell-based assays. (Journal of Biomolecular Screening 2009:371-380)
Cooke D., Bloom S.: The obesity pipeline: current strategies in the development of anti-obesity drugs. Nat Rev Drug Discov2006;5:919-931.
2.
Padwal RS, Majumdar SR: Drug treatments for obesity: orlistat, sibutramine, and rimonabant . Lancet2007;369:71-77.
3.
Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Porrino LJ: Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology2004;47(suppl 1):345-358.
4.
Brown AJ: Novel cannabinoid receptors. Br J Pharmacol2007;152:567-575.
5.
Matias I., Bisogno T., Di Marzo V.: Endogenous cannabinoids in the brain and peripheral tissues: regulation of their levels and control of food intake. Int J Obes2006;30:S7-S12.
6.
Di Marzo V.: The endocannabinoid system in obesity and type 2 diabetes. Diabetologia2008;51:1356-1367.
7.
Pagotto U., Pasquali R.: Endocannabinoids and energy metabolism. J Endocrinol Invest2006;29:66-76.
8.
Lim WK: GPCR drug discovery: novel ligands for CNS receptors. Recent Patents CNS Drug Discov2007;2:107-112.
9.
Bohn LM: Constitutive trafficking: more than just running in circles?Mol Pharmacol2007;71:957-958.
10.
Milligan G. : Principles: extending the utility of [35S]GTP gamma S binding assays. Trends Pharmacol Sci2003;24:87-90.
11.
Harrison C. , Traynor JR: The [35S]GTPγS binding assay: approaches and applications in pharmacology. Life Sci2003;74:489-508.
12.
Heding A.: Use of the BRET 7TM receptor/β-arrestin assay in drug discovery and screening . Expert Rev Mol Diagn2004;4:403-411.
13.
Milligan G. : Applications of bioluminescence- and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors . Eur J Pharm Sci2004;21:397-405.
14.
Vrecl M., Jorgensen R., Pogacnik A., Heding A.: Development of a BRET2 screening assay using β-arrestin 2 mutants . J Biomol Screen2004;9:322-333.
15.
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.
16.
Bakshi K., Mercier RW, Pavlopoulos S.: Interaction of a fragment of the cannabinoid CB1 receptor C-terminus with arrestin-2. FEBS Lett2007;581:5009-5016.
17.
Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG: Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-β-arrestin complexes after receptor endocytosis . J Biol Chem2001;276:19452-19460.
18.
Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS: Differential affinities of visual arrestin, βarrestin1, and βarrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem2000;275:17201-17210.
19.
Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG: Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization . J Biol Chem1999;274:32248-32257.
20.
Hamdan FF, Audet M., Garneau P., Pelletier J., Bouvier M.: High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based β-arrestin2 recruitment assay. J Biomol Screen2005;10:463-475.
21.
Heding A., Vrecl M., Bogerd J., McGregor A., Sellar R., Taylor PL, et al: Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem1998;273:11472-11477.
22.
Heding A., Vrecl M., Hanyaloglu AC, Sellar R., Taylor PL, Eidne KA: The rat gonadotropin-releasing hormone receptor internalizes via a β-arrestin-independent, but dynamin-dependent, pathway: addition of a carboxyl-terminal tail confers β-arrestin dependency. Endocrinology2000;141:299-306.
23.
Hanyaloglu AC, Vrecl M., Kroeger KM, Miles LE, Qian H., Thomas WG, et al: Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to β-arrestin-dependent internalization. J Biol Chem2001;276:18066-18074.
24.
Liu JJ, Hartman DS, Bostwick JR: An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis. Anal Biochem2003;318:91-99.
25.
Kostenis E. , Martini L., Ellis J., Waldhoer M., Heydorn A., Rosenkilde MM, et al: A highly conserved glycine within linker I and the extreme C terminus of G protein α subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity. J Pharmacol Exp Ther2005;313:78-87.
26.
Vrecl M., Drinovec L., Elling C., Heding A.: Opsin oligomerization in a heterologous cell system. J Recept Signal Transduct Res2006;26:505-526.
27.
Mukhopadhyay S., Shim JY, Assi AA, Norford D., Howlett AC: CB(1) cannabinoid receptor-G protein association: a possible mechanism for differential signaling . Chem Phys Lipids2002;121:91-109.
28.
Bertrand L. , Parent S., Caron M., Legault M., Joly E., Angers S., et al: The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). J Recept Signal Transduct Res2002;22:533-541.
29.
Hsieh C., Brown S., Derleth C., Mackie K.: Internalization and recycling of the CB1 cannabinoid receptor. J Neurochem1999;73:493-501.
30.
Qian H., Pipolo L., Thomas WG: Association of β-arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization. Mol Endocrinol2001;15:1706-1719.
31.
Ghosh RN, DeBiasio R., Hudson CC, Ramer ER, Cowan CL, Oakley RH: Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology. J Biomol Screen2005;10:476-484.
32.
Jensen RT, Battey JF, Spindel ER, Benya RV: International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev2008;60:1-42.
33.
Choi G., Guo J., Makriyannis A.: The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism. Biochim Biophys Acta2005;1668:1-9.34.
34.
Nie J., Lewis DL: The proximal and distal C-terminal tail domains of the CB1 cannabinoid receptor mediate G protein coupling. Neuroscience2001 ;107:161-167.
35.
Celver J., Vishnivetskiy SA, Chavkin C., Gurevich VV: Conservation of the phosphate-sensitive elements in the arrestin family of proteins. J Biol Chem2002;277:9043-9048.
36.
Kubale V., Abramovic Z., Pogacnik A., Heding A., Sentjurc M., Vrecl M.: Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2. Cell Tissue Res2007;330:231-245.
37.
Leterrier C. , Bonnard D., Carrel D., Rossier J., Lenkei Z.: Constitutive endocytic cycle of the CB1 cannabinoid receptor. J Biol Chem2004;279:36013-36021.
38.
Pertwee RG: Inverse agonism and neutral antagonism at cannabinoid CB1 receptors . Life Sci2005;76:1307-1324.
39.
Milligan G. : Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective. Mol Pharmacol2003;64:1271-1276.
40.
Kostenis E. , Waelbroeck M., Milligan G.: Techniques: promiscuous Gα proteins in basic research and drug discovery. Trends Pharmacol Sci2005;26:595-602.
