Abstract
G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington’s disease (HD). Decreasing type 1 cannabinoid receptor (CB1) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB1 levels and/or signaling may be beneficial for HD patients while decreasing CB1 levels and/or signaling may be detrimental. Recent studies have found that Gαi/o-biased CB1 agonists activate extracellular signal-regulated kinase (ERK), increase CB1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB1 agonists that are β-arrestin1-biased were found to reduce CB1 protein levels and cell viability. Measuring agonist bias of known and novel CB1 agonists will provide important data that predict CB1-specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Labbadia J, Morimoto RI (2013) Huntington’s disease: underlying molecular mechanisms and emerging concepts. Trends Biochem Sci 38:378–385
Ross CA, Aylward EH, Wild EJ et al (2014) Huntington disease: natural history, biomarkers and prospects for therapeutics. Nat Rev Neurol 10:204–216
Bachoud-Levi AC, Deglon N, Nguyen JP et al (2000) Neuroprotective gene therapy for Huntington’s disease using a polymer encapsulated BHK cell line engineered to secrete human CNTF. Hum Gene Ther 11:1723–1729
Bloch J, Bachoud-Levi AC, Deglon N et al (2004) Neuroprotective gene therapy for Huntington’s disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study. Hum Gene Ther 15:968–975
Ramaswamy S, Kordower JH (2012) Gene therapy for Huntington’s disease. Neurobiol Dis 48:243–254
Bartus RT, Johnson EM (2016) Clinical tests of neurotrophic factors for human neurodegenerative diseases: Part 1. Where have we been and what have we learned? Neurobiol Dis 97:156–168
Yang W, Tu Z, Sun Q, Li XJ (2016) CRISPR/Cas9: implications for modeling and therapy of neurodegenerative diseases. Front Mol Neurosci 28:9–30
Adam OR, Jankovic J (2008) Symptomatic treatment of Huntington disease. Neurotherapeutics 51:81–97
Frankc F (2014) Treatment of Huntington’s disease. Neurotherapeutics 11:153–160
Mason SL, Barker RA (2016) Advancing pharmacotherapy for treating Huntington’s disease: a review of the existing literature. Expert Opin Pharmacol 17:41–52
Lagerström MC, Schiöth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357
Oldham WM, Hamm HE (2008) Heterotrimeric G protein activation by protein-coupled receptors. Nat Rev Mol Cell Biol 9:60–71
Rosenbaum DM, Rasmussen SG, Kobilka BK (2009) The structure and function of G-protein-coupled receptors. Nature 459:356–363
Millar RP, Newton CL (2010) The year in G protein-coupled receptor research. Mol Endocrinol 24:261–274
Kenakin T, Watson C, Muniz-Medina V et al (2012) A simple method for quantifying functional selectivity and agonist bias. ACS Chem Neurosci 3:193–203
Pertwee RG (2008) Ligands that target cannabinoid receptors in the brain: from THC to anandamide and beyond. Addict Biol 13:147–159
Matsuda LA, Lolait SJ, Brownstein MJ et al (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561–564
Tharp WG, Lee YH, Maple RL, Pratley RE (2008) The cannabinoid CB1 receptor is expressed in pancreatic delta-cells. Biochem Biophys Res Commun 372:595–600
Cota D (2007) CB1 receptors: emerging evidence for central and peripheral mechanisms that regulate energy balance, metabolism, and cardiovascular health. Diabetes Metab Res Rev 23:507–517
Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61–65
Núñez E, Benito C, Pazos MR et al (2004) Cannabinoid CB2 receptors are expressed by perivascular microglia cells in the human brain: an immunohistochemical study. Synapse 53:208–213
Fernández-Ruiz J, Romero J, Velasco G et al (2006) Cannabinoid CB2 receptor: a new target for controlling neural cell survival. Trends Pharmacol Sci 28:39–45
Rodriguez de Fonseca F, Del Arco I, Bermudez-Silva FJ et al (2005) The endocannabinoid system: physiology and pharmacology. Alcohol 40:2–14
Di Marzo V, Fontana A, Cadas H et al (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372:686–691
Stella N, Piomelli D (2001) Receptor-dependent formation of endogenous cannabinoids in cortical neurons. Eur J Pharmacol 425:189–196
Devane WA, Hanus L, Breuer A et al (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258:1946–1949
Mechoulam R, Ben-Shabat S, Hanus L et al (1995) Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 50:83–90
Howlett AC (2005) Cannabinoid receptor signaling. Handb Exp Pharmacol 168:53–79
Turu G, Hunyady L (2010) Signal transduction of the CB1 cannabinoid receptor. J Mol Endocrinol 44:75–85
Ranieri R, Laezza C, Bifulco M et al (2016) Endocannabinoid system in neurological disorders. Recent Pat CNS Drug Discov 10:90–112
Denovan-Wright EM, Robertson HA (2000) Cannabinoid receptor messenger RNA levels decrease in a subset of neurons of the lateral striatum, cortex and hippocampus of transgenic Huntington’s disease mice. Neuroscience 98:705–713
Glass M, Dragunow M, Faull RL (2000) The pattern of neurodegeneration in Huntington’s disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington’s disease. Neuroscience 97:505–519
Sagredo O, Pazos MR, Valdeolivas S, Fernandez-Ruiz J (2012) Cannabinoids: novel medicines for the treatment of Huntington’s disease. Recent Pat CNS Drug Discov 7:41–48
Naydenov AV, Sepers MD, Swinney K et al (2014a) Genetic rescue of CB1 receptors on medium spiny neurons prevents loss of excitatory striatal synapses but not motor impairment in HD mice. Neurobiol Dis 71:140–150
Chiarlone A, Bellocchio L, Blázquez C et al (2014) A restricted population of CB1 cannabinoid receptors with neuroprotective activity. Proc Natl Acad Sci U S A 111:8257–8262
Blázquez C, Chiarlone A, Sagredo O et al (2011) Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease. Brain 134:119–136
Blázquez C, Chiarlone A, Bellocchio L et al (2015) The CB1 cannabinoid receptor signals striatal neuroprotection via a PI3K/Akt/mTORC1/BDNF pathway. Cell Death Differ 22:1618–1629
Kloster E, Saft C, Epplen JT, Arning L (2013) CNR1 variation is associated with the age at onset in Huntington disease. Eur J Med Genet 56:416–419
Mievis S, Blum D, Ledent C (2011) Worsening of Huntington disease phenotype in CB1 receptor knockout mice. Neurobiol Dis 42:524–529
McIntosh BT, Hudson B, Yegorova S et al (2007) Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells. Br J Pharmacol 152:1111–1120
Laprairie RB, Bagher AM, Kelly ME et al (2014) Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons. J Biol Chem 289:24845–24862
Khajehali E, Malone DT, Glass M et al (2015) Biased agonism and biased allosteric modulation at the CB1 cannabinoid receptor. Mol Pharmacol 88:368–379
Laprairie RB, Bagher AM, Kelly ME, Denovan-Wright EM (2016) Biased type 1 cannabinoid receptor signaling influences neuronal viability in a cell culture model of Huntington disease. Mol Pharmacol 89:364–375
Dowie MJ, Howard ML, Nicholson LF et al (2010) Behavioural and molecular consequences of chronic cannabinoid treatment in Huntington’s disease transgenic mice. Neuroscience 170:324–336
Violin JD, Crombie AL, Soergel DG, Lark MW (2014) Biased ligands at G-protein-coupled receptors: promise and progress. Trends Pharmacol Sci 2014(35):308–316
Kenakin T, Christopoulos A (2013) Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov 12:205–216
Black JW, Leff P (1983) Operational models of pharmacological agonism. Proc R Soc Lond B Biol Sci 220:141–162
Stahl EL, Zhou L, Ehlert FJ, Bohn LM (2015) A novel method for analyzing extremely biased agonism at G protein-coupled receptors. Mol Pharmacol 87:866–877
Chen H, Kovar J, Sissons S et al (2005) A cell based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. Anal Biochem 338:136–142
Wong S (2004) A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors. Anal Biochem 333:265–272
Boveia V, Schutz-Geschwender A (2015) Quantitative analysis of signal transduction with in-cell western immunofluorescence assays. Methods Mol Biol 1314:115–130
Daigle TL, Kearn CS, Mackie K (2008) Rapid CB1 cannabinoid receptor desensitization defines the time course of ERK1/2 MAP kinase signaling. Neuropharmacology 54:36–44
Hudson BD, Hébert TE, Kelly ME (2010) Physical and functional interaction between CB1 cannabinoid receptors and beta2-adrenoceptors. Br J Pharmacol 160:627–642
Laprairie RB, Kelly ME, Denovan-Wright EM (2013) Cannabinoids increase type 1 cannabinoid receptor expression in a cell culture model of striatal neurons: implications for Huntington’s disease. Neuropharmacology 72:47–57
Bagher AM, Laprairie RB, Kelly ME, Denovan-Wright EM (2013) Co-expression of the human cannabinoid receptor coding region splice variants (hCB1) affects the function of hCB1 receptor complexes. Eur J Pharmacol 721:341–354
Bagher AM, Laprairie RB, Kelly ME, Denovan-Wright EM (2016) Antagonism of dopamine receptor 2 long affects cannabinoid receptor 1 signaling in a cell culture model of striatal medium spiny projection neurons. Mol Pharmacol 89:652–666
Miller JW (2004) Tracking G protein-coupled receptor trafficking using odyssey imaging. http://www.licor.com/bio/PDF/Miller_GPCR.pdf. Accessed 1 Mar 2010
Griffin MT, Figueroa KW, Liller S, Ehlert FJ (2007) Estimation of agonist activity at G protein-coupled receptors: analysis of M2 muscarinic receptor signaling through Gi/o, Gs, and G15. J Pharmacol Exp Ther 321:1193–1207
Ehlert FJ, Suga H, Griffin MT (2011) Quantifying agonist activity at G protein-coupled receptors. J Vis Exp (58):e3179
Ehlert FJ (2015) Functional studies cast light on receptor states. Trends Pharmacol Sci 36:596–604
Kenakin T (2015) The measurement of receptor signaling bias. Methods Mol Biol 1335:163–176
Lauckner JE, Hille B, Mackie K (2005) The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins. Proc Natl Acad Sci U S A 102:19144–19149
Milligan G, Unson CG, Wakeman DJO (1989) Cholera toxin treatment produces down-regulation of the α-subunit of the stimulatory guanine-nucleotide-binding protein (Gs). Biochem J 262:643–649
Obara Y, Okano Y, Ono S et al (2008) βγ subunits of G(i/o) suppress EGF-induced ERK5 phosphorylation, whereas ERK1/2 phosphorylation is enhanced. Cell Signal 20:1275–1283
Rives ML, Rossillo M, Liu-Chen LY, Javitch JA (2012) 6′-Guanidinonaltrindole (6′-GNTI) is a G protein-biased κ-opioid receptor agonist that inhibits arrestin recruitment. J Biol Chem 287:27050–27054
Wu H, Wacker D, Mileni M et al (2012) Structure of the human κ-opioid receptor in complex with JDTic. Nature 485:327–332
Acknowledgments
This work was supported by a Bridge Funding Grant from Dalhousie University to EMD-W. A.M.B. was supported by studentships from Dalhousie University and King Abdulaziz University, Jeddah, Saudi Arabia. R.B.L. was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bagher, A.M., Laprairie, R.B., Kelly, M.E.M., Denovan-Wright, E.M. (2018). Methods to Quantify Cell Signaling and GPCR Receptor Ligand Bias: Characterization of Drugs that Target the Endocannabinoid Receptors in Huntington’s Disease. In: Precious, S., Rosser, A., Dunnett, S. (eds) Huntington’s Disease. Methods in Molecular Biology, vol 1780. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7825-0_25
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7825-0_25
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7824-3
Online ISBN: 978-1-4939-7825-0
eBook Packages: Springer Protocols