Skip to main content

Methods to Quantify Cell Signaling and GPCR Receptor Ligand Bias: Characterization of Drugs that Target the Endocannabinoid Receptors in Huntington’s Disease

Part of the Methods in Molecular Biology book series (MIMB,volume 1780)

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.

Keywords

  • GPCRs
  • CB1
  • Cannabinoids bias
  • In-Cell Western™
  • On-Cell Western™
  • ERK
  • CREB
  • PLC
  • Akt
  • CB1 localization
  • CB1 expression
  • Operational model

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-7825-0_25
  • Chapter length: 23 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   189.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-7825-0
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   249.99
Price excludes VAT (USA)
Hardcover Book
USD   349.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Labbadia J, Morimoto RI (2013) Huntington’s disease: underlying molecular mechanisms and emerging concepts. Trends Biochem Sci 38:378–385

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  2. Ross CA, Aylward EH, Wild EJ et al (2014) Huntington disease: natural history, biomarkers and prospects for therapeutics. Nat Rev Neurol 10:204–216

    CrossRef  CAS  PubMed  Google Scholar 

  3. 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

    CrossRef  CAS  PubMed  Google Scholar 

  4. 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

    CrossRef  CAS  PubMed  Google Scholar 

  5. Ramaswamy S, Kordower JH (2012) Gene therapy for Huntington’s disease. Neurobiol Dis 48:243–254

    CrossRef  CAS  PubMed  Google Scholar 

  6. 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

    CrossRef  CAS  PubMed  Google Scholar 

  7. 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

    Google Scholar 

  8. Adam OR, Jankovic J (2008) Symptomatic treatment of Huntington disease. Neurotherapeutics 51:81–97

    Google Scholar 

  9. Frankc F (2014) Treatment of Huntington’s disease. Neurotherapeutics 11:153–160

    CrossRef  CAS  Google Scholar 

  10. Mason SL, Barker RA (2016) Advancing pharmacotherapy for treating Huntington’s disease: a review of the existing literature. Expert Opin Pharmacol 17:41–52

    CrossRef  CAS  Google Scholar 

  11. Lagerström MC, Schiöth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357

    CrossRef  CAS  PubMed  Google Scholar 

  12. Oldham WM, Hamm HE (2008) Heterotrimeric G protein activation by protein-coupled receptors. Nat Rev Mol Cell Biol 9:60–71

    CrossRef  CAS  PubMed  Google Scholar 

  13. Rosenbaum DM, Rasmussen SG, Kobilka BK (2009) The structure and function of G-protein-coupled receptors. Nature 459:356–363

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  14. Millar RP, Newton CL (2010) The year in G protein-coupled receptor research. Mol Endocrinol 24:261–274

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  15. 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

    CrossRef  CAS  PubMed  Google Scholar 

  16. Pertwee RG (2008) Ligands that target cannabinoid receptors in the brain: from THC to anandamide and beyond. Addict Biol 13:147–159

    CrossRef  CAS  PubMed  Google Scholar 

  17. 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

    CrossRef  CAS  PubMed  Google Scholar 

  18. 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

    CrossRef  CAS  PubMed  Google Scholar 

  19. 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

    CrossRef  CAS  PubMed  Google Scholar 

  20. Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61–65

    CrossRef  CAS  PubMed  Google Scholar 

  21. 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

    CrossRef  CAS  PubMed  Google Scholar 

  22. 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

    CrossRef  CAS  PubMed  Google Scholar 

  23. Rodriguez de Fonseca F, Del Arco I, Bermudez-Silva FJ et al (2005) The endocannabinoid system: physiology and pharmacology. Alcohol 40:2–14

    CrossRef  CAS  Google Scholar 

  24. Di Marzo V, Fontana A, Cadas H et al (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372:686–691

    CrossRef  PubMed  Google Scholar 

  25. Stella N, Piomelli D (2001) Receptor-dependent formation of endogenous cannabinoids in cortical neurons. Eur J Pharmacol 425:189–196

    CrossRef  CAS  PubMed  Google Scholar 

  26. 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

    CrossRef  CAS  PubMed  Google Scholar 

  27. 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

    CrossRef  CAS  PubMed  Google Scholar 

  28. Howlett AC (2005) Cannabinoid receptor signaling. Handb Exp Pharmacol 168:53–79

    CrossRef  CAS  Google Scholar 

  29. Turu G, Hunyady L (2010) Signal transduction of the CB1 cannabinoid receptor. J Mol Endocrinol 44:75–85

    CrossRef  CAS  PubMed  Google Scholar 

  30. Ranieri R, Laezza C, Bifulco M et al (2016) Endocannabinoid system in neurological disorders. Recent Pat CNS Drug Discov 10:90–112

    CrossRef  CAS  PubMed  Google Scholar 

  31. 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

    CrossRef  CAS  PubMed  Google Scholar 

  32. 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

    CrossRef  CAS  PubMed  Google Scholar 

  33. 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

    CrossRef  CAS  PubMed  Google Scholar 

  34. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  35. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  36. 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

    CrossRef  PubMed  Google Scholar 

  37. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  38. 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

    CrossRef  PubMed  Google Scholar 

  39. Mievis S, Blum D, Ledent C (2011) Worsening of Huntington disease phenotype in CB1 receptor knockout mice. Neurobiol Dis 42:524–529

    CrossRef  CAS  PubMed  Google Scholar 

  40. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  41. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  42. 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

    CrossRef  CAS  PubMed  Google Scholar 

  43. 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

    CrossRef  CAS  PubMed  Google Scholar 

  44. 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

    CrossRef  CAS  PubMed  Google Scholar 

  45. 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

    CrossRef  CAS  Google Scholar 

  46. Kenakin T, Christopoulos A (2013) Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov 12:205–216

    CrossRef  CAS  PubMed  Google Scholar 

  47. Black JW, Leff P (1983) Operational models of pharmacological agonism. Proc R Soc Lond B Biol Sci 220:141–162

    CrossRef  CAS  PubMed  Google Scholar 

  48. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  49. 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

    CrossRef  CAS  PubMed  Google Scholar 

  50. Wong S (2004) A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors. Anal Biochem 333:265–272

    CrossRef  CAS  PubMed  Google Scholar 

  51. Boveia V, Schutz-Geschwender A (2015) Quantitative analysis of signal transduction with in-cell western immunofluorescence assays. Methods Mol Biol 1314:115–130

    CrossRef  PubMed  Google Scholar 

  52. 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

    CrossRef  CAS  PubMed  Google Scholar 

  53. Hudson BD, Hébert TE, Kelly ME (2010) Physical and functional interaction between CB1 cannabinoid receptors and beta2-adrenoceptors. Br J Pharmacol 160:627–642

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  54. 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

    CrossRef  CAS  PubMed  Google Scholar 

  55. 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

    CrossRef  CAS  PubMed  Google Scholar 

  56. 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

    CrossRef  CAS  PubMed  Google Scholar 

  57. 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

  58. 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

    CrossRef  CAS  PubMed  Google Scholar 

  59. Ehlert FJ, Suga H, Griffin MT (2011) Quantifying agonist activity at G protein-coupled receptors. J Vis Exp (58):e3179

    Google Scholar 

  60. Ehlert FJ (2015) Functional studies cast light on receptor states. Trends Pharmacol Sci 36:596–604

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  61. Kenakin T (2015) The measurement of receptor signaling bias. Methods Mol Biol 1335:163–176

    CrossRef  PubMed  Google Scholar 

  62. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  63. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  64. 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

    CrossRef  CAS  PubMed  Google Scholar 

  65. 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

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  66. Wu H, Wacker D, Mileni M et al (2012) Structure of the human κ-opioid receptor in complex with JDTic. Nature 485:327–332

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

Download references

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

Authors

Corresponding author

Correspondence to Eileen M. Denovan-Wright .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

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