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

  • Amina M. Bagher
  • Robert B. Laprairie
  • Melanie E. M. Kelly
  • Eileen M. Denovan-WrightEmail author
Protocol
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 

Notes

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.

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Copyright information

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

Authors and Affiliations

  • Amina M. Bagher
    • 1
    • 2
  • Robert B. Laprairie
    • 1
    • 3
  • Melanie E. M. Kelly
    • 1
    • 4
  • Eileen M. Denovan-Wright
    • 1
    Email author
  1. 1.Department of PharmacologyDalhousie UniversityHalifaxCanada
  2. 2.Department of Pharmacology and ToxicologyKing Abdulaziz UniversityJeddah, KSASaudi Arabia
  3. 3.College of Pharmacy and NutritionUniversity of SaskatchewanSaskatoonCanada
  4. 4.Department of Ophthalmology and Visual SciencesDalhousie UniversityHalifaxCanada

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