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

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 Gα_i/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.