(1093) Elucidation of Cannabinoid Receptors G-protein Coupling Switch from gαi to gαs

Objectives: The primary objective of this study investigate and better understand the mechanisms of action and potential differences in signaling pathways associated with cannabinoid receptors (CB1 and CB2), when exposed to different ligands, including synthetic cannabinoid receptor agonists (SCRAs). We will evaluate SCRA-CB1/CB2 Receptors Gi to Gs signaling switch by using G protein activation BRET biosensor using varying receptor densities in HEK293 Cells.

Composed of mainly CB1R and CB2R, cannabinoid receptors are G-protein coupled receptors (GPCR) located in the brain and periphery and are responsible for producing various neuromodulatory effects. These are the same receptors that are the target for Δ9-tetrahydrocannabinol (Δ9-THC), which is the main component of cannabis used recreationally and therapeutically. In an attempt to take advantage of its role in many physiological effects, more than 250 synthetic cannabinoid receptor agonists (SCRAs) have been developed in the last 10 years. Unfortunately, along with the development of SCRAs came its association with their illicit use and severe adverse effects such as seizures, tachycardia, and most notably witnessed in a 2016 “zombie” outbreak in New York. As it can be seen, these illicit SCRAs have shown to be more potent and have shown differences in effects and mechanisms. Δ9-THC is a partial agonist that has a high affinity for CB1R and activates its receptor to its canonical Gαi-mediated signaling pathway similarly to other cannabinoids. On the other hand, there are studies that suggest certain SCRAs show “superagonism”, meaning they are more efficacious than typical full agonists to certain CB receptors. Although SCRAs are also known to undergo Gαi-mediated signaling pathway, depending on the SCRA and its environmental conditions, they can preferentially activate different signaling pathways known as signaling bias. Due to its highly efficacious nature and its potential for functional selectivity, SCRA may trigger the unique effects compared to Δ9-THC and other structurally different cannabinoids scaffolds. Considering these physiological importance in mind, by elucidating the different G-protein mechanisms shown from CB1/CB2 receptors, the coupling switch from Gαi to Gαs may be an explanation to many of the clinical differences that are shown in SCRA abuse and cancer cells. Due to SCRA’s superagonism, we hypothesize that its transition in Gαi to Gαs coupling may be more pronounced in the presence of higher receptor density and therefore will evaluate the relationship between receptor expression and CB receptor signaling with 3 distinct CB agonists for CB1R and CB2R, the latter being the negative control. Considering the rise of Δ9-THC products nationwide as well as the development of SCRAs in both recreational and potential therapeutic purposes, this study will be crucial in enhancing our understanding of SCRA mechanism of action and toxicity as well as CB receptor’s potential impact on diseases.

Methods: Using the Flp-In System, which takes advantage of the DNA recombination system to express a particular gene of interest, CB1R and CB2R expressing stable cell lines are made. After the selection of stable cell lines, we will establish varying inducible CB1R and CB2R densities of HEK293 cell lines and measure G-protein subunit interactions through BRET experiments. The ligands that will be tested are Δ9-THC, CP55 and MDMB-FUBINACA, which represents a common component: a cannabis, a full CB1R agonist, and a SCRA superagonist. By transfecting and fusing Renilla luciferase and Venus into its respective G-protein subunits to its cells, we will measure BRET levels to evaluate the bias between Gαi and Gαs depending on its various receptor densities. This particular series of experiments, when completed, will be the first to use a G-protein activation BRET and study the influence of receptor density on CB receptor G-protein activity.

Results: N/A

Conclusions: N/A