374Differential activation of G proteins by synthetic cannabinoid receptor agonists, utilizing the CAMYEL BRET biosensor

Introduction

Cannabinoid Receptor 1 (CB1) agonists
Synthetic cannabinoid receptor agonists (SCRAs) have been extensively used as tools to study the two cannabinoid G protein-coupled receptors (GPCR’s), CB1 and CB2. Unfortunately, SCRAs now represent the most rapidly growing class of new psychoactive substances associated with drug misuse. SCRAs pose serious health risks with adverse effects such as severe vomiting, chest pain, increased heart rate, kidney damage, seizures/unconsciousness and even death. The molecular mechanisms through which SCRA’s exert their toxic effects remains unclear.¹

GPCR’s in general, span the outer membrane of a cell and are coupled to a trimeric G protein complex (G_a, G_β, G_γ) inside the cell. Upon agonist activation of the GPCR, the trimeric G protein complex dissociates releasing Ga (of which there are four major subtypes: Ga_i, Ga_s, Ga_q, Ga_12/13) and Gβ_γ proteins within the cell. Many cellular responses can be initiated through the Ga proteins, depending on which of the four major subtypes is present in a particular cells. Most relevant here is Gas which stimulates adenyl cyclase activity leading to an increase in cAMP, which in turn initiates further downstream
responses. In contrast Ga_i, inhibits adenyl cyclase leading to a decrease in cAMP production.

Previous studies have shown that SCRAs are agonists at both CB1 and CB2 receptors, with the psychoactive effects attributed to CB1 activation. It is also known that cannabinoid receptor G protein signaling for SCRAs is predominantly through the Ga_i protein family.

In this study², a representative group of sixteen cannabinoids was chosen (twelve confirmed in patients admitted to emergency departments with SCRA toxicity and 4 reference compounds) and determined whether these cannabinoids signal via the Gi and/or Gs pathways, using a Bioluminescence Resonance Energy Transfer (BRET) biosensor.

Assay Principle

The CAYMEL BRET biosensor³ is composed of the EPAC protein bound to both a luciferase (RLuc) and yellow fluorescent protein (YFP) as shown in fig. 1. In the absence of cAMP, the conformation of EPAC allows Rluc and YFP to be in close proximity. A BRET signal can therefore occur by addition of Coelenterazine (RLuc substrate). As cAMP is formed in the cell, it binds EPAC, which in turn causes a conformational change that diminishes the BRET signal as the concentration of cAMP increases.

Materials & Methods

• 96-well microplate, white, clear bottom, poly D lysine coated (Sigma-Aldrich)
• PHERAstar FS, BRET 1 optic module
• HEK293 Flpln cells stably expressing CB1
• pcDNA3L-His-CAMYEL plasmid

Transfection Procedure
Human HEK293 cells, stably expressing CB1 receptor (HEKCB1)² were transfected with pcDNA3L-His-CAMYEL (5ug) plasmid using the linear polyethylenimine (PEI) protocol. The PEI/DNA complex mixture was sequentially added in a 1:6 ratio and cells incubated at 37 °C and 5% CO₂ for 24h.

GPCR activation and cAMP measurement
To determine whether the tested cannabinoids signal via Ga_s and Ga_i pathways, transfected cells stably expressing CB1 and CAMYEL were treated/not treated with Pertussis Toxin (PTX). PTX inactivates the Ga_i signaling pathway but has no effect on Gas proteins. With PTX mediated inactivation of Ga_i
signaling, the sole effect of potential agonists on Gas can be investigated. Both populations of cells were plated to a density of 100,000 cells per well. On the day of the assay, Forskolin was prepared (3µM final concentration). It is used to raise levels of cAMP so inhibition can be measured. Luciferase substrate (Coelenterazine H, 5µM) was added and the response to the subsequently added cannabinoid was measured at a final concentration of 10uM. CAMP levels were monitored in real time and in live cells using the CAYMEL BRET biosensor. Changes in the BRET ratio were measured with a PHERAstar plate reader at 37°C in kinetic mode, every 40 s for 20 min.

Instrument Settings

Results & Discussion

We initially measured CB1-mediated activation of Ga_s proteins by different cannabinoids (PTX treated cells). Typical signal curves are shown in fig. 2A.

A reverse BRET ratio (475/535) was used so that an increase in ratio corresponded to an increase in cAMP. The area under the total signal curve was used for quantification. Sixteen cannabinoids were initially tested at a concentration of 10μM. It was found that all tested cannabinoids, increased cAMP levels (12-45%) above that produced by FSK alone (fig. 2B).

In contrast, THC did not significantly alter levels over FSK. Concentration response curves were then constructed for five structurally different compounds to determine EC₅₀ and E_max values for activation of both the Ga_s and Ga_i signaling pathways. All the SCRAs tested activated CB1 via both pathways (tab.1) however all compounds were much more potent for Ga_i as compared to Gα_s pathways. The activation of CB1-Ga_s by different cannabinoids showed a wide
variation in Emax values and there was a significant difference in efficacy between the different compounds. The rank order of efficacy for stimulation via Gas was: AB-FUBINACA~PB22 >5F-MDMB-PICA> XLR-11>JWH-018. In contrast, all cannabinoids showed similar maximal activity at Gα_i.

Table 1: EC50 and Emax values for selected agents

Conclusion