Biased Agonism of Three Different Cannabinoid Receptor Agonists in Mouse Brain Cortex

Abstract

Cannabinoid receptors are able to couple to different families of G proteins when activated by an agonist drug. It has been suggested that different intracellular responses may be activated depending on the ligand. The goal of the present study was to characterize the pattern of G protein subunit stimulation triggered by three different cannabinoid ligands, Δ9-THC, WIN55212-2, and ACEA in mouse brain cortex. Stimulation of the [35S]GTPγS binding coupled to specific immunoprecipitation with antibodies against different subtypes of G proteins (Gαi1, Gαi2, Gαi3, Gαo, Gαz, Gαs, Gαq/11, and Gα12/13), in the presence of Δ9-THC, WIN55212-2 and ACEA (submaximal concentration 10 μM) was determined by scintillation proximity assay (SPA) technique in mouse cortex of wild type, CB1 knock-out, CB2 knock-out and CB1/CB2 double knock-out mice. Results show that, in mouse brain cortex, cannabinoid agonists are able to significantly stimulate not only the classical inhibitory Gαi/o subunits but also other G subunits like Gαz, Gαq/11, and Gα12/13. Moreover, the specific pattern of G protein subunit activation is different depending on the ligand. In conclusion, our results demonstrate that, in mice brain native tissue, different exogenous cannabinoid ligands are able to selectively activate different inhibitory and non-inhibitory Gα protein subtypes, through the activation of CB1 and/or CB2 receptors. Results of the present study may help to understand the specific molecular pathways involved in the pharmacological effects of cannabinoid-derived drugs.

Biased Receptor Signaling in Drug Discovery

“…For example, dopamine receptor selectivity between Ga i1 , Ga i2 , Ga i3 , Ga o , plus Gb 1 and Gg 2 has been reported using GTPgS assays (Gazi et al, 2003). Similarly, selectivity within Ga i1 , Ga i2 , Ga i3 , Ga o , Ga z , Ga q/11 , and Ga 12/13 has been reported with cannabinoid receptor agonism (Diez-Alarcia et al, 2016). Recently, the application of sensitive BRET probes to directly measure G protein activation in living cells has been applied to the measurement of G protein subunit selectivity (Lohse et al, 2012;Saulière et al, 2012).…”

“…7TMRs generally can interact with members spanning across these general groupings despite the fact that the G protein functional outcomes can be very different (Michal et al, 2007;Inoue et al, 2012;Saulière et al, 2012). For instance, cannabinoid receptors interact with Ga z , Ga q/11 , and Ga 12/13 (Diez-Alarcia et al, 2016;Laprairie et al, 2017). Ligand-stabilized receptor conformations drive the selection of G protein interactions and these selections can be discerned within G protein subunit families; for instance, studies with bioluminescence resonance energy transfer (BRET) biosensors have shown that oxytocin analogs differentiate between individual G i/o family members (Ga q , Ga i1 , Ga i2 , Ga i3 , Ga oA , Ga oB ; Busnelli et al, 2012).…”

Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays

“…This procedure can mitigate concerns related to activation or inhibition of multiple G proteins and reduces the variability in sensitivity by different G proteins (Milligan, 2003; Strange, 2010). This assay has since been used to identify specific CB 1 receptor–G protein interactions (Blume, Eldeeb, Bass, Selley, & Howlett, 2015; Diez-Alarcia et al, 2016; Eldeeb et al, 2016; Erdozain, Diez-Alarcia, Meana, & Callado, 2012). …”

When orexins meet cannabinoids: Bidirectional functional interactions

“…Both cannabinoid CB1R and CB2R are mainly coupled to Gi/o resulting in the inhibition of the adenylate cyclase/cAMP cascade and voltage-gated Ca2+ channels, as well as the stimulation of inwardly rectifying K+ currents, and of mitogen-activated protein kinase (MAPK) activity [18]. Interestingly, a growing amount of evidence shows that CB1R can couple with different subunits of the classic inhibitory Gi/o proteins, but also with Gαz, Gαq/11, and Gα12/13 [19,20], and that under specific circumstances, CB1R can shift toward activation of Gs and potentiation of neurotransmission [21,22]. In addition, activation of CB1R in astrocytes increases intracellular calcium probably mediated by Gq proteins [23], and in the cardiovascular system, CB1R can bind to Gs or Gq/11 to modulate vasoconstriction and hypertension [24].…”