CB₁ cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gi/o function

Abstract

Introduction CB1 cannabinoid receptors (CB1Rs) stimulate Gi/o-dependent signaling pathways. CB1R-mediated cAMP increases were proposed to result from Gs activation, but CB1R-stimulated GTPγS binding to Gs has not heretofore been investigated. Methods Three models of CB1R- stimulated cAMP production were tested: Pertussis toxin disruption of Gi/o in N18TG2 cells; L341A/A342L-CB1R expressed in CHO cells; and CB1 and D2 dopamine receptors endogenously co-expressed in MN9D cells. cAMP was assayed by [3H]cAMP binding competition. G protein activation was assayed by antibody-targeted Scintillation Proximity Assay (SPA). Results In L341A/A342L-CB1-CHO cells, cannabinoid agonists significantly stimulated cAMP accumulation over vehicle; CP55940-stimulated [35S]GTPγS binding to Gi1/2/3 was reversed, whereas binding to Gs was not different from CB1R. In MN9D cells, CB1 agonist HU210 or D2 agonist quinpirole alone inhibited forskolin-activated cAMP accumulation, whereas HU210 plus quinpirole increased cAMP accumulation above basal. HU210 alone stimulated [35S]GTPγS binding to Gi1/2/3, whereas co-stimulation with quinpirole reversed HU210-stimulated [35S]GTPγS binding to Gi1/2/3. Conclusions CB1R couples to Gs but with low efficacy compared to Gi/o. The L341A/A342L mutation in CB1R reversed CP55940 activation of Gi to an inhibition, but had no effect on Gs. Combined CB1 plus D2 agonists in MN9D cells converted the CB1 agonist-mediated activation of Gi to inhibition of Gi. In these models, the CB1 agonist response was converted to an inverse agonist response at Gi activation. Cannabinoid agonist-stimulated cAMP accumulation can be best explained as reduced activation of Gi, thereby attenuating the tonic inhibitory influence of Gi on the major isoforms of adenylyl cyclase.

Gαs signalling of the CB1 receptor and the influence of receptor number

“…An obvious but inherent limitation of this endpoint, however, is that it is not equipped to distinguish contributions of the different G protein subtypes to the total signal, a problem that originates from the mutually antagonistic effects that Gα i and Gα s activity exert on the cAMP pathway. Indeed, recent work by Eldeeb et al () would suggest that nett cAMP signalling does not necessarily reflect CB 1 receptor‐mediated G protein activity, as Gα s protein cycling was elicited concentration‐dependently following agonist stimulation under conditions where the nett cAMP effect was inhibitory. The use of PTX to irreversibly and specifically inactivate Gα i proteins is a widely used approach that aids in cAMP phenotype characterization.…”

Cannabinoid Receptors in the Central Nervous System: Their Signaling and Roles in Disease

“…Upon ligand binding and receptor activation, CB 1 receptors are primarily coupled to pertussis toxin (PTX)-sensitive Gi/o type G proteins which leads to a rapid decrease in levels of cAMP by inhibiting adenylate cyclase activity (Figure 1A; Howlett et al, 2004). Coupling to other G proteins including Gs, albeit with low efficacy, can also stimulate adenylate cyclase (Glass and Felder, 1997; Glass and Northup, 1999; Varga et al, 2008; Bosier et al, 2010) though the extent of accumulation of cAMP is not necessarily a good indicator of G protein coupling (Eldeeb et al, 2016). Evidence of promiscuous coupling to different G proteins, signaling roles mediated by β-arrestins and signaling from intracellular compartments (Figure 1B) adds yet another level of complexity making these receptors, like other GPCRs, pluridimentional (Bosier et al, 2010).…”

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

“…When selected and subcloned for thioguanine resistance, N18TG2 and N4TG1 cells were useful for hybridization with either the rat C6BU1 bromouridine deoxyribose (BUdR)-resistant glioma (NG108-15) (Gilman & Minna, 1973; Hamprecht, 1977; Klee & Nirenberg, 1974), and these hybrid cells have been utilized for studies of CB 1 receptors (Devane, Spain, Coscia, & Howlett, 1986; Howlett, Qualy, & Khachatrian, 1986; Mackie, Devane, & Hille, 1993). N18TG2 cells have also been used to “immortalize” neurons by hybridization: with mesencephalic neurons (MN9D) (Choi et al, 1991), which have been used for studies of interactions between CB 1 cannabinoid and D 2 dopamine receptors (Calipari et al, 2014; Eldeeb, Leone-Kabler, & Howlett, 2016); dorsal root ganglia cells (F-11) (Cruciani, Dvorkin, Morris, Crain, & Makman, 1993; Francel et al, 1987; Platika, Boulos, Baizer, & Fishman, 1985), which have been used for investigation of the endocannabinoid system (Fan et al, 2011; Fioravanti et al, 2008; Rimmerman et al, 2008; Ross et al, 2001) and other reports; and spinal cord neurons (NSC-34) (Cashman et al, 1992), which have been used for studies of neuroprotection by cannabinoids (Moreno-Martet et al, 2012). A 2017 PubMed search shows that since their initial characterization, cloned cell lines from the C1300 tumor have been the model system of choice for nearly 2000 publications.…”

“…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). …”