The CB1 cannabinoid receptor‐mediated inhibition of cAMP accumulation in neuroblastoma cells: role of different Gi/o protein subtypes

Abstract

The CB1 cannabinoid receptor (CB1R) is a G protein coupled receptor with robust expression in the nervous system. CB1R signaling is mediated by pertussis toxin (PTX)‐sensitive Gi /o proteins, and leads to inhibition of adenylyl cyclase. Because of the importance of the cyclic AMP pathway in differentiated neuronal functions, the investigation of G protein subtypes involved in the inhibition of adenylyl cyclase activity is of particular interest. The aim of the present study is to investigate the effect of individual Gi/o proteins on CB1R‐promoted inhibition of forskolin‐ stimulated cAMP accumulation in N18TG2 neuroblastoma cells. We utilized [3H]cAMP binding competition assay and cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) as previously described ( Blume et al 2015 and Eldeeb et al 2017).The CB1R agonist, CP55940 (100 nM) inhibited forskolin‐stimulated cAMP accumulation to 34±8% of forskolin alone. Pretreatment with 100 ng/ml of PTX for 20 hrs attenuated CP55940 induced inhibition to 85±24% of forskolin control. In N18TG2 cells that were stably expressing either PTX‐resistant Gαo, Gαi1, Gαi2 or Gαi3 proteins, treatment of PTX‐pretreated with CP55940 (100 nM) inhibited cAMP accumulation to 38±3% Gαo, 53±4 Gαi1, 76±24 Gαi2 and 28±15% Gαi3 of forsklin alone. Using SPA, CP55940 (100 nM) stimulated [35S]GTPγS binding mediated by Gi/o protein subtypes, Gαo, Gαi1, Gαi2 and Gαi3 in N18TG2 Gαi3 PTX‐resistant cell membranes. In contrast, in PTX‐pretreated membranes CP55940 was able to stimulate [35S] GTPγS binding mediated by Gαi3 only. In conclusion, these data show that Gi/o proteins contribute differently to CB1R‐promoted inhibition of cAMP in N18TG2 cells, with greater contribution by Gαi3. This is consistent with our earlier findings of the high level of Gαi3 expression and its contribution toward total G protein activation in N18TG2 membranes ( Eldeeb et al 2017). The contribution of different G protein subtypes to CB1R signaling is necessary to consider as we evaluate CB1R functions in the nervous system.Support or Funding InformationNIH grants R01‐DA042157, P50‐DA006634