Fig. 2
Signaling in HEK-293T cells expressing NMDA-CB2R heteromers. HEK-293T cells transfected with the cDNAs for two protomers of the NMDA receptor: GluN1 (1 μg) and GluN2B (0.75 μg) and/or with the cDNA for the CB2R (1 μg), and were treated with selective agonists (15 μM NMDA for NMDAR and/or 100 nM JWH-133 for CB2R). When indicated cells were pretreated with selective receptor antagonists (1 μM MK-801 for NMDA or 1 μM SR-144528 for CB2R). A–C Intracellular cAMP levels were determined by TR-FRET as described in Methods. As Gi coupling was assessed, decreases in cAMP levels were determined in cells previously treated with 0.5 μM forskolin (15 min). Values are the mean ± S.E.M. of 6 independent experiments performed in triplicates. In cAMP one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment). ANOVA summary: A F: 67.6, p<0.001; B F: 238.0, p<0.001; C F: 62.5 p<0.001. D–F ERK1/2 phosphorylation was analyzed using an AlphaScreen®SureFire® kit (Perkin Elmer). Values are the mean ± S.E.M. of 5 independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle treatment). ANOVA summary: D F: 5.4, p<0.005; E F: 8.3, p<0.001; F F: 3.8, p<0.005