FIGURE 4.
a, 2H-labeled cannabinoid agonist CP-55,940-d6. b, 2H MAS NMR spectra of CP-55,940-d6 in CB2 receptor proteoliposomes containing 1 mol % of POPC-d4 as an internal intensity standard. Spectra were recorded at 20 °C and a MAS frequency of 14.5 kHz. Spectra without (black) and with (blue) a 10-fold excess of unlabeled CP-55,940 were recorded. The inset shows the same spectra with intensity adjustment to match signal intensities of the ligand. The asterisk denotes a weak natural abundance 2H signal from the terminal methyl groups of lipid hydrocarbon chains. c, activation of G protein by agonist-bound CB2 receptor reconstituted into liposomes. A plasma membrane preparation of E. coli cells expressing CB2 receptor-130 fusion protein was used as control. Its CB2 receptor content was determined to be 2.5 μg/mg of total protein. An average of 7,560 counts per minute (cpm) for the [35S] GTPγS·Gαi1 complex per ng of CB2 receptor, after subtraction of background reactivity without CB2 receptor, was measured as control and is reported as 100%. The data shown in c are the average of four G protein activation assays conducted on one set of samples (n = 4).