Extended Data Figure 6. Signalling profile of PZM21 and other μOR agonists.

a, PZM21 is an efficacious G_i and G_o agonist in a GTP γS assay. b, Like other μOR agonists, PZM21 induces a dose-dependent decrease in cAMP levels that is sensitive to pertussis toxin, confirming G_i/o mediated signalling. c, Herkinorin is a G_i/o agonist and robustly recruits arrestin in a BRET assay performed in the absence of GRK2 overexpression. TRV130 or PZM21 show undetectable levels of arrestin recruitment in the same experiement. d, PZM21 and other opioids show no activity in a calcium-release assay, indicating no G_q-mediated second messenger signalling. The positive control TFLLR-NH2 efficiently activates the G_q coupled receptor PAR-1. e, PZM21 and TRV130 induce much decreased receptor internalization versus DAMGO and even morphine. f, Herkinorin and TRV130 are potent agonists of the κOR. PZM21 is a κOR antagonist (see Extended Data Fig. 4). g, In HEK293 cells, GRK2 expression levels have minimal effect on the potency and efficacy of the unbiased agonist DAMGO in a G_i/o activation assay. Increased GRK2 levels change the basal cAMP signal due to increased desentization of μOR, which lowers receptor basal activity and leads to elevated isoproterenol-induced cAMP. In an arrestin-recruitment BRET assay, increased GRK2 expression increases both the potency and maximal efficacy of the unbiased agonist DAMGO. This is likely because GRK2 mediated phosphorylation is required for efficient β-arrestin recruitment. h, G_i activation and arrestin recruitment in cells co-expressing 1.0 μg/15 cm² of GRK2. Notably, PZM21 induces a higher maximal level of arrestin recruitment as compared to U2OS cells, which express very low levels of GRK2, but this level is significantly lower than morphine. Despite the lower efficacy for arrestin recruitment observed for morphine, TRV130 and PZM21 compared to DAMGO, a formal calculation of bias by the operational models fails to show that this effect is significant. i, Table of pEC₅₀ values and E_max values for various signalling assays. All data are mean ± s.e.m. of results (n = 2–6 measurements).