Figure 2. Effect on α_2A adrenergic receptor (α_2A AR)–mediated cyclic adenosine monophosphate (cAMP) inhibition by GNAO1 mutants.

(A–C) Dose–response curves of representative GNAO1 mutants. (A) Dose–response curves of loss-of-function (LOF) and partial loss-of-function (PLOF) mutants (G40R, L199P, N270H, F275S, A227V, Y231C) show changes in cAMP production in response to the adenylate cyclase activator forskolin and α₂ AR agonist UK14,304, compared to the positive control (wild-type [WT]) and negative control (pcDNA). (B) Dose–response curves of functioning Gα_o mutants show changes in cAMP production in response to the α₂ AR agonist UK14,304. All dose–response curves are shown in comparison with WT and G184S. (C) G42R displays a biphasic dose–response curve with cAMP inhibition at low concentrations (gain of function [GOF]), followed by enhancement of cAMP levels at higher concentrations of UK14,304. (D) Quantification of EC₅₀ of functioning Gα_o mutants. G42R, G203R, and E246K exhibit significantly increased potency for α_2A AR–mediated cAMP inhibition similar to the known GOF mutation G184S. *p < 0.05, **p < 0.01, ***p < 0.001 using paired t test between WT and each mutant separately (figure e-4). (E) Percentage of maximum inhibition (n = 5) was normalized to pcDNA (0%; resulting in activation of cAMP) and WT (100%). ****p < 0.0001 using 1-way analysis of variance with Bonferroni post hoc test for pairwise comparison. Note that the maximum inhibition of G42R was calculated at UK14,304 of 15.8 nM. NF = normal function; PTX = pertussis toxin.