Extended Data Fig. 7.

G_i-coupled receptors accommodate the C terminus of nucleotide-decoupled G_s heterotrimers yet cannot activate wild-type G_s. A) Agonist-dependent BRET between Rluc8-tagged G_i-coupled receptors α₂AR (top) and M₄R (bottom) and G_s 4A heterotrimers is nearly abolished when Gα_s 4A is truncated by removing 5 amino acids from the C terminus (Δ5); mean ± S.D. n=3–5. B) Neither of these receptors is capable of stimulating adenylyl cyclase by activating wild-type G_s. Cyclic AMP (cAMP) was monitored using an EPAC-based BRET sensor; α₂AR was stimulated with 1 μM UK 14,304 (UK) and M₄R was stimulated with 10 μM oxotremorine M (OXO) agonist-induced activation; responses to 10 μM forskolin (FSK) are shown for comparison; *****P < 0.05, one-way ANOVA with Dunnett’s multiple comparisons; ns, not significant.