Figure 6.

ER/K linker length tunes basal GPCR-Gi protein downstream response in live cells. (a) Schematic of the GPCR-G protein fusions. Color represents cognate GPCR-G protein pair and corresponding downstream response (red: α2-AR, Gs, and cAMP levels; green: A₁R, α2-AR, Gi, and cAMP inhibition). (b–e) Fusion type is indicated on the top left. FRET Ratios (525 nm/475 nm; open black circles, right y-axis) and basal (ligand-free) downstream response (filled colored circles, left y-axis) are compared to ER/K linker length (nm). (b) Percent inhibition of 1 μM forskolin induced cAMP production for canonical Gi coupled adenosine type 1 receptor (A₁R). (c) Basal cAMP levels for A₁R-Gs fusions. (d) % cAMP inhibition for Gi fusions and (e) cAMP levels for Gs fusions to α2-AR, a promiscuous Gs/Gi coupled receptor²⁸. (b,d) Representative experiment with at least three replicates per condition. (c,e) Data are derived from at least three independent experiments, with at least three replicates per condition. (b–e) One-way ANOVA with a Tukey’s post-test was performed for FRET measurements (black) and downstream response (colored). Data are represented as mean ± S.E.M. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). For additional statistical analysis see Supplementary Table 4.