Fig. 3.

Assessment of functional impact of RGS14 somatic mutations on GPCR-G protein activation and G protein binding. (A) Schematic representation of kinetic BRET experiments. HEK 293 cells were transfected with 200 ng of α2A‐AR, 200 ng of Venus‐Gβ1 200 ng of Venus‐Gγ2, 200ng of mas‐GRK3ct‐Luc, 1000 ng of mutant Gαo (PTX insensitive), and 0 or 200ng of WT Flag-RGS or 200ng of Flag tagged RGS mutant. (B) Structural view of selected mutated tolerant (red) and intolerant (blue) residues in RGS14. Average whole traces of BRET signal over time (n = 3) are shown comparing WT and somatic mutations. 3DMTR identified residues that led to loss of function are tolerant residues Flag-S127P (C), and intolerant mutated residues Flag-R173C (G) and Flag-R173H (I). Tolerant residue Flag-D137Y (E) did not lead to change of function. BRET amplitude observed at 100s was compared between 0ng RGS14, 200ng WT RGS14, and mutants Flag-S127P (D), Flag-D137Y (F), Flag-R173C (H), and Flag-R173H (J). Error bars represent mean ± S.D. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (**P < 0.005). (K) Coimmunoprecipitation studies show that RGS14 mutants S127P, R173C and R173H blocked binding to Gαo-AlF4.