Fig. 4. Selection of RGS protein mutants for validation in mammalian cells using a BRET-based G protein activity biosensor.

(A) Left: Selection of a representative set of 9 RGS-box cancer-associated mutations from the 49 that were assessed in yeast. Criteria for selection (shown in the table) included sampling mutations with various degrees of defective GAP activity in yeast, as well as position within and outside the G protein interface, among others. Right: The protein structure depicts the positions of the selected mutations (colored yellow or red according to the increasing frequency of mutation at that coordinate) are shown relative to the Gα interface (green). (B) Description of the assay to assess RGS protein GAP activity in mammalian cells using a BRET-based biosensor. Agonist-induced GPCR activation leads to the dissociation of Venus-tagged Gβγ from Gα, which in turn binds to the c-terminal domain of GRK3 fused to nanoluciferase (Nluc) resulting in increased BRET. Subsequent addition of a GPCR antagonist causes a decline of the BRET signal upon G protein inactivation, which can be accelerated by the GAP activity of an RGS protein. Comparison of the acceleration of the deactivation rate in the presence of WT or mutant RGS proteins was used to determine their relative GAP activities.