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. Author manuscript; available in PMC: 2020 Aug 4.
Published in final edited form as: Sci Signal. 2020 Feb 4;13(617):eaax8620. doi: 10.1126/scisignal.aax8620

Fig. 3. Systematic assessment of cancer-associated RGS protein mutations on GAP activity using a scalable yeast-based assay.

Fig. 3.

(A) Criteria for selecting a representative set of 49 cancer-associated mutations in RGS domains to screen for potential disruptive effects. (B) Diagram of the yeast-based β-galactosidase (β-gal) assay used to assess the function of mutant RGS proteins in inhibiting human G protein signaling. AGS1 stimulates the G protein–dependent activation of the Fus1 promoter in yeast lacking pheromone-responsive GPCRs through Gβγ. This activation response is suppressed by the GAP activity of RGS proteins, which accelerate the deactivation of human Gαi3. (C) Representative readout of the β-gal assay described in (B). Inhibition of AGS1-dependent G protein activation by a WT or mutant RGS protein was determined with a fluorogenic β-galactosidase substrate in a 96-well format. (D) Effects of different RGS8 mutants on G protein activation in yeast. β-gal activity (normalized to the percentage of maximal activation, “AGS1 only”) was used to calculate the relative GAP activity using the inset equation. Data are means ± SEM of six experiments. **P < 0.01 and ***P < 0.001 as compared to “AGS1 only” (black asterisks) or to “AGS1 + RGS8 WT” (red asterisks) by one-way ANOVA with Bonferroni multiple-comparisons test. (E) Effects of the selected 49 RGS protein cancer-associated mutations on GAP activity in the yeast-based G protein activity assay. Relative GAP activity was determined relative to the respective RGS WT protein for each mutant as described for (D). Background is shaded yellow, orange, and red, respectively, to indicate mild (75 to 50% of WT), moderate (50 to 25% of WT), and severe (25 to 0% of WT) reductions in GAP activity. Results are means ± SEM of at least six experiments. (F) Percentage of screened mutations that severely, moderately, or mildly affected GAP function as described for (E) when considered in aggregate (“All mutants”) or when stratified as “Interface” (within 5Å of Gα) or “Non-interface” positions.