Figure 3.
Effect of the Q152A point mutation in Gαq-RC on the ability of GRK2 to inhibit inositol phosphate production. (A) HEK-293 cells were transfected with 0.1 μg of the constitutively active Gαq-R183C or Gαq-Q152A-RC and 0.2 μg of myc, His-tagged Gβ and 0.1 μg of Gγ and increasing amounts of GRK2-K220R and empty vector up to a total of 1.0 μg of DNA. 24 hrs after transfection the cells were labeled with 2 μCi/ml myo-[3H]inositol and 16 hours later inositol phosphate production was determined, as described in Experimental Procedures. The results shown are averages from five independent experiments each done in triplicate and displayed as percent control ± S.D. The control is the inositol phosphate production stimulated by Gαq-R183C or Gαq- Q152A-RC in the absence of any co-expressed GRK2-K220R. A (⋆) denotes a statistically significant difference (p < 0.05) by two-way ANOVA followed by a Bonferroni post-test, between the indicated Gαq-Q152A-RC bar and the Gαq-RC bar transfected with the same amount of GRK2-K220R. A (#) indicates a statistically significant difference (p < 0.05) by one-way ANOVA followed by a Dunnett post-test, between the indicated bar and the control, either Gαq-RC or Gαq-Q152A-RC in the absence of cotransfected GRK2-K220R. (B) Western blots of total cellular lysates from a representative inositol phosphate experiment from (A) probed with the EE monoclonal antibody showing that increasing GRK2-K220R expression does not effect expression of Gαq-RC or Gαq-Q152A-RC. The bottom panel of Figure 4 B shows the level of GRK2- K220R overexpression. The bands corresponding to 10, 25 and 100 ng of GRK2-K220R transfected can be seen after very short exposures; however, the GRK2 band corresponding to 5 ng of cDNA transfected is barely visible, even after long exposures, suggesting that comparatively low levels of GRK2 expression can significantly inhibit Gαq signaling.