FIGURE 5.
Angiotensin-induced protein synthesis is dependent upon β-arrestin2 expression and Mnk1 activity. A, HEK-293 cells stably expressing the AT1AR were transfected with either control siRNA (CTL), β-arrestin2 siRNA (β-arr2), or Mnk1 siRNA (Mnk1) for 48 h. Cells were serum-starved for an additional 24 h to arrest cycling. Phosphate-buffered saline (nonstimulated), AngII (100 nm), or SII-AngII (10 μm) along with 3H-labeled leucine was added to the medium; 24 h later, cells were harvested as described under “Experimental Procedures” and radiolabeled proteins were precipitated and quantitated. Data have been normalized such that the control siRNA, nonstimulated is set to 100%. Results are representative of nine independent experiments each performed, at a minimum, in duplicate. Statistical significance was calculated using one-way ANOVA with post test CTL NS versus CTL AngII (***, p < 0.001) or CTL SII (*, p < 0.05) and CTL AngII versus β-arr2 (**, p < 0.01) AngII and Mnk1 AngII (**, p < 0.01). B, HEK-293 cells either stably expressing HA-AT1AR or transiently expressing HA-AT1ARDRY/AAY (as indicated) were serum-starved for 24 h and then pretreated for 1 h with either Me2SO (DMSO) or 30 μm CGP57380, the Mnk1 inhibitor (reported IC50 2.2 μm). Cells were then stimulated with AngII in the presence of [3H] leucine for 24 h, and protein synthesis was calculated as described in A. Results depicted represent the mean of four independent experiments ± S.E. Using a one-way ANOVA with post test, the Me2SO AngII-stimulated condition is significantly greater (*, p < 0.05) than the CGP AngII condition for each receptor.