Fig. 5.

Effect of Rab6Q72L on the cell-surface expression, subcellular localization and signaling of AT1R. A. Effect of Rab6Q72L on AT1R transport to the cell surface. HEK293T cells were cultured and transfected with the pcDNA3 vector or FLAG-Rab6Q72L and the cell surface expression of AT1R was determined by ligand [³H]-Ang II binding as described under "Experimental procedures." Nonspecific binding was obtained in the presence of 10 µM nonradioactive Ang II. The mean values of specific ligand binding were 12730 ± 1562 cpm (n = 3, each in triplicate) from cells transfected with AT1R with the pcDNA3 vector. The data shown are the percentage of the mean value obtained from cells expressing AT1R alone and are presented as the means ± S.E. *, p < 0.05 versus control. B. Effect of Rab6Q72L on the subcellular localization of AT1R. HEK293T cells were transfected with AT1R-GFP and the pcDNA3 vector (control) or FLAG-Rab6Q72L and the subcellular localization of AT1R was revealed by fluorescence microscopy detecting GFP. The data shown are representative images of at least three independent experiments. Scale bar, 10 µm. C. Effect of Rab6Q72L on AT1R-mediated IP accumulation. HEK293T cells were transfected with AT1R-GFP and the pcDNA3.1 vector or FLAG-Rab6Q72L. The transfected cells were split into 60-mm culture dishes, incubated with myo-[³H]inositol, and stimulated with Ang II at 1 µM. IP production was measured as described under "Experimental procedures." The basal levels of IP production in untransfected HEK293T cells and in HEK293T cells transfected with AT1R-GFP and pcDNA3.1 or FLAG-Rab6Q72L were 2643 ± 312, 2544 ± 221 and 1805 ± 342 cpm, respectively. The data are shown as the -fold increase over the respective basal levels of IP production in response to Ang II stimulation and represent the means ± S.E. of three experiments. *, p < 0.05 versus cells transfected with AT1R-GFP alone.