Fig. 1. Effect of transient expression of Rab2Q65L and Rab6Q72L on the cell-surface expression of α_2B-AR and β₂-AR. A. Western blot analysis of GFP-tagged Rab2Q65L expression. HEK293T cells cultured on 6-well plates were transfected with the pEGFP-C1 vector (control) or GFP-Rab2Q65L mutant. Cell homogenates were separated by 12% SDS-PAGE and expression of Rab2Q65L was detected by Western blotting using anti-Rab2 (upper panel) and anti-GFP antibodies (middle panel). B. Western blot analysis of FLAG-tagged Rab6Q72L expression. HEK293T cells cultured on 6-well plates were transfected with the pcDNA3 vector (control) or FLAG-Rab6Q72L mutant and expression of Rab6Q72L was detected by Western blotting using anti-Rab6 (upper panel) and anti-FLAG M2 antibodies (middle panel). β-actin expression is shown in lower panels as a loading control. C. The subcellular distribution of GFP-Rab2Q65L and FLAG-Rab6Q72L. HEK293T cells cultured on coverslips were transfected with GFP-conjugated Rab2Q65L or FLAG-Rab6Q72L. The subcellular distribution of GFP-Rab2Q65L was revealed by fluorescence microscopy detecting GFP and FLAG-Rab6Q72L by immunostaining with anti-FLAG antibodies as described under “Experimental procedures.” Scale bar, 10 μm. D. Inhibition of the cell-surface expression of α_2B-AR and β₂-AR by Rab2Q65L and Rab6Q72L. HEK293T cells were transfected with GFP-conjugated α_2B-AR or β₂-AR together with the pEGFP-C1 vector (GFP), GFP-tagged Rab2Q65L, the pcDNA3 vector or FLAG-Rab6Q72L. The expression of α_2B-AR or β₂-AR at the cell surface was determined by intact cell ligand binding using [³H]-RX821002 and [³H]-CGP12177, respectively, as described in the “Experimental procedures.” The mean values of specific ligand binding were 43,371 ± 6368, 44,247 ± 3979, 49,014 ± 8433 and 51,385 ± 2879 cpm (n = 3, each in triplicate) from cells transfected with α_2B-AR with pEGFP-C1, α_2B-AR with pcDNA3, β₂-AR with pEGFP-C1 or β₂-AR with pcDNA3, respectively. The data shown are percentages of the mean value obtained from cells transfected with individual receptor and the pcDNA3 or pEGFP-C1 vector and are presented as the means ± S.E. of three experiments. , p < 0.05 versus the cells transfected with respective receptor and the pcDNA3 or pEGFP-C1 vector. E. Effect of Rab2Q65L and Rab6Q72L on total expression of α_2B-AR and β₂-AR. HEK293T cells were transfected with GFP-conjugated α_2B-AR or β₂-AR together with the pcDNA3 vector, FLAG-Rab6Q72L or Rab2Q65L in the pcDNA3 vector. The overall receptor expression was determined by measuring GFP fluorescence using a flow cytometer as described in the “Experimental procedures.” F. Specific [³H]-RX821002 binding to membrane fractions prepared from the cells transfected with α_2B-AR and Rab2Q65L or Rab6Q72L. HEK293T cells were transiently transfected with α_2B-AR together with the pcDNA3 vector, FLAG-Rab6Q72L or Rab2Q65L. The membrane preparation (15 μg of protein) was incubated with increasing concentrations of [³H]-RX821002 (5–160 nM) for 30 min. Specific binding was determined in duplicate, and non-specific binding was determined in the presence of 10 μM rauwolscine as described under “Experimental procedures.” The data shown are representative of at least three separate experiments, each with similar results.

Fig. 2. Effect of Rab2Q65L and Rab6Q72L on the subcellular distribution of α_2B-AR and β₂-AR. A. HEK293T cells cultured on coverslips were transfected with α_2B-AR-GFP or β₂-AR-GFP (100 ng) together with the pcDNA3 vector (control), Rab2Q65L or Rab6Q72L (400 ng). The subcellular distribution of the receptors was revealed by detecting GFP fluorescence as described under “Experimental procedures.” B. Co-localization of β₂-AR-GFP with ERGIC53. HEK293T cells cultured on coverslips were transfected with β₂-AR-GFP and Rab2Q65L and stained with anti-ERGIC53 antibodies (1:200 dilution). C. Co-localization of β₂-AR-GFP with GM130. HEK293T cells cultured on coverslips were transfected with β₂-AR-GFP and Rab6Q72L and stained with anti-GM130 antibodies (1:200 dilution). Green, β₂-AR-GFP; red, ERGIC53 (B) and GM130 (C); blue, DNA staining by 4,6-diamidino-2-phenylindole (nucleus); yellow, co-localization of β₂-AR with ERGIC53 (B) and GM130 (C). The data shown are representative images of at least three independent experiments. Scale bars, 10 μm.

Fig. 3. Effect of siRNA-mediated knockdown of Rab2 and Rab6 on the cell-surface expression of α_2B-AR and β₂-AR. A. HEK293T cells cultured on 6-well dishes were transiently transfected with control siRNA, Rab2 or Rab6 siRNA as described under “Experimental procedures.” At 48 h after transfection, total homogenate protein was separated by 12% SDS-PAGE, and expression of Rab2 (upper panel), Rab6 (middle panel) and Rab1 (lower panel) was detected by Western blotting using isoform-specific antibodies. B. Effect of Rab2 and Rab6 siRNA on their expression. The data are expressed as percentages of Rab protein expression in cells transfected with control siRNA and presented as the means ± S.E. of three individual experiments. , p < 0.05 versus their respective control siRNA. C. Effect of siRNA-mediated depletion of Rab2 and Rab6 on the cell-surface expression of α_2B-^{AR and β}₂^{-AR. HEK293T cells were transfected with GFP-conjugated} α_2B^{-AR or β}₂^{-AR together with} the control or Rab siRNA as described in the “Experimental procedures.” The cell-surface expression of the receptors was determined by intact cell ligand binding as described in the legend of Fig. 1. The data shown are percentages of the mean value obtained from cells transfected with individual receptor with control siRNA and are presented as the mean ± S.E. of three experiments. , p < 0.05 versus the cells transfected with respective receptor and control siRNA. D. Effect of Rab2 and Rab6 siRNA on total expression of α_2B-AR and β₂-AR by measuring GFP fluorescence as described in the legend of Fig. 1.

Fig. 4. Effect of Rab2 and Rab6 on signaling of α_2B-AR and β₂-AR. A. Effect of Rab2 and Rab6 on ERK1/2 activation by α_2B-AR. HEK293T cells were transfected with GFP-tagged α_2B-AR together the pEGFP-C1 vector (GFP), GFP-Rab2Q65L, the pcDNA3 vector, FLAG-Rab6Q72L, Rab2 control siRNA, Rab2 siRNA, Rab6 control siRNA or Rab6 siRNA as described under “Experimental procedures.” The cells were stimulated with UK14304 at a concentration of 1 μM for 5 min at 37 °C. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies (ERK1/2-P). Upper panel, representative blots of ERK1/2 activation; lower panel, total ERK1/2 expression. B. Quantitative data expressed as percentages of ERK1/2 activation obtained from cells transfected with α_2B-AR and a separate control and stimulated with 1 μM UK14304 and presented as the means ± S.E. of three experiments. C. Effect of Rab2 and Rab6 on cAMP production by the β₂-AR agonist ISO. HEK293T cells were cultured in 100-mm plates and transfected with β₂-AR as described in (A) for α_2B-AR and then stimulated with ISO (10 μM) for 10 min. cAMP concentrations were determined by using cAMP enzymeimmunoassay system as described under “Experimental procedures.” The data are presented as fold increase in response to ISO stimulation over the basal values and as the means ± S.E. of three experiments. , p < 0.05 versus their respective controls.

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.” Non-specific binding was obtained in the presence of 10 μM nonradioactive Ang II. The mean values of specific ligand binding were 12,730 ± 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.

Fig. 6. Effect of the dominant-negative mutant Rab6N126I on the cell-surface expression of α_2B-AR, β₂-AR and AT1R. A and B. Western blot analysis of expression (A) and the subcellular distribution of FLAG-Rab6N126I (B) as described in the legend of Fig. 1 for FLAG-Rab6Q72L. C. Effect of FLAG-Rab6N126I on cell-surface expression of α_2B-AR, β₂-AR and AT1R. HEK293T cells were transfected with GFP-conjugated α_2B-AR, β₂-AR or AT1R together with the pcDNA3 vector (control) or FLAG-Rab6N126I. The expression of α_2B-AR and β₂-AR at the cell surface was determined as described in the legend of Fig. 1 and the cell-surface AT1R measured as in Fig. 5. The data shown are percentages of the mean value obtained from cells transfected with individual receptor and the pcDNA3 vector and are presented as the means ± S.E. of three experiments. , p < 0.05 versus control.