Fig 1.
α1B-Adrenergic receptor action and desensitization.
Panel A: Cells expressing wild-type (black symbols) or DsRed-tagged (red symbols) human α1B-adrenergic receptors were pre-incubated for 15 min in the absence or presence or 1 μM phorbol myristate acetate (PMA) and then were challenged with 10 noradrenaline (NA, arrow), 1 μM PMA or 1 μM bradykinin (BK) and intracellular calcium concentration was recorded. Plotted in the left figure are the means and vertical lines that represent the S.E.M. of 4–6 experiments using different cell preparations. *p < 0.001 vs. NA action in cells pre-incubated without PMA. Middle and right figures are representative tracings. Panel B: Cells expressing wild-type (black symbols) or DsRed-tagged (red symbols) human α1B-adrenergic receptors were pre-incubated for 15 min in the absence or presence or 1 μM sphingosine 1-phosphate (S1P) and then were challenged with 10 noradrenaline (NA, arrow), 1 μM S1P or 1 μM bradykinin (BK) and intracellular calcium concentration was recorded. In the left figure the means and vertical lines are plotted, that represent he S.E.M. of 4–6 experiments using different cell preparations. *p < 0.001 vs. NA action in cells pre-incubated without S1P. Middle and right figures are representative tracings.
Fig 2.
α1B-Adrenergic receptor phosphorylation and internalization.
Panel A: Cells expressing wild-type (black symbols) or DsRed-tagged (red symbols) human α1B-adrenergic receptors were incubated in the absence of any agent (B, baseline, 30 min), 10 μM noradrenaline (NA, 15 min) or 1 μM sphingosine 1-phosphate (S1P, 30 min) to study receptor phosphorylation state. Plotted are the means and vertical lines representing the S.E.M. of 3–4 experiments using different cell preparations. *p < 0.005 vs. B (their respective cell line baseline value), **p < 0.05 vs. B (their respective baseline value). Representative autoradiographs are presented. Panel B: Confocal microscopy images of cells expressing DsRed α1B-adrenergic receptors incubated for 15 min in the absence of any agent (B, baseline), 10 μM noradrenaline (NA) or 1 μM sphingosine 1-phosphate (S1P). Panel C: Confocal microscopy images of cells expressing wild-type α1B-adrenergic receptors incubated for 15 min in the absence of any agent (B, baseline), 10 μM noradrenaline (NA) or 1 μM sphingosine 1-phosphate (S1P). Scale bars: 15 μm.
Fig 3.
DsRed-tagged α1B-adrenergic receptor and membrane-directed EGFP-tagged β-arrestin 2 colocalization.
Panel A: Representative image of cells expressing DsRed-tagged human α1B-adrenergic receptors and membrane-directed EGFP-tagged β-arrestin 2; colocalization is indicated in white. Panel B: Cells were incubated for the times indicated in the presence of 10 μM noradrenaline (NA) or 1 μM sphingosine 1-phosphate (S1P) and images were obtained. Plasma membrane fluorescence was deleted and intracellular colocalization is indicated in white. Panel C: Images obtained as indicated in Panel B were analyzed for intracellular colocalization of adrenergic receptors and β-arrestin. Plotted are the means and vertical lines representing the S.E.M. of 3–4 experiments using different cell preparations and 4–5 images were obtained and analyzed, from each condition. Scale bars: 15 μm.
Fig 4.
α1B-Adrenergic receptor-early endosome antigen 1 interaction.
Images of cells coexpressing α1B-adrenergic receptors tagged with the DsRed fluorescent protein (α1B-AR) and the early endosomes antigen 1 (EEA1) tagged with the enhanced green fluorescent protein (EGFP). Cells were incubated for 15 min in the absence of any agent (B, upper row) or presence of 10 μM noradrenaline (plus 1 μM propranolol) (NA, middle row) or 1 μM sphingosine 1-phosphate (S1P, lower row). Cells were fixed and observed in a fluorescence confocal microscope. The following images are presented: EGFP fluorescence (EGFP was excited and its fluorescence recorded; first column), DsRed fluorescence (DsRed was excited and its fluorescence recorded; second column), “FRET channel” (EGFP was excited, the laser to excite DsRed remained off, and DsRed fluorescence was recorded; third column) and “FRET index” (images processed with the "FRET and Colocalization Analizer", fourth column). Scale bars: 15 μm.
Fig 5.
α1B-Adrenergic receptor-Rab 5 interaction.
Images of cells coexpressing α1B-adrenergic receptors tagged with the DsRed fluorescent protein (α1B-AR) and Rab 5 tagged with the enhanced green fluorescent protein (EGFP). Other indications as in Fig. 1. Panel A, wild-type Rab 5 (WT); panel B, dominant-negative Rab 5 (Rab 5-GDP); and panel C, constitutively active Rab 5 (Rab 5-GTP). In panel D, the quantitative analysis of the FRET index is presented. Plotted are the means and vertical lines representing the S.E.M of 5–7 experiments using different cell preparations. * p< 0.001 vs. wild-type baseline (B) and stimulated with sphingosine 1-phosphate (S1P). ** p< 0.001 vs. Rab 5-GTP baseline (B) and stimulated with sphingosine 1-phosphate (S1P). Scale bars: 15 μm.
Fig 6.
α1B-Adrenergic receptor-Rab 4 and Rab 11 interactions.
Images of cells coexpressing α1B-adrenergic receptors tagged with the DsRed fluorescent protein (α1B-AR) and Rab 4 (panel A) or Rab 11 (panel B) tagged with the enhanced green fluorescent protein (EGFP). Scale bars: 15 μm.Other indications as in Fig. 1.
Fig 7.
α1B-Adrenergic receptor-Rab 7 interaction.
Images of cells coexpressing α1B-adrenergic receptors tagged with the DsRed fluorescent protein (α1B-AR) and Rab 7 tagged with the enhanced green fluorescent protein (EGFP). Scale bars: 15 μm. Other indications as in Fig. 1.
Fig 8.
α1B-Adrenergic receptor-Rab 9 interaction.
Images of cells coexpressing α1B-adrenergic receptors tagged with the DsRed fluorescent protein (α1B-AR) and Rab 9 tagged with the enhanced green fluorescent protein (EGFP). Other indications as in Fig. 1. Panel A, wild-type Rab 9 (WT); panel B, dominant-negative Rab 9 (Rab 9-GDP); and panel C, constitutively-active Rab 9 (Rab 9-GTP). In panel D, the quantitative analysis of the FRET index is presented. Plotted are the means and vertical lines representing the S.E.M of 5–10 experiments using different cell preparations. * p< 0.001 vs. Rab 9-GTP baseline (B); ** p< 0.001 vs. wild-type baseline (B) and wild-type stimulated with noradrenaline (NA). Scale bars: 15 μm.
Fig 9.
Panel A: plotted are the data obtained from cells incubated for 15 min in the absence of any agent (open bars) or with 10 μM noradrenaline (plus 1 μM propranolol)(NA, blue bars). Panel B: plotted are the data obtained from cells incubated for 15 min in the absence of any agent (open bars) or with 1 μM sphingosine 1-phosphate (S1P, brown bars). In the abscissa the protein tagged with EGFP is indicated. Plotted are the means with vertical lines representing the S.E.M. of 5 to 7 determinations using different cell preparations. *p< 0.05 vs. absence of stimuli.
Fig 10.
Time-course of the α1B-adrenergic receptor-Rab 5 interaction.
Panel A: FRET index images of cells coexpressing Ds-Red-tagged α1B-adrenergic receptors and EGFP-tagged Rab 5 treated for the times indicated with 10 μM noradrenaline (NA) or sphingosine 1-phosphate (S1P). Scale bars: 15 μm. Panel B: Quantitative analysis of the data presented in Panel A. Plotted are the means and vertical lines representing the S.E.M of 5–7 experiments using different cell preparations. Noradrenaline, blue line and symbols; sphingosine 1-phosphate, brown line and symbols.
Fig 11.
Time-course of the α1B-adrenergic receptor-Rab 9 interaction.
Panel A: FRET index images of cells coexpressing Ds-Red-tagged α1B-adrenergic receptors and EGFP-tagged Rab 9 treated for the times indicated with 10 μM noradrenaline (NA) or sphingosine 1-phosphate (S1P). Scale bars: 15 μm. Panel B: Quantitative analysis of the data presented in Panel A. Plotted are the means and vertical lines representing the S.E.M of 5–7 experiments using different cell preparations. Noradrenaline, blue line and symbols; sphingosine 1-phosphate, brown line and symbols.
Fig 12.
Representative emission spectra obtained from cells coexpressing Ds-Red-tagged α1B-adrenergic receptors and EGFP-tagged Rab proteins 5 or 9.
The emission spectrum was captured at time zero (baseline, continuous lines), then cells were incubated for 15 min with 10μM noradrenaline (NA, blue lines) or with 1 μM sphingosine 1-phosphate (S1P, brown lines) and then, the emission spectrum was captured again (black dotted lines).
Fig 13.
Coinmunoprecipitation of α1B-adrenergic receptors and the marker proteins.
Rab 5 (panel A) and Rab 9 (panel B) were studied. Cells were incubated for 15 min in the absence of any agent or presence of 10 μM noradrenaline plus 1 μM propranolol (NA) or 1 μM sphingosine 1-phosphate (S1P). Cell lysates were obtained and the proteins were inmmunoprecipitated using an anti-EGFP antibody. The resulting immunocomplexes were analyzed by Western blots. The signal ratio (DsRed / EGFP) is plotted in the upper part of the panels; plotted are the means with vertical lines representing the S.E.M. of 3 experiments performed using different cell preparations. Representative blots are presented.
Fig 14.
Models for α1B-adrenergic-Rab protein interaction.
α1B-Adrenergic internalization and recycling back to the plasma membrane during homologous (panel A) and heterologous (panel B) desensitization is depicted. NA, noradrenaline; α1B-AR, α1B-adrenergic receptor; S1P, sphingosine 1-phosphate; S1P1, sphingosine 1-phosphate receptor type 1. Rab proteins are shown as membrane anchored proteins.