Efecto de la activación del receptor para el factor de crecimiento similar a la insulina tipo I (IGF-1, insulin like growth factor 1) sobre el receptor alfa-1B adrenérgico y su función.

Tesis (Ingeniería Biotecnológica), Instituto Politécnico Nacional, UPIBI, 2006, 1 archivo PDF, (50 páginas). tesis.ipn.m

Role of cysteine residues in the carboxyl-terminus of the follicle-stimulating hormone receptor in intracellular traffic and postendocytic processing

Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these cysteine residues are S-palmitoylated, the data presented emphasize on this posttranslational modification as an important factor for both upward and downward trafficking of this receptor

α_1B-Adrenergic receptor-Rab 5 interaction.

<p>Images of cells coexpressing α_1B-adrenergic receptors tagged with the DsRed fluorescent protein (α_1B-AR) and Rab 5 tagged with the enhanced green fluorescent protein (EGFP). Other indications as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121165#pone.0121165.g001" target="_blank">Fig. 1</a>. 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.</p

α_1B-Adrenergic receptor-Rab 4 and Rab 11 interactions.

<p>Images of cells coexpressing α_1B-adrenergic receptors tagged with the DsRed fluorescent protein (α_1B-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121165#pone.0121165.g001" target="_blank">Fig. 1</a>.</p

Representative emission spectra obtained from cells coexpressing Ds-Red-tagged α1B-adrenergic receptors and EGFP-tagged Rab proteins 5 or 9.

<p>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).</p

α_1B-Adrenergic receptor action and desensitization.

<p>Panel A: Cells expressing wild-type (black symbols) or DsRed-tagged (red symbols) human α_1B-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 α_1B-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.</p

α_1B-Adrenergic receptor phosphorylation and internalization.

<p>Panel A: Cells expressing wild-type (black symbols) or DsRed-tagged (red symbols) human α_1B-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 α_1B-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 α_1B-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.</p

DsRed-tagged α1B-adrenergic receptor and membrane-directed EGFP-tagged β-arrestin 2 colocalization.

<p>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.</p

α_1B-Adrenergic receptor-Rab 9 interaction.

<p>Images of cells coexpressing α_1B-adrenergic receptors tagged with the DsRed fluorescent protein (α_1B-AR) and Rab 9 tagged with the enhanced green fluorescent protein (EGFP). Other indications as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121165#pone.0121165.g001" target="_blank">Fig. 1</a>. 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.</p