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α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

Castillo-Badillo JA, Sánchez-Reyes OB, Alfonzo-Méndez MA, Romero-Ávila MT, Reyes-Cruz G, García-Sáinz JA - PLoS ONE (2015)

Bottom Line: Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi.It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7.In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ap. Postal 70-248, México D.F. 04510, Mexico.

ABSTRACT
Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0121165.g002: 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.

Mentions: Noradrenaline was unable to increase intracellular calcium concentration in untransfected HEK293 cells but induced an immediate and robust response, in cells expressing human wild type or DsRed-tagged α1B-adrenergic receptors (Fig. 1, panel A). As expected, in these cells, activation of protein kinase C with phorbol myristate acetate essentially abolished the effect of noradrenaline on this parameter, without altering baseline intracellular calcium or bradykinin action (Fig. 1, panel A). Sphingosine 1-phosphate induced an immediate, robust increase in intracellular calcium concentration (Fig. 1, panel B). When cells were incubated with this bioactive lipid for 15 min, the effect of noradrenaline was markedly desensitized but the action of bradykinin was not altered (Fig. 1, panel B). These data indicate that sphingosine 1-phosphate induced heterologous α1B-adrenergic desensitization and that the effect is not due to intracellular calcium depletion. The data were essentially identical when the wild type or DsRed-tagged receptors were used and are entirely consistent with data obtained using EGFP-tagged α1B-adrenergic receptors [42]. Similarly, we observed that wild type and DsRed-tagged α1B-adrenergic receptors are phosphoproteins whose phosphorylation state is increased by agents that induce desensitization such as noradrenaline (homologous) or sphingosine 1-phosphate (heterologous) (Fig. 2, panel A). Internalization of Wild-type and DsRed-tagged α1B-adrenergic receptors after 15 min stimulation with noradrenaline (10 μM) or sphingosine 1-phophate is depicted in Fig. 2 (panels B and C, respectively). Internalization was mainly evidenced by a decrease in the delineation of the plasma membrane by fluorescence and by an increase in the number and intensity of fluorescent vesicles, which is consistent with what has been observed with EGFP-tagged α1B-adrenergic receptors [42].


α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

Castillo-Badillo JA, Sánchez-Reyes OB, Alfonzo-Méndez MA, Romero-Ávila MT, Reyes-Cruz G, García-Sáinz JA - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4370394&req=5

pone.0121165.g002: 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.
Mentions: Noradrenaline was unable to increase intracellular calcium concentration in untransfected HEK293 cells but induced an immediate and robust response, in cells expressing human wild type or DsRed-tagged α1B-adrenergic receptors (Fig. 1, panel A). As expected, in these cells, activation of protein kinase C with phorbol myristate acetate essentially abolished the effect of noradrenaline on this parameter, without altering baseline intracellular calcium or bradykinin action (Fig. 1, panel A). Sphingosine 1-phosphate induced an immediate, robust increase in intracellular calcium concentration (Fig. 1, panel B). When cells were incubated with this bioactive lipid for 15 min, the effect of noradrenaline was markedly desensitized but the action of bradykinin was not altered (Fig. 1, panel B). These data indicate that sphingosine 1-phosphate induced heterologous α1B-adrenergic desensitization and that the effect is not due to intracellular calcium depletion. The data were essentially identical when the wild type or DsRed-tagged receptors were used and are entirely consistent with data obtained using EGFP-tagged α1B-adrenergic receptors [42]. Similarly, we observed that wild type and DsRed-tagged α1B-adrenergic receptors are phosphoproteins whose phosphorylation state is increased by agents that induce desensitization such as noradrenaline (homologous) or sphingosine 1-phosphate (heterologous) (Fig. 2, panel A). Internalization of Wild-type and DsRed-tagged α1B-adrenergic receptors after 15 min stimulation with noradrenaline (10 μM) or sphingosine 1-phophate is depicted in Fig. 2 (panels B and C, respectively). Internalization was mainly evidenced by a decrease in the delineation of the plasma membrane by fluorescence and by an increase in the number and intensity of fluorescent vesicles, which is consistent with what has been observed with EGFP-tagged α1B-adrenergic receptors [42].

Bottom Line: Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi.It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7.In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ap. Postal 70-248, México D.F. 04510, Mexico.

ABSTRACT
Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).

No MeSH data available.


Related in: MedlinePlus