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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

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.
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pone.0121165.g010: 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.

Mentions: As expected, when the dominant negative mutant of Rab 9 (Rab 9-GDP) was employed we were unable to detect FRET under any of the conditions studied (Fig. 8, panel B). To our surprise in cells coexpressing α1B-adrenergic receptors and the Rab 9 constitutively active mutant (Rab 9-GTP), some signal in the “FRET channel” was observed emitting from the red fluorescent protein, DsRed, under baseline conditions, i. e., in the absence of added stimulus, although the increase was variable and it was not statistically significant (Fig. 8, panel C). Such baseline FRET was further increased by both noradrenaline and sphingosine 1-phosphate (Fig. 8, panel C). The FRET index analysis is presented in Fig. 8 (panel D).The time-course of the effects of noradrenaline and sphingosine 1-phosphate on FRET index was studied using cells coexpressing α1B-adrenergic receptors and either Rab 5 (Fig. 10) or Rab 9 (Fig. 11). It can be observed that noradrenaline induced a marked progressive increase in FRET in cell expressing Rab 5 but hardly had any effect on cells expressing Rab 9 (Figs. 10 and 11). In contrast, sphingosine 1-phosphate induced a small rapid increase in FRET (2 min) in cells expressing Rab 5, which vanished at later times (Fig. 10) and a rapid and progressive increase in FRET in cells expressing Rab 9 (Fig. 11).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4370394&req=5

pone.0121165.g010: 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.
Mentions: As expected, when the dominant negative mutant of Rab 9 (Rab 9-GDP) was employed we were unable to detect FRET under any of the conditions studied (Fig. 8, panel B). To our surprise in cells coexpressing α1B-adrenergic receptors and the Rab 9 constitutively active mutant (Rab 9-GTP), some signal in the “FRET channel” was observed emitting from the red fluorescent protein, DsRed, under baseline conditions, i. e., in the absence of added stimulus, although the increase was variable and it was not statistically significant (Fig. 8, panel C). Such baseline FRET was further increased by both noradrenaline and sphingosine 1-phosphate (Fig. 8, panel C). The FRET index analysis is presented in Fig. 8 (panel D).The time-course of the effects of noradrenaline and sphingosine 1-phosphate on FRET index was studied using cells coexpressing α1B-adrenergic receptors and either Rab 5 (Fig. 10) or Rab 9 (Fig. 11). It can be observed that noradrenaline induced a marked progressive increase in FRET in cell expressing Rab 5 but hardly had any effect on cells expressing Rab 9 (Figs. 10 and 11). In contrast, sphingosine 1-phosphate induced a small rapid increase in FRET (2 min) in cells expressing Rab 5, which vanished at later times (Fig. 10) and a rapid and progressive increase in FRET in cells expressing Rab 9 (Fig. 11).

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