The β2ARs (β2-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some β2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a β2AR–Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His6-tagged Rab11 protein and β2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of β2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the β2AR interacts preferentially with the GDP-bound form of Rab11. Arg333 and Lys348 in the C-terminal tail of the β2AR were identified as crucial determinants for Rab11 binding. A β2AR construct with these two residues mutated to alanine, β2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of β2AR RK/AA was drastically reduced when compared with wild-type β2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the β2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the β2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.
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Research Article|
January 28 2009
Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction
Audrey Parent;
Audrey Parent
1Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, QC, Canada, J1H 5N4, and Centre de Recherche Clinique-Etienne Lebel, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada, J1H 5N4
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Emilie Hamelin;
Emilie Hamelin
2Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, QC, Canada, J1H 5N4, and Centre de Recherche Clinique-Etienne Lebel, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada, J1H 5N4
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Pascale Germain;
Pascale Germain
3Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, QC, Canada, J1H 5N4, and Centre de Recherche Clinique-Etienne Lebel, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada, J1H 5N4
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Jean-Luc Parent
Jean-Luc Parent
1
4Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, QC, Canada, J1H 5N4, and Centre de Recherche Clinique-Etienne Lebel, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada, J1H 5N4
1To whom correspondence should be addressed (email jean-luc.parent@usherbrooke.ca).
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Publisher: Portland Press Ltd
Received:
April 30 2008
Revision Received:
October 16 2008
Accepted:
November 04 2008
Accepted Manuscript online:
November 04 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 418 (1): 163–172.
Article history
Received:
April 30 2008
Revision Received:
October 16 2008
Accepted:
November 04 2008
Accepted Manuscript online:
November 04 2008
Citation
Audrey Parent, Emilie Hamelin, Pascale Germain, Jean-Luc Parent; Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction. Biochem J 15 February 2009; 418 (1): 163–172. doi: https://doi.org/10.1042/BJ20080867
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