Journal
BIOCHEMICAL JOURNAL
Volume 418, Issue -, Pages 163-172Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20080867
Keywords
beta(2)-adrenergic receptor (beta 2AR); G-protein-coupled receptor (GPCR); Rab11; recycling; trafficking
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Funding
- Fonds de la Recherche en Sante du Quebec
- Canadian Institutes of Health Research [MOP-690851]
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The beta 2ARs (beta(2)-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 beta 2ARs 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 beta 2AR-Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His(6)-tagged Rab11 protein and beta 2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of beta 2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the beta 2AR interacts preferentially with the GDP-bound form of Rab11. Arg(333) and Lys(348) in the C-terminal tail of the beta 2AR were identified as crucial determinants for Rab11 binding. A beta 2AR construct with these two residues mutated to alanine, beta 2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of beta 2AR RK/AA was drastically reduced when compared with wild-type beta 2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the beta 2AR 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 beta 2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.
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