Rab11 regulates the recycling of the beta2-adrenergic receptor through a direct interaction.
The beta2ARs (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 beta2ARs 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 beta2AR-Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His(6)-tagged Rab11 protein and beta2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of beta2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the beta2AR interacts preferentially with the GDP-bound form of Rab11. Arg(333) and Lys(348) in the C-terminal tail of the beta2AR were identified as crucial determinants for Rab11 binding. A beta2AR construct with these two residues mutated to alanine, beta2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of beta2AR RK/AA was drastically reduced when compared with wild-type beta2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the beta2AR 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 beta2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.
Parent
Audrey
A
Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, QC, Canada, J1H 5N4.
Hamelin
Emilie
E
Germain
Pascale
P
Parent
Jean-Luc
JL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D018343:Receptors, Adrenergic, beta-2; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins