Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction

Abstract

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.

Interaction of the Human Prostacyclin Receptor with Rab11

“…The Rab11 subfamily of GTPases, consisting of Rab11a, Rab11b, and the epithelial Rab25 (or Rab11c), along with their many specific effectors, have emerged as principal regulators of membrane traffic from the endosomal recycling compartment (ERC) and from apical recycling endosomes in polarized cells (5,6). In addition to interacting with specific effectors, in the case of certain GPCRs, it is increasingly recognized that Rab11 members may interact directly with the receptor cargo to regulate its intracellular trafficking and signaling (7)(8)(9). Moreover, Rab25, a GTPase linked to aggressiveness and metastasis of ovarian and breast cancers (10,11), interacts directly with the ␣5␤1 integrin to regulate invasive tumor migration (12).…”

Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain

“…Recently, Rabs have been shown to directly associate with the proteins being trafficked through recycling endosomes (43,44). We demonstrate that Rab35 immunoprecipitates KCa2.3 confirming these proteins are part of the same complex.…”

Regulation of β2-Adrenergic Receptor Maturation and Anterograde Trafficking by an Interaction with Rab Geranylgeranyltransferase

“…The PCR fragment was digested with EcoRI and XhoI and ligated into the pcDNA3-FLAG vector (31) digested with the same enzymes. The ␤ 2 AR C-terminal constructs were subcloned as described previously (22). Deletion and site-directed mutagenesis were carried out by PCR.…”

“…We recently showed that an interaction between GPCRs and Rab11a directed receptor trafficking (22,23,32). A higher molecular mass form of Rab11a was detected after longer exposure of Western blot membranes, and this form was increased when GPCRs were co-expressed in HEK293 cells.…”

“…Rab-mediated vesicular transport is well known to regulate membrane receptor trafficking, but less is known about whether membrane receptors conversely regulate the Rab-trafficking machinery. We and others showed that GPCRs interact with Rabs resulting in the regulation of receptor trafficking (22)(23)(24)(25)(26)(27)(28)(29). The task of unraveling whether membrane receptors interact with other elements of the Rab-associated machinery to direct their own trafficking remains.…”