Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the ␤-isoform of the thromboxane A 2 receptor (TP␤) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TP␤ association occurring in the absence of agonist, which is not modulated by stimulation of TP␤. We show with purified TP␤ intracellular domains fused to GST and HISRab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TP␤. Amino acids 335-344 of the TP␤ C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TP␤ interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.

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.

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the b isoform of the thromboxane A 2 receptor (TPb). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPb-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPb. The TPb-RACK1 association was further confirmed by co-immunoprecipitation studies in HEK293 cells and was not modulated by stimulation of the receptor. We observed that cell surface expression of TPb was increased when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by small interfering RNA. Confocal microscopy confirmed the impaired cell surface expression of TPb and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPb-RACK1 association takes place in the ER. The effect of RACK1 on receptor trafficking to the cell surface appears to be selective to some G protein-coupled receptors (GPCRs) because inhibition of RACK1 expression also affected cell surface targeting of the angiotensin II type 1 receptor and CXCR4 but not of b 2 -adrenergic and prostanoid DP receptors. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of the transport of a membrane receptor from the ER to the cell surface.

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP␣ and TP␤) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP␤ is regulated by agonistinduced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP␤. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP␤ in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP␤ was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP␤ to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.