We and others have shown that trafficking of G-protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Here, we show by overexpression and depletion studies that HACE1, a HECTdomain-containing ubiquitin ligase, promotes the recycling of the b 2 -adrenergic receptor (b 2 AR), a prototypical G-protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the b 2 AR in conjunction with HACE1 triggered ubiquitylation of Rab11a, as observed by western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitylated on Lys145. A Rab11a-K145R mutant failed to undergo b 2 AR-HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the b 2 AR. Rab11a, but not Rab11a-K145R, was activated by b 2 AR-HACE1, indicating that ubiquitylation of Lys145 is involved in activation of Rab11a. Co-expression of b 2 AR-HACE1 also potentiated ubiquitylation of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases through their ubiquitylation, with associated functional effects demonstrated on Rab11a. This suggests a new pathway whereby a cargo protein, such as a Gprotein-coupled receptor, can regulate its own trafficking by inducing the ubiquitylation and activation of a Rab GTPase.

With over 30% of current medications targeting this family of proteins, G-protein–coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

The GPCR DP1 promotes the activity of L-PGDS, the enzyme that produces the DP1 agonist PGD2, while at the same time L-PGDS promotes the export and activity of DP1 in response to PGD2.

The delta opioid receptor (DOPr) is known to be mainly expressed in intracellular compartments. It remains unknown why DOPr is barely exported to the cell surface, but it seems that a substantial proportion of the immature receptor is trapped within the endoplasmic reticulum (ER) and the Golgi network. In the present study, we performed LC-MS/MS analysis to identify putative protein partners involved in the retention of DOPr. Analysis of the proteins co-immunoprecipitating with Flag-DOPr in transfected HEK293 cells revealed the presence of numerous subunits of the coatomer protein complex I (COPI), a vesicle-coating complex involved in recycling resident proteins from the Golgi back to the ER. Further analysis of the amino acid sequence of DOPr identified multiple consensus di-lysine and di-arginine motifs within the intracellular segments of DOPr. Using cell-surface ELISA and GST pulldown assays, we showed that DOPr interacts with COPI through its intracellular loops 2 and 3 (ICL2 and ICL3, respectively) and that the mutation of the KAK (ICL2) or KEK (ICL3) putative COPI binding sites increased the cell-surface expression of DOPr in transfected cells. Altogether, our results indicate that COPI is a binding partner of DOPr and provide a putative mechanism to explain why DOPr is highly retained inside the cells.

Background:Interacting partners and regulation of Rab geranylgeranyltransferase are poorly characterized. Mechanisms of GPCR maturation and anterograde trafficking are not fully understood. Results: RGGTA interacts with a dileucine motif in the ␤ 2 AR to regulate ␤ 2 AR maturation/anterograde trafficking and ␤ 2 ARmediated Rab geranylgeranylation. Conclusion: RGGTA and the ␤ 2 AR interact functionally. Significance: This is the first demonstration of a functional interaction between RGGTA and a transmembrane receptor. Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases.Here, we show that the ␤ 2 -adrenergic receptor (␤ 2 AR) interacts with the Rab geranylgeranyltransferase ␣-subunit (RGGTA). Confocal microscopy showed that ␤ 2 AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L 339 L 340 motif in the ␤ 2 AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the ␤ 2 AR, which requires the interaction of RGGTA with the ␤ 2 AR L 339 L 340 motif. Furthermore, the ␤ 2 AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the ␤ 2 AR was dependent on the RGGTA-interacting L 339 L 340 motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted ␤ 2 AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases.

A direct and functional interaction between a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex and G protein–coupled receptor (GPCRs) is shown. Evidence is provided that distinct nascent GPCRs can undergo alternative folding pathways and that CCT/TRiC is critical in preventing aggregation of some GPCRs and in promoting their proper maturation and expression.

Thromboxane A 2 (TXA 2 ) is an important lipid mediator whose function in apoptosis is the subject of conflicting reports. Here, a yeast two-hybrid screen for proteins that interact with the C-terminus of the TXA 2 receptor (TP) identified Siva1 as a new TP-interacting protein. Contradictory evidence suggests pro-and anti-apoptotic roles for Siva1. We show that a cisplatin treatment induces TXA 2 synthesis in HeLa cells. We demonstrate that endogenous TP stimulation promotes cisplatin-induced apoptosis of HeLa cells and that such modulation requires the expression of Siva1, as evidenced by inhibiting its endogenous expression using siRNAs. We reveal that, upon stimulation of TP, degradation of Siva1 is impeded, resulting in an accumulation of the protein, which translocates from the nucleus to the cytosol. Translocation of Siva1 correlates with its reduced interaction with Mdm2 (an inhibitor of p53 signalling), as well as with its increased interaction with TRAF2 and XIAP (known to enhance pro-apoptotic signalling). Our data provide a model that reconciles the pro-and anti-apoptotic roles that were reported for Siva1 and identify a new mechanism for promoting apoptosis by G protein-coupled receptors. Our findings may have implications in the use of cyclo-oxygenase inhibitors during cisplatin chemotherapy and might provide a target to reduce cisplatin toxicity on non-cancerous tissues.

Accumulating evidence indicates that G protein-coupled receptors (GPCRs) interact with Rab GTPases during their intracellular trafficking. How GPCRs recruit and activate the Rabs is unclear. Here, we report that depletion of endogenous L-type prostaglandin D synthase (L-PGDS) in HeLa cells inhibited recycling of the prostaglandin D 2 (PGD 2) DP1 receptor (DP1) to the cell surface after agonist-induced internalization and that L-PGDS overexpression had the opposite effect. Depletion of endogenous Rab4 prevented L-PGDS-mediated recycling of DP1, and L-PGDS depletion inhibited Rab4-dependent recycling of DP1, indicating that both proteins are mutually involved in this pathway. DP1 stimulation promoted its interaction through its intracellular C terminus with Rab4, which was increased by L-PGDS. Confocal microscopy revealed that DP1 activation induces L-PGDS/Rab4 co-localization. L-PGDS/ Rab4 and DP1/Rab4 co-immunoprecipitation levels were increased by DP1 agonist treatment. Pulldown assays with purified GST-L-PGDS and His 6-Rab4 indicated that both proteins interact directly. L-PGDS interacted preferentially with the inactive, GDP-locked Rab4S22N variant rather than with WT Rab4 or with constitutively active Rab4Q67L proteins. Overexpression and depletion experiments disclosed that L-PGDS partakes in Rab4 activation following DP1 stimulation. Experiments with deletion mutants and synthetic peptides revealed that amino acids 85-92 in L-PGDS are involved in its interaction with Rab4 and in its effect on DP1 recycling. Of note, GTP␥S loading and time-resolved FRET assays with purified proteins suggested that L-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how L-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation. This work was supported by a grant from the Canadian Institutes of Health Research (to J. L. P.). The authors declare that they have no conflicts of interest with the contents of this article. 1 Doctoral salary award from the Fonds de Recherche Québec-Santé (FRQS). 2 Doctoral salary support from FRQS and from the National Sciences and Engineering Research Council of Canada (NSERC). 3 M.Sc. salary support from FRQS.