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 maturation and folding of G protein-coupled receptors are governed by mechanisms that remain poorly understood. In an effort to characterize these biological events, we optimized a novel, gel-free proteomic approach to identify partners of the β2-adrenergic receptor (β2AR). In addition to a number of known interacting proteins such as heterotrimeric G protein subunits, this allowed us to identify proteins involved in endoplasmic reticulum (ER) QC of the receptor. Among β2AR-associated proteins is Ring finger protein 5 (RNF5), an E3 ubiquitin ligase anchored to the outer membrane of the ER. Coimmunoprecipitation assays confirmed, in a cellular context, the interaction between RNF5 and the β2AR as well as the prostaglandin D2 receptor (DP). Confocal microscopy revealed that DP colocalized with RNF5 at the ER. Coexpression of RNF5 with either receptor increased levels of their expression, whereas small interfering RNA-mediated knockdown of endogenous RNF5 promoted the opposite. RNF5 did not modulate the ubiquitination state of β2AR or DP. Instead, RNF5 ubiquitinated JNK-associated membrane protein (JAMP), a protein that recruits the proteasome to the ER membrane and that is negatively regulated by RNF5-mediated ubiquitination. JAMP coimmunoprecipitated with both β2AR and DP and decreased total receptor protein levels through proteasomal degradation. Expression of DP, a receptor largely retained in the ER, promoted proteasome recruitment by JAMP. Degradation of both receptors via JAMP was increased when RNF5 was depleted. Our data suggest that RNF5 regulates the turnover of specific G protein-coupled receptors by ubiquitinating JAMP and preventing proteasome recruitment.

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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.