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L-type prostaglandin D synthase regulates the trafficking of the PGD2 DP1 receptor by interacting with the GTPase Rab4

  • Chantal Binda
    Footnotes
    Affiliations
    Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Samuel Génier
    Footnotes
    Affiliations
    Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Jade Degrandmaison
    Footnotes
    Affiliations
    Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Samuel Picard
    Footnotes
    Affiliations
    Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Louis Fréchette
    Footnotes
    Affiliations
    Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Steve Jean
    Affiliations
    Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Eric Marsault
    Affiliations
    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Jean-Luc Parent
    Correspondence
    To whom correspondence should be addressed: Dépt. de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Ave. Nord, Sherbrooke, Québec J1H 5N4, Canada. Tel.:819-821-8000 (ext. 75283)
    Affiliations
    Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

    Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Author Footnotes
    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.
    5 The abbreviations used are:PGD2prostaglandin D2COXcyclooxygenasePGDSprostaglandin D synthaseH- and L-PGDShematopoietic and lipocalin-type PGDS, respectivelyGPCRG protein–coupled receptorDsiRNADicer-substrate siRNAHATUhexafluorophosphate azabenzotriazole tetramethyl uroniumDIPEAN,N-diisopropylethylamineTIPStriisopropylsilaneEDT1,2-ethanedithiolGAPGTPase-activating proteinGEFguanine nucleotide exchange factorβ2ARβ2-adrenergic receptorGSTGSH-S-transferaseICLintracellular loopGTPγSguanosine 5′-3-O-(thio)triphosphateTRtime-resolvedDMEMDulbecco's modified Eagle's mediumTBSTris-buffered salineFmocN-(9-fluorenyl)methoxycarbonylHAhemagglutininGAPDHglyceraldehyde-3-phosphate dehydrogenase.
Open AccessPublished:October 01, 2019DOI:https://doi.org/10.1074/jbc.RA119.008233
      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 D2 (PGD2) 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 His6-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, GTPγS 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.

      Introduction

      Prostaglandin D2 (PGD2)
      The abbreviations used are: PGD2
      prostaglandin D2
      COX
      cyclooxygenase
      PGDS
      prostaglandin D synthase
      H- and L-PGDS
      hematopoietic and lipocalin-type PGDS, respectively
      GPCR
      G protein–coupled receptor
      DsiRNA
      Dicer-substrate siRNA
      HATU
      hexafluorophosphate azabenzotriazole tetramethyl uronium
      DIPEA
      N,N-diisopropylethylamine
      TIPS
      triisopropylsilane
      EDT
      1,2-ethanedithiol
      GAP
      GTPase-activating protein
      GEF
      guanine nucleotide exchange factor
      β2AR
      β2-adrenergic receptor
      GST
      GSH-S-transferase
      ICL
      intracellular loop
      GTPγS
      guanosine 5′-3-O-(thio)triphosphate
      TR
      time-resolved
      DMEM
      Dulbecco's modified Eagle's medium
      TBS
      Tris-buffered saline
      Fmoc
      N-(9-fluorenyl)methoxycarbonyl
      HA
      hemagglutinin
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase.
      is a lipid mediator involved in numerous physiological processes, such as bronchoconstriction, vasodilatation (
      • Hardy C.C.
      • Robinson C.
      • Tattersfield A.E.
      • Holgate S.T.
      The bronchoconstrictor effect of inhaled prostaglandin D2 in normal and asthmatic men.
      ), sleep (
      • Ueno R.
      • Honda K.
      • Inoué S.
      • Hayaishi O.
      Prostaglandin D2, a cerebral sleep-inducing substance in rats.
      ), and pain (
      • Eguchi N.
      • Minami T.
      • Shirafuji N.
      • Kanaoka Y.
      • Tanaka T.
      • Nagata A.
      • Yoshida N.
      • Urade Y.
      • Ito S.
      • Hayaishi O.
      Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice.
      ). It is also implicated in inflammatory responses, such as asthma (
      • Matsuoka T.
      • Hirata M.
      • Tanaka H.
      • Takahashi Y.
      • Murata T.
      • Kabashima K.
      • Sugimoto Y.
      • Kobayashi T.
      • Ushikubi F.
      • Aze Y.
      • Eguchi N.
      • Urade Y.
      • Yoshida N.
      • Kimura K.
      • Mizoguchi A.
      • et al.
      Prostaglandin D2 as a mediator of allergic asthma.
      ) and atherosclerosis (
      • Ishizuka T.
      • Matsui T.
      • Okamoto Y.
      • Ohta A.
      • Shichijo M.
      Ramatroban (BAY u 3405): a novel dual antagonist of TXA2 receptor and CRTh2, a newly identified prostaglandin D2 receptor.
      ). PGD2 exhibits anti-inflammatory properties as well (
      • Gilroy D.W.
      • Colville-Nash P.R.
      • Willis D.
      • Chivers J.
      • Paul-Clark M.J.
      • Willoughby D.A.
      Inducible cyclooxygenase may have anti-inflammatory properties.
      ,
      • Ianaro A.
      • Ialenti A.
      • Maffia P.
      • Pisano B.
      • Di Rosa M.
      Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.
      • Vong L.
      • Ferraz J.G.P.
      • Panaccione R.
      • Beck P.L.
      • Wallace J.L.
      A pro-resolution mediator, prostaglandin D2, is specifically up-regulated in individuals in long-term remission from ulcerative colitis.
      ) and can promote bone formation (
      • Gallant M.A.
      • Chamoux E.
      • Bisson M.
      • Wolsen C.
      • Parent J.-L.
      • Roux S.
      • de Brum-Fernandes A.J.
      Increased concentrations of prostaglandin D2 during post-fracture bone remodeling.
      ,
      • Gallant M.A.
      • Samadfam R.
      • Hackett J.A.
      • Antoniou J.
      • Parent J.-L.
      • de Brum-Fernandes A.J.
      Production of prostaglandin D2 by human osteoblasts and modulation of osteoprotegerin, RANKL, and cellular migration by DP and CRTH2 receptors.
      ). PGD2 is formed from arachidonic acid through the action of cyclooxygenases (COXs). COXs convert arachidonic acid released from membranes to PGH2, which is metabolized by two types of PGD2 synthase (PGDS). The hematopoietic PGDS (H-PGDS) is GSH-dependent (
      • Urade Y.
      • Eguchi N.
      Lipocalin-type and hematopoietic prostaglandin D synthases as a novel example of functional convergence.
      ) and is mainly expressed in mast cells (
      • Urade Y.
      • Ujihara M.
      • Horiguchi Y.
      • Igarashi M.
      • Nagata A.
      • Ikai K.
      • Hayaishi O.
      Mast cells contain spleen-type prostaglandin D synthetase.
      ), megakaryocytes (
      • Fujimori K.
      • Kanaoka Y.
      • Sakaguchi Y.
      • Urade Y.
      Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells: roles of the oct-1 element in the 5′-flanking region and the AP-2 element in the untranslated exon 1.
      ), and T-helper 2 lymphocytes (
      • Tanaka K.
      • Ogawa K.
      • Sugamura K.
      • Nakamura M.
      • Takano S.
      • Nagata K.
      Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets.
      ). On the other hand, the lipocalin-type PGDS (l-PGDS) is GSH-independent and is expressed abundantly in the central nervous system (
      • Urade Y.
      • Fujimoto N.
      • Hayaishi O.
      Purification and characterization of rat brain prostaglandin D synthetase.
      ,
      • Blödorn B.
      • Mäder M.
      • Urade Y.
      • Hayaishi O.
      • Felgenhauer K.
      • Brück W.
      Choroid plexus: the major site of mRNA expression for the β-trace protein (prostaglandin D synthase) in human brain.
      ), the heart (
      • Eguchi Y.
      • Eguchi N.
      • Oda H.
      • Seiki K.
      • Kijima Y.
      • Matsu-ura Y.
      • Urade Y.
      • Hayaishi O.
      Expression of lipocalin-type prostaglandin D synthase (β-trace) in human heart and its accumulation in the coronary circulation of angina patients.
      ), the retina (
      • Beuckmann C.T.
      • Gordon W.C.
      • Kanaoka Y.
      • Eguchi N.
      • Marcheselli V.L.
      • Gerashchenko D.Y.
      • Urade Y.
      • Hayaishi O.
      • Bazan N.G.
      Lipocalin-type prostaglandin D synthase (β-trace) is located in pigment epithelial cells of rat retina and accumulates within interphotoreceptor matrix.
      ), and the genital organs (
      • Gerena R.L.
      • Eguchi N.
      • Urade Y.
      • Killian G.J.
      Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis.
      ). l-PGDS is also the only enzyme among the members of the lipocalin gene family and binds small lipophilic substances like retinoic acid (
      • Tanaka T.
      • Urade Y.
      • Kimura H.
      • Eguchi N.
      • Nishikawa A.
      • Hayaishi O.
      Lipocalin-type prostaglandin D synthase (β-trace) is a newly recognized type of retinoid transporter.
      ), bilirubin (
      • Beuckmann C.T.
      • Aoyagi M.
      • Okazaki I.
      • Hiroike T.
      • Toh H.
      • Hayaishi O.
      • Urade Y.
      Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.
      ), and gangliosides (
      • Mohri I.
      • Taniike M.
      • Okazaki I.
      • Kagitani-Shimono K.
      • Aritake K.
      • Kanekiyo T.
      • Yagi T.
      • Takikita S.
      • Kim H.-S.
      • Urade Y.
      • Suzuki K.
      Lipocalin-type prostaglandin D synthase is up-regulated in oligodendrocytes in lysosomal storage diseases and binds gangliosides.
      ).
      PGD2 activates two different G protein–coupled receptors (GPCRs), the D prostanoid receptor (DP1) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells, also known as DP2). DP1 is a member of the family of prostanoid receptors (
      • Boie Y.
      • Sawyer N.
      • Slipetz D.M.
      • Metters K.M.
      • Abramovitz M.
      Molecular cloning and characterization of the human prostanoid DP receptor.
      ). On the other hand, CRTH2 is a member of the chemoattractant receptor family, sharing higher sequence homology with the fMLP and C5a receptors than with the prostanoid receptor family (
      • Hirai H.
      • Tanaka K.
      • Yoshie O.
      • Ogawa K.
      • Kenmotsu K.
      • Takamori Y.
      • Ichimasa M.
      • Sugamura K.
      • Nakamura M.
      • Takano S.
      • Nagata K.
      Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2.
      ). GPCRs, which are among the most abundant membrane proteins, respond to a host of stimuli, including light, hormones, lipids, neurotransmitters, and odorants, to induce various physiological responses (
      • Lebon G.
      • Tate C.G.
      [G protein-coupled receptors in the spotlight].
      ). They share a common molecular topology constituting of a hydrophobic core of seven transmembrane α-helices, three intracellular loops, three extracellular loops, an extracellular N terminus, and an intracellular C terminus. GPCRs are generally delivered to the plasma membrane in a ligand-responsive and signaling-competent form. Following agonist stimulation, the majority of GPCRs internalize into endosomes from which they can undergo recycling to the cell surface or lysosomal degradation (
      • Lachance V.
      • Degrandmaison J.
      • Marois S.
      • Robitaille M.
      • Génier S.
      • Nadeau S.
      • Angers S.
      • Parent J.-L.
      Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
      • Costanzi S.
      • Tikhonova I.G.
      • Harden T.K.
      • Jacobson K.A.
      Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands.
      ,
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ,
      • Ritter S.L.
      • Hall R.A.
      Fine-tuning of GPCR activity by receptor-interacting proteins.
      • Magalhaes A.C.
      • Dunn H.
      • Ferguson S.S.
      Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins.
      ).
      We and others have shown that GPCRs interact with small Rab GTPases (Rabs) to control their trafficking (
      • Lachance V.
      • Degrandmaison J.
      • Marois S.
      • Robitaille M.
      • Génier S.
      • Nadeau S.
      • Angers S.
      • Parent J.-L.
      Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
      ,
      • Esseltine J.L.
      • Dale L.B.
      • Ferguson S.S.G.
      Rab GTPases bind at a common site within the angiotensin II type I receptor carboxyl-terminal tail: evidence that Rab4 regulates receptor phosphorylation, desensitization, and resensitization.
      • Hamelin E.
      • Thériault C.
      • Laroche G.
      • Parent J.-L.
      The intracellular trafficking of the G protein-coupled receptor TPβ depends on a direct interaction with Rab11.
      ,
      • Parent A.
      • Hamelin E.
      • Germain P.
      • Parent J.-L.
      Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction.
      ,
      • Dateyama I.
      • Sugihara Y.
      • Chiba S.
      • Ota R.
      • Nakagawa R.
      • Kobayashi T.
      • Itoh H.
      RABL2 positively controls localization of GPCRs in mammalian primary cilia.
      ,
      • Seachrist J.L.
      • Ferguson S.S.G.
      Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases.
      ,
      • Dong C.
      • Yang L.
      • Zhang X.
      • Gu H.
      • Lam M.L.
      • Claycomb W.C.
      • Xia H.
      • Wu G.
      Rab8 interacts with distinct motifs in α2B- and β2-adrenergic receptors and differentially modulates their transport.
      • Li C.
      • Wei Z.
      • Fan Y.
      • Huang W.
      • Su Y.
      • Li H.
      • Dong Z.
      • Fukuda M.
      • Khater M.
      • Wu G.
      The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.
      ). However, the interacting partners involved in the assembly of GPCR-Rab complexes and in the activation of Rabs that are important for the correct routing of a given GPCR remain poorly characterized. With over 60 known members, Rabs form the largest branch of the Ras-related small GTPase family (
      • Cherfils J.
      • Zeghouf M.
      Regulation of small GTPases by GEFs, GAPs, and GDIs.
      • Hutagalung A.H.
      • Novick P.J.
      Role of Rab GTPases in membrane traffic and cell physiology.
      ,
      • Stenmark H.
      Rab GTPases as coordinators of vesicle traffic.
      • Wandinger-Ness A.
      • Zerial M.
      Rab proteins and the compartmentalization of the endosomal system.
      ). Rabs have been identified as key regulators of numerous cellular processes that determine, for example, cell shape, motility, differentiation, and growth. They are also involved at almost every level of vesicle-mediated transport (
      • Bhuin T.
      • Roy J.K.
      Rab proteins: the key regulators of intracellular vesicle transport.
      ,
      • Wang G.
      • Wu G.
      Small GTPase regulation of GPCR anterograde trafficking.
      ). Depending on their cellular function, each Rab has a distinct subcellular localization, enabling the efficient transport of cargo proteins between compartments (
      • Bhuin T.
      • Roy J.K.
      Rab proteins: the key regulators of intracellular vesicle transport.
      ,
      • Pfeffer S.R.
      Rab GTPases: specifying and deciphering organelle identity and function.
      ). To accomplish their roles, Rabs alternate between a GDP-bound inactive and a GTP-bound active form (
      • Christoforidis S.
      • Zerial M.
      Purification and identification of novel Rab effectors using affinity chromatography.
      ,
      • Zhen Y.
      • Stenmark H.
      Cellular functions of Rab GTPases at a glance.
      ). When Rabs are activated, they can anchor to a specific membrane or vesicle, where they regulate its trafficking (
      • Blümer J.
      • Rey J.
      • Dehmelt L.
      • Mazel T.
      • Wu Y.-W.
      • Bastiaens P.
      • Goody R.S.
      • Itzen A.
      RabGEFs are a major determinant for specific Rab membrane targeting.
      ,
      • Pfeffer S.R.
      Rab GTPase regulation of membrane identity.
      ). GTPase-activating proteins (GAPs) are needed to counteract the slow intrinsic GTPase activity of Rabs and to allow rapid cycling to their inactive states by favoring GTP hydrolysis (
      • Pfeffer S.R.
      Rab GTPase regulation of membrane identity.
      ,
      • Segev N.
      Ypt/rab gtpases: regulators of protein trafficking.
      ). In their inactive forms, Rabs are tightly bound to GDP, and the dissociation of the nucleotide is a slow process. Guanine nucleotide exchange factors (GEFs) accelerate the conversion of Rab GTPases from the inactive to the active form (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ). Because there is a higher cytosolic concentration of GTP than GDP, GTP binds quickly after the GDP is dislodged, which in turn displaces the GEF to yield the active GTP-bound form (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ,
      • Ishida M.
      • Oguchi M.E.
      • Fukuda M.
      Multiple types of guanine nucleotide exchange factors (GEFs) for Rab small GTPases.
      ). Other mechanisms of Rab GTPase activation have been reported, such as phosphorylation and ubiquitination (
      • Lachance V.
      • Degrandmaison J.
      • Marois S.
      • Robitaille M.
      • Génier S.
      • Nadeau S.
      • Angers S.
      • Parent J.-L.
      Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
      ,
      • Yudowski G.A.
      • Puthenveedu M.A.
      • Henry A.G.
      • von Zastrow M.
      Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.
      ).
      We have shown previously that DP1 is recycled back to the plasma membrane following internalization via Rab4-positive recycling endosomes (
      • Gallant M.A.
      • Slipetz D.
      • Hamelin E.
      • Rochdi M.D.
      • Talbot S.
      • de Brum-Fernandes A.J.
      • Parent J.-L.
      Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
      ). Rab4 plays a pivotal role in the rapid recycling of numerous key cargo proteins back to the cell surface, such as integrins, receptors, ubiquitin ligases, proteases, and channels (
      • Wandinger-Ness A.
      • Zerial M.
      Rab proteins and the compartmentalization of the endosomal system.
      ). Accumulating evidence indicates that Rab4 is required for cancer cell invasion (
      • Ishida M.
      • Oguchi M.E.
      • Fukuda M.
      Multiple types of guanine nucleotide exchange factors (GEFs) for Rab small GTPases.
      ,
      • Koch D.
      • Rai A.
      • Ali I.
      • Bleimling N.
      • Friese T.
      • Brockmeyer A.
      • Janning P.
      • Goud B.
      • Itzen A.
      • Müller M.P.
      • Goody R.S.
      A pull-down procedure for the identification of unknown GEFs for small GTPases.
      • Müller M.P.
      • Goody R.S.
      Molecular control of Rab activity by GEFs, GAPs and GDI.
      ,
      • Yoshimura S.
      • Gerondopoulos A.
      • Linford A.
      • Rigden D.J.
      • Barr F.A.
      Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.
      ,
      • Do M.T.
      • Chai T.F.
      • Casey P.J.
      • Wang M.
      Isoprenylcysteine carboxylmethyltransferase function is essential for RAB4A-mediated integrin β3 recycling, cell migration and cancer metastasis.
      • Barbarin A.
      • Frade R.
      Procathepsin L secretion, which triggers tumour progression, is regulated by Rab4a in human melanoma cells.
      ) by regulating the recycling of furin (
      • Arsenault D.
      • Lucien F.
      • Dubois C.M.
      Hypoxia enhances cancer cell invasion through relocalization of the proprotein convertase furin from the trans-Golgi network to the cell surface.
      ), β3 integrin, and MT1-MMP, leading to invadosome formation, degradation, and remodeling of the extracellular matrix (
      • Frittoli E.
      • Palamidessi A.
      • Marighetti P.
      • Confalonieri S.
      • Bianchi F.
      • Malinverno C.
      • Mazzarol G.
      • Viale G.
      • Martin-Padura I.
      • Garré M.
      • Parazzoli D.
      • Mattei V.
      • Cortellino S.
      • Bertalot G.
      • Di Fiore P.P.
      • Scita G.
      A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination.
      ). Given its biological importance, it is surprising that the mechanisms of Rab4 activation are still largely unknown.
      Our prior work showed that l-PGDS interacts with DP1 and directs the anterograde transport of the receptor by acting as a co-factor of the Hsp90 molecular chaperone (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). The purpose of the present study was to investigate the possible role of l-PGDS in Rab4-dependent recycling of DP1. Our intriguing findings indicate that l-PGDS, an enzyme that synthesizes PGD2, regulates the recycling of the DP1 receptor for PGD2 by recruiting and activating Rab4.

      Results

      l-PGDS regulates the recycling of DP1

      We previously reported that l-PGDS interacts directly with DP1 and is involved in the anterograde trafficking of the receptor (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). As such, we were interested in determining whether it also takes part in other aspects of DP1 trafficking. Two cell types were used throughout our experiments: 1) HeLa cells because they endogenously express DP1, l-PGDS, and Rab4 and produce PGD2 (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ) and 2) human embryonic kidney 293 (HEK293) cells for studies that involved l-PGDS overexpression because they express low levels of endogenous l-PGDS protein. We assessed the involvement of l-PGDS in agonist-induced internalization of DP1. HeLa cells stably expressing FLAG-tagged DP1 and transfected with control or l-PGDS–specific siRNAs (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ) were used to perform cell-surface ELISAs (
      • Lachance V.
      • Degrandmaison J.
      • Marois S.
      • Robitaille M.
      • Génier S.
      • Nadeau S.
      • Angers S.
      • Parent J.-L.
      Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
      ,
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      • Génier S.
      • Degrandmaison J.
      • Moreau P.
      • Labrecque P.
      • Hébert T.E.
      • Parent J.-L.
      Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.
      ,
      • Roy S.J.
      • Glazkova I.
      • Fréchette L.
      • Iorio-Morin C.
      • Binda C.
      • Pétrin D.
      • Trieu P.
      • Robitaille M.
      • Angers S.
      • Hébert T.E.
      • Parent J.-L.
      Novel, gel-free proteomics approach identifies RNF5 and JAMP as modulators of GPCR stability.
      • Lachance V.
      • Cartier A.
      • Génier S.
      • Munger S.
      • Germain P.
      • Labrecque P.
      • Parent J.-L.
      Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.
      ). Time-course analyses showed that the depletion of l-PGDS significantly increases the agonist-induced internalization of DP1 by roughly 50% (Fig. 1A). These results were confirmed with the use of a second l-PGDS siRNA (Fig. 1B). Cell-surface detection assays of DP1 were also conducted using HEK293 cells. Time-course analyses showed that the overexpression of l-PGDS results in a ∼30% decrease in DP1 agonist-induced internalization after 120 min of stimulation (Fig. 1C). Parallel experiments were conducted to verify whether l-PGDS acts in a similar fashion with respect to the β2-adrenergic receptor (β2AR), the prototypical GPCR. Overexpression of l-PGDS had no effect on the agonist-induced internalization of FLAG-tagged β2AR in HEK293 cells (Fig. 1D).
      Figure thumbnail gr1
      Figure 1DP1 recycling is increased by L-PGDS. A and B, HeLa cells stably expressing FLAG-DP1 were transfected with an siRNA targeting l-PGDS (s11446 in A and CDS4/5 in B) or a negative control siRNA. 72 h post-transfection, cells were stimulated with 1 μm PGD2 for the indicated times in A or for 60 min in B. Receptor cell-surface expression was measured by ELISA, and the percentage of receptor internalization was calculated. Cells were harvested as described under “Experimental procedures” to assess protein levels by Western blotting using l-PGDS, Rab4, and GAPDH antibodies. C, HEK293 cells were transfected with pcDNA3-FLAG-DP1 alone or in combination with pcDNA3-l-PGDS-HA. 48 h post-transfection, cells were stimulated with 1 μm PGD2 for the indicated times. Receptor cell-surface expression was measured by ELISA, and the percentage of receptor internalization was calculated. D, HEK293 cells were transfected with pcDNA3-FLAG-β2AR alone or in combination with pcDNA3-l-PGDS-HA. 48 h post-transfection, cells were stimulated with 10 μm isoproterenol for the indicated times. Receptor cell-surface expression was measured by ELISA, and the percentage of receptor internalization was calculated. E, HEK293 cells were co-transfected with pcDNA3-FLAG-DP1 and pcDNA3-l-PGDS or pcDNA3-l-PGDS-W43A/G47A, an l-PGDS mutant with reduced binding to Hsp90. Cells were treated with 1 μm PGD2 for 30 min at 37 °C and then incubated in DMEM for the indicated time periods to prevent further internalization and to allow receptor recycling. Cell-surface expression of the receptor was detected by ELISA, and the percentage of receptor recycling was calculated. F, HEK293 cells were transfected with pcDNA3-FLAG-DP1 alone or in combination with pcDNA3-l-PGDS-HA or pcDNA3-l-PGDS-W43A/G47A. 48 h post-transfection, cells were stimulated with 1 μm PGD2 for 60 min. Receptor cell-surface expression was measured by ELISA, and the percentage of receptor internalization was calculated. Results are means ± S.E. (error bars) or means ± S.D. (error bars) (for B and F) of at least three separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.
      Given that an increase in receptor recycling can decrease the detected percentage of receptor internalization, recycling time-course experiments were conducted to determine whether l-PGDS regulates DP1 recycling. Cells were stimulated with 1 μm PGD2 for 30 min to promote receptor internalization and were then incubated in agonist-free culture medium for various times to allow receptor recycling. l-PGDS increased DP1 recycling 2-fold after 15 min of agonist removal (Fig. 1E). We previously showed that l-PGDS is involved in the assembly of an Hsp90–l-PGDS–DP1 complex that is required for receptor export to the cell surface (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). We thus tested the l-PGDS W43A/G47A mutant, defective in binding to Hsp90 (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ), to determine whether the involvement of l-PGDS in DP1 recycling is due to its Hsp90 co-factor activity. The l-PGDS W43A/G47A mutant promoted DP1 recycling (Fig. 1E) and reduced agonist-induced internalization of DP1 (Fig. 1F) similarly to WT l-PGDS, suggesting that l-PGDS plays a role in the recycling of DP1 that is independent of its association with Hsp90.

      l-PGDS and Rab4 play mutually dependent roles in regulating DP1 recycling

      GPCRs are internalized by vesicles at the plasma membrane, which deliver them to early endosomes. The receptors are then either targeted to degradation pathways or are recycled back to the cell membrane for further activation via Rab4-positive endosomes or Rab11-positive endosomes (
      • Lachance V.
      • Cartier A.
      • Génier S.
      • Munger S.
      • Germain P.
      • Labrecque P.
      • Parent J.-L.
      Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.
      • Dale L.B.
      • Seachrist J.L.
      • Babwah A.V.
      • Ferguson S.S.G.
      Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases.
      ,
      • Hammad M.M.
      • Kuang Y.-Q.
      • Morse A.
      • Dupré D.J.
      Rab1 interacts directly with the β2-adrenergic receptor to regulate receptor anterograde trafficking.
      ,
      • Mulvaney E.P.
      • O'Meara F.
      • Khan A.R.
      • O'Connell D.J.
      • Kinsella B.T.
      Identification of α-helix 4 (α4) of Rab11a as a novel Rab11-binding domain (RBD): interaction of Rab11a with the prostacyclin receptor.
      • Langemeyer L.
      • Nunes Bastos R.
      • Cai Y.
      • Itzen A.
      • Reinisch K.M.
      • Barr F.A.
      Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation.
      ). Our previous work showed that DP1 is recycled via Rab4-positive endosomes but not by the Rab11-dependent recycling pathway (
      • Gallant M.A.
      • Slipetz D.
      • Hamelin E.
      • Rochdi M.D.
      • Talbot S.
      • de Brum-Fernandes A.J.
      • Parent J.-L.
      Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
      ). We thus investigated the role of l-PGDS in the Rab4-dependent recycling of DP1. First, we performed a cell-surface expression assay using HEK293 cells that had been stimulated with PGD2 for 2 h and calculated the percentage of internalized receptors when l-PGDS and Rab4 were co-expressed alone or together (Fig. 2A). The overexpression of l-PGDS decreased the agonist-induced internalization of DP1 by ∼30% compared with the internalization of DP1 alone (Fig. 2A, column 2 versus column 1), similar to what was observed in Fig. 1. The expression of Rab4 decreased DP1 internalization by 27% (Fig. 2A, column 3 versus column 1). Interestingly, the co-expression of l-PGDS and Rab4 resulted in a 52% decrease in DP1 internalization (Fig. 2A, column 4 versus column 1).
      Figure thumbnail gr2
      Figure 2Mutually dependent roles of L-PGDS and Rab4 in regulating DP1 recycling. A, HEK293 cells were transfected with pcDNA3-FLAG-DP1, pcDNA3-HA-Rab4, pcDNA3-l-PGDS-MYC, or a combination of constructs as indicated. 48 h post-transfection, cells were stimulated with 1 μm PGD2 for the indicated times. Receptor cell-surface expression was measured by ELISA, and the percentage of receptor internalization was calculated. B and C, HEK293 cells were transfected with a DsiRNA targeting Rab4 (HSC.RNAI.N004578.12.9 in B and HSC.RNAI.N004578.13.3 in C) or a DsiRNA negative control. 24 h after DsiRNA transfection, cells were transiently transfected with pcDNA3-FLAG-DP1 and pcDNA3-l-PGDS-HA. 48 h after the second transfection, cells were either stimulated with 1 μm PGD2 for 2 h to measure receptor cell-surface expression by ELISA and calculate the percentage of receptor internalization or harvested as described under “Experimental procedures” to assess protein levels by Western blotting using Rab4, HA, and GAPDH antibodies. D, HeLa cells stably expressing FLAG-DP1 were transfected with siRNA s11446 targeting l-PGDS or a siRNA negative control. 24 h after siRNA transfection, cells were transiently transfected with pcDNA3-HA-Rab4WT. 48 h after the second transfection, cells were stimulated with 1 μm PGD2 for 2 h to measure receptor cell-surface expression by ELISA and calculate the percentage of receptor internalization. E, HEK293 cells were transfected with a DsiRNA targeting Rab4 or a DsiRNA negative control. 24 h after DsiRNA transfection, cells were transiently transfected with pcDNA3-FLAG-DP1. 48 h after the second transfection, cells were treated with 1 μm PGD2 for 30 min at 37 °C and then incubated in DMEM for the indicated time periods to prevent further internalization and to allow receptor recycling. Cell-surface expression of the receptor was detected by ELISA, and the percentage of receptor recycling was calculated. The mean of the data obtained in the presence of the DsiRNA control was set to 100%. Results are means ± S.D. (error bars) of at least three separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.
      To determine whether the involvement of l-PGDS in DP1 recycling is Rab4-dependent, we transfected HEK293 cells with control or Rab4-specific DsiRNAs. The cells were stimulated with PGD2 for 2 h prior to cell-surface expression assays. Knockdown of Rab4 expression reduces DP1 recycling, leading to the detection of increased agonist-induced internalization of the receptor. As can be seen in Fig. 2B (column 3 versus column 1), Rab4 depletion increased DP1 agonist-induced internalization by 26% compared with the control. Furthermore, the ability of l-PGDS to decrease DP1 internalization by 31% (Fig. 2B, column 2 versus column 1) in the presence of the control DsiRNA was abrogated by Rab4 depletion (Fig. 2B, column 4 versus column 3). Similar data were obtained with a second Rab4 DsiRNAs (Fig. 2C). These results suggest that Rab4 is required for l-PGDS to play its role in DP1 recycling.
      We then verified whether the effect of Rab4 on DP1 recycling depended on l-PGDS. Using an l-PGDS–specific siRNA, we assessed the functional involvement of endogenous l-PGDS in the internalization of DP1 in HeLa cells stably expressing the FLAG-tagged receptor that had been transfected with Rab4. Depletion of l-PGDS increased the percentage of internalized receptors by 26% compared with the control siRNA (Fig. 2D, column 3 versus column 1), confirming the results shown in Fig. 1A. The promotion of DP1 recycling by the co-expression of Rab4 reduced agonist-induced receptor internalization by 21% in the presence of a control siRNA (Fig. 2D, column 2 versus column 1), which was abrogated by the depletion of l-PGDS (Fig. 2D, column 4 versus column 3). Finally, it can be seen in Fig. 2E that Rab4 depletion reduces DP1 recycling after agonist-induced internalization, confirming the data we obtained before (
      • Gallant M.A.
      • Slipetz D.
      • Hamelin E.
      • Rochdi M.D.
      • Talbot S.
      • de Brum-Fernandes A.J.
      • Parent J.-L.
      Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
      ). Taken together, these results indicate that l-PGDS and Rab4 play mutually dependent roles in the regulation of DP1 recycling.

      Rab4 co-localizes with l-PGDS upon DP1 stimulation

      Our previous confocal microscopy studies showed that DP1 and l-PGDS are present in vesicular structures in the cytoplasm and mainly co-localize in the perinuclear region (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ) and that DP1 displayed strong co-localization with Rab4 following PGD2 stimulation of DP1 (
      • Gallant M.A.
      • Slipetz D.
      • Hamelin E.
      • Rochdi M.D.
      • Talbot S.
      • de Brum-Fernandes A.J.
      • Parent J.-L.
      Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
      ). Confocal microscopy performed in HeLa cells revealed that l-PGDS and Rab4 weakly co-localize in basal conditions as depicted in the top panels of Fig. 3. l-PGDS is mainly localized in vesicular structures “surrounding” the Rab4-positive compartments, reflected by the fluorogram showing clear distinction between the red and green pixels. Stimulation of DP1 with PGD2 induced a redistribution of l-PGDS and enhanced noticeably its co-localization with Rab4 (Fig. 3, bottom panels) as shown by the increase in co-localizing yellow pixels in the insets.
      Figure thumbnail gr3
      Figure 3L-PGDS co-localizes intracellularly with Rab4 upon DP1 stimulation. HeLa cells were transiently transfected with pcDNA3-l-PGDS-MYC and pEGFP-C2-Rab4 for 48 h. The cells were then incubated with vehicle (top) or with 1 μm PGD2 (bottom) for 60 min. The cells were then fixed and prepared for confocal microscopy as indicated under “Experimental procedures.” EGFP-Rab4 was visualized using a 488-nm emission laser line and an EGFP detection filter (green). l-PGDS was labeled using a MYC-specific polyclonal primary antibody and an Alexa Fluor 633-conjugated anti-rabbit IgG secondary antibody. l-PGDS was visualized using a 633-nm emission laser line and an Alexa Fluor 633 detection filter (red). Overlays of the staining patterns of the green fluorescent EGFP-Rab4 and the red-labeled l-PGDS (merge) and the corresponding fluorograms are presented. The areas with a high degree of co-localization appear yellow. All laser intensities and acquisition parameters were conserved among the different conditions to allow comparison. The images shown are single confocal slices and are representative of ∼200 observed cells over three independent experiments. Bars, 10 μm.

      l-PGDS promotes the interaction between Rab4 and DP1

      We and others have shown that trafficking of GPCRs can be mediated by their interactions with Rabs (
      • Esseltine J.L.
      • Dale L.B.
      • Ferguson S.S.G.
      Rab GTPases bind at a common site within the angiotensin II type I receptor carboxyl-terminal tail: evidence that Rab4 regulates receptor phosphorylation, desensitization, and resensitization.
      ,
      • Hamelin E.
      • Thériault C.
      • Laroche G.
      • Parent J.-L.
      The intracellular trafficking of the G protein-coupled receptor TPβ depends on a direct interaction with Rab11.
      • Parent A.
      • Hamelin E.
      • Germain P.
      • Parent J.-L.
      Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction.
      ,
      • Seachrist J.L.
      • Ferguson S.S.G.
      Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases.
      ,
      • Dong C.
      • Yang L.
      • Zhang X.
      • Gu H.
      • Lam M.L.
      • Claycomb W.C.
      • Xia H.
      • Wu G.
      Rab8 interacts with distinct motifs in α2B- and β2-adrenergic receptors and differentially modulates their transport.
      • Li C.
      • Wei Z.
      • Fan Y.
      • Huang W.
      • Su Y.
      • Li H.
      • Dong Z.
      • Fukuda M.
      • Khater M.
      • Wu G.
      The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.
      ,
      • Lachance V.
      • Cartier A.
      • Génier S.
      • Munger S.
      • Germain P.
      • Labrecque P.
      • Parent J.-L.
      Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.
      • Dale L.B.
      • Seachrist J.L.
      • Babwah A.V.
      • Ferguson S.S.G.
      Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases.
      ,
      • Hammad M.M.
      • Kuang Y.-Q.
      • Morse A.
      • Dupré D.J.
      Rab1 interacts directly with the β2-adrenergic receptor to regulate receptor anterograde trafficking.
      • Mulvaney E.P.
      • O'Meara F.
      • Khan A.R.
      • O'Connell D.J.
      • Kinsella B.T.
      Identification of α-helix 4 (α4) of Rab11a as a novel Rab11-binding domain (RBD): interaction of Rab11a with the prostacyclin receptor.
      ). To investigate the interaction between DP1, l-PGDS, and Rab4 in a cellular context, we performed immunoprecipitation assays on lysates of HEK293 cells expressing FLAG-DP1, l-PGDS-MYC, or HA-Rab4 with a FLAG-specific mAb (Fig. 4A). The co-immunoprecipitation of Rab4 with DP1 was detected in both the absence and presence of l-PGDS co-expression and was promoted by PGD2 stimulation over time. Of note, the co-expression of l-PGDS increased the DP1-Rab4 interaction by 75 and 65% after 60 and 120 min of stimulation, respectively (Fig. 4A, top panel and densitometry graph). As we reported previously (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ), the DP1–l-PGDS interaction was not modulated by agonist stimulation (Fig. 4A, second panel from top).
      Figure thumbnail gr4
      Figure 4The Rab4-DP1 interaction is promoted by L-PGDS. A, HEK293 cells transiently transfected with pcDNA3-FLAG-DP1, pcDNA3-HA-Rab4, pcDNA3-l-PGDS-MYC, or a combination of constructs were stimulated for the indicated times with 1 μm PGD2. Immunoprecipitation (IP) of the receptor was performed using a FLAG-specific mAb, and immunoblotting (IB) was performed with FLAG-specific polyclonal, peroxidase-conjugated anti-HA or anti-MYC antibodies. The graph shows densitometry analyses performed on four different experiments. Rab4 pixels were normalized on DP1 pixels, and results are presented as the ratio of these values (means ± S.E. (error bars)). B, binding assays were carried out using purified GSH-Sepharose–bound GST-DP1-CT and intracellular loops (ICL) incubated with His6-Rab4. Rab4 binding to the receptor domains was detected by immunoblotting using an anti-His antibody, and the GST fusion proteins present in the binding reaction were detected using an anti-GST antibody. C, binding assays were carried out using purified GSH-Sepharose–bound GST-DP1-CT incubated with His6-l-PGDS and a cellular lysate of cells transfected with pcDNA3-HA-Rab4. Rab4 binding to the receptor domains was detected by immunoblotting using an anti-HA antibody, l-PGDS was detected using an anti-His antibody, and the GST fusion proteins were detected using an anti-GST antibody. Blots shown are representative of four independent experiments. ****, p < 0.0001.
      We then determined whether the DP1-Rab4 interaction could be direct. We performed in vitro binding assays using purified DP1 intracellular domains fused to GSH-S-transferase (GST) and purified Rab4 fused to a hexahistidine tag (His6-Rab4). As indicated by the results presented in Fig. 4B, Rab4 mainly interacts directly with the C terminus of DP1 and, more weakly, with the first intracellular loop (ICL1), but not with ICL2 or ICL3. We then investigated the possibility that l-PGDS modulates this interaction because our previous work revealed that l-PGDS can also interact directly with the DP1 C terminus (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). We used the purified GST-DP1-C terminus construct to conduct in vitro binding assays using cell lysates of HEK293 cells expressing HA-Rab4 in the presence or absence of purified His6-l-PGDS. Fig. 4C shows that the interaction between Rab4 and the DP1-C terminus is augmented in the presence of l-PGDS. Taken together, these results indicate that the DP1-Rab4 interaction can be direct and is increased by the agonist stimulation of DP1 and by the presence of l-PGDS.

      l-PGDS interacts directly with Rab4

      Given the involvement of l-PGDS in the DP1-Rab4 interaction, we performed immunoprecipitation assays using lysates from HEK293 cells expressing l-PGDS-MYC, HA-Rab4, or FLAG-DP1 and a MYC-specific mAb to determine whether l-PGDS interacts with Rab4. The co-immunoprecipitation of Rab4 was detected by Western blotting using an HA antibody. Interestingly, the interaction between Rab4 and l-PGDS was strongly increased over time when DP1 was stimulated with PGD2 (Fig. 5A), in agreement with the l-PGDS–Rab4 co-localization data from Fig. 3. The l-PGDS–Rab4 interaction was confirmed at native level, where endogenous Rab4 co-immunoprecipitated following the immunoprecipitation of endogenous l-PGDS from HeLa cells (Fig. 5B), which produce PGD2 and express DP1 intrinsically (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). We also performed in vitro binding assays using purified l-PGDS fused to GST together with purified His6-Rab4. Fig. 5C reveals that Rab4 bound to GST-l-PGDS but not to GST, showing that l-PGDS can interact directly with Rab4.
      Figure thumbnail gr5
      Figure 5L-PGDS interacts directly with Rab4. A, HEK293 cells transiently transfected with pcDNA3-FLAG-DP1, pcDNA3-HA-Rab4, and pcDNA3-l-PGDS-MYC were stimulated for the indicated times with 1 μm PGD2. Immunoprecipitation (IP) of l-PGDS was performed using a MYC-specific mAb, and immunoblotting (IB) was performed with a peroxidase-conjugated anti-MYC or anti-HA or a FLAG-specific polyclonal antibody. B, immunoprecipitation was performed in HeLa cells using l-PGDS–specific monoclonal or rat isotypic control IgG antibodies, and immunoblotting was done using l-PGDS–specific polyclonal or Rab4-specific polyclonal antibodies. C, binding assays were carried out using purified GSH-Sepharose–bound GST-l-PGDS incubated with His6-Rab4. The binding of Rab4 to l-PGDS was detected by immunoblotting using an anti-His antibody, and GST-l-PGDS was detected using an anti-GST antibody. D, binding assays were carried out using purified GSH-Sepharose-bound GST-l-PGDS incubated with cellular lysates of HEK293 cells transfected with pcDNA3-HA-Rab4WT, pcDNA3-HA-Rab4S22N, or pcDNA3-HA-Rab4Q67L. Rab4 binding to l-PGDS was detected by immunoblotting using an anti-HA antibody. l-PGDS was detected using an anti-GST antibody. E, HEK293 cells were transiently transfected with pcDNA3-l-PGDS-MYC and the indicated pcDNA3-HA-Rab constructs. Immunoprecipitation of l-PGDS was performed using a MYC-specific mAb, and immunoblotting was performed with a peroxidase-conjugated anti-MYC or anti-HA. Graphs show densitometry analyses performed on three different experiments. Rab4 pixels were normalized on l-PGDS pixels, and results are presented as -fold of these values (means ± S.D. (error bars)). ***, p < 0.001.
      Because Rab4 is a GTPase that cycles between inactive GDP-bound and active GTP-bound forms, we were interested in determining whether l-PGDS interacts preferentially with one of the two forms. We performed in vitro GST-l-PGDS pulldown assays using lysates of HEK293 cells expressing HA-Rab4WT, HA-Rab4S22N (GDP-locked, inactive mutant), or HA-Rab4Q67L (GTPase-deficient, constitutively active mutant). The pulldown of Rab4 was detected by Western blotting using an HA antibody (Fig. 5D). Interestingly, l-PGDS displays a strong preference for binding to Rab4S22N over WT Rab4 or Rab4Q67L. Following this result, we questioned whether l-PGDS could interact with GDP-locked mutants of other Rabs. We performed immunoprecipitation assays using a MYC-specific mAb on lysates from HEK293 cells expressing l-PGDS–MYC and various HA-Rabs (Rab4S22N, Rab5S34N, Rab11S25N, Rab1S25N, Rab6T27N, and Rab8T22N). Fig. 5E shows that, among the Rabs that were tested, l-PGDS interacts only with the GDP-locked form of Rab4. These results suggest that the interaction between Rab4 and l-PGDS can be modulated by the agonist stimulation of DP1 and can occur endogenously by a direct protein-protein interaction, preferentially with the GDP-bound form of Rab4.

      l-PGDS increases the levels of activated Rab4

      We next studied whether DP1 activates Rab4 and if l-PGDS takes part in this mechanism. Rabaptin is an effector of Rab4 that interacts with the GTP-bound form of Rab4, which can be used in pulldown experiments to detect Rab4 activation (
      • Langemeyer L.
      • Nunes Bastos R.
      • Cai Y.
      • Itzen A.
      • Reinisch K.M.
      • Barr F.A.
      Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation.
      ,
      • Kanie T.
      • Jackson P.K.
      Guanine nucleotide exchange assay using fluorescent MANT-GDP.
      ). We performed in vitro binding assays using purified GST-rabaptin and lysates of HEK293 cells stably expressing DP1 that also expressed HA-Rab4WT alone or in combination with l-PGDS–MYC following a time course of PGD2 stimulation. Fig. 6A shows that Rab4 was weakly activated after 0 and 15 min of DP1 stimulation (lanes 1 and 2), followed by a gradual increase in Rab4 activation after 30 min of PGD2 treatment (lanes 3–5). A densitometric analysis (Fig. 6A, bottom panel) indicated that there is a marked increase in Rab4 activation at the basal level and following stimulation of DP1 with PGD2 when l-PGDS is co-expressed (Fig. 6A, lanes 6–10). l-PGDS does not interact with rabaptin (Fig. 6A, second panel, IB: MYC). We then verified whether depletion of endogenous l-PGDS reduces Rab4 activation. GST-rabaptin pulldown assays were performed on lysates of HeLa cells stably expressing DP1 that were transfected with the indicated combinations of HA-Rab4WT, control, or l-PGDS–specific siRNAs (Fig. 6B). There was basal activation of Rab4 (Fig. 6B, lane 1) that was strongly increased after 60 and 120 min of DP1 stimulation (lanes 2 and 3) when cells were treated with the control siRNA. On the other hand, basal activation of Rab4 was abrogated (Fig. 6B, lane 4), and PGD2-induced Rab4 activation was greatly reduced (Fig. 6B, lanes 5 and 6) when l-PGDS was depleted. We then carried out in vitro GTPγS-loading experiments with purified His6-Rab4 in the absence or presence of purified l-PGDS for times ranging from 0 to 15 min. GTP loading of Rab4 was detected as described above by GST-rabaptin pulldown and Western blotting analyses. Interestingly, l-PGDS increased the GTPγS loading of Rab4 (Fig. 6C), as indicated by enhanced Rab4 binding to rabaptin.
      Figure thumbnail gr6
      Figure 6Activation of Rab4 is promoted by L-PGDS. A, HEK293 cells stably expressing pcDNA3-FLAG-DP1 were transiently transfected with pcDNA3-HA-Rab4, pcDNA3-l-PGDS-MYC, or a combination of constructs and stimulated for the indicated times with 1 μm PGD2 48 h post-transfection. Binding assays were carried out using purified GSH-Sepharose–bound GST-rabaptin incubated with these cellular lysates. Rab4 and l-PGDS binding to rabaptin were detected by immunoblotting (IB) using a peroxidase-conjugated anti-HA or anti-MYC antibody, respectively. GST-rabaptin was detected using an anti-GST antibody. B, HeLa cells stably expressing FLAG-DP1 were transfected with siRNA s11446 targeting l-PGDS or a negative control siRNA. 72 h post-transfection, cells were stimulated with 1 μm PGD2 for the indicated times. Binding assays were carried out using purified GSH-Sepharose–bound GST-rabaptin incubated with these cellular lysates, and binding of active Rab4 was detected by immunoblotting using a peroxidase-conjugated anti-HA antibody. GST-rabaptin was detected using an anti-GST antibody. C, GTPγS loading of Rab4 was performed as described under “Experimental procedures” for the indicated periods of time. Binding assays were carried out using purified GSH-Sepharose–bound GST-rabaptin incubated with His6-Rab4, His6-l-PGDS, or a combination of both. The binding of active Rab4 and l-PGDS was detected by immunoblotting using an anti-Rab4 or anti-l-PGDS antibody, and GST-rabaptin was detected using an anti-GST antibody. Graphs show densitometry analyses performed on at least three different experiments. Rab4 pixels were normalized on rabaptin pixels (means ± S.E. (error bars) in A and C, means ± S.D. in B). **, p < 0.01; ****, p < 0.0001.
      To further study whether l-PGDS can participate in Rab4 activation, we used purified l-PGDS and Rab4 proteins and performed GDP-GTP exchange experiments. The classic assay using Mant-GDP (
      • Langemeyer L.
      • Nunes Bastos R.
      • Cai Y.
      • Itzen A.
      • Reinisch K.M.
      • Barr F.A.
      Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation.
      ,
      • Kanie T.
      • Jackson P.K.
      Guanine nucleotide exchange assay using fluorescent MANT-GDP.
      ) could not be used, as l-PGDS strongly binds Mant-GDP nonspecifically. This could be due to the Mant moiety, because excess of GDP could not compete with Mant-GDP binding on l-PGDS (data not shown). Therefore, we turned to a Transcreener GDP time-resolved (TR)-FRET assay. The latter bases its principle on the displacement of a GDP HiLyte647 tracer initially bound to a GDP antibody-terbium conjugate by the GDP released by a small GTPase (Fig. 7A). GDP-GTP exchange on the small GTPase leads to the release of GDP in the reaction that displaces the tracer, resulting in a decrease in the TR-FRET signal. Fig. 7B shows that the addition of increasing concentrations of purified l-PGDS to GDP-loaded Rab4 causes a dose-dependent decrease in TR-FRET signals, consistent with the idea that l-PGDS promotes Rab4 activation and GDP release. Time-course assays revealed that the half-time of the GDP-GTP exchange reaction is accelerated in the presence of l-PGDS (t½ = 42.8 min) compared with Rab4 alone (t½ = 64.4 min), further suggesting that there is increased Rab4 activation in the presence of l-PGDS (Fig. 7C). Together, these results indicate that l-PGDS can partake in Rab4 activation, but further experiments will be needed to determine the nature of the mechanism involved.
      Figure thumbnail gr7
      Figure 7L-PGDS increases nucleotide exchange on Rab4. A, schematic representation of the TR-FRET reaction. GDP released by the enzyme reaction displaces a GDP HiLyte647 tracer initially bound to a GDP antibody conjugated to terbium (Tb), thus generating a decrease in TR-FRET signal. B, titration of l-PGDS ranging from 1 nm to 10 μm was performed in the presence of a fixed Rab4 concentration of 80 nm as described under “Experimental procedures.” C, time-course assays were carried out for the indicated periods of time. Final concentrations of 80 nm Rab4 and 320 nm l-PGDS were used, and enzyme reactions were performed and stopped, and TR-FRET signal was measured as described under “Experimental Procedures.” Graphs are represented as TR-FRET ratios (665/615), and results are means ± S.E. (error bars) of three separate experiments. Data were analyzed with GraphPad Prism using a four-parameter nonlinear regression curve fitting.

      Identification of the Rab4-binding domain on l-PGDS

      Our next aim was to identify the l-PGDS domain involved in the interaction with Rab4. To this end, we produced several l-PGDS deletion mutant constructs that were fused to GST (Fig. 8A) and used them in in vitro pulldown assays with purified His6-Rab4. The binding reactions were analyzed by immunoblotting using an anti-His mAb to detect the binding of Rab4. As summarized in Fig. 8A, there were no differences among the first five mutants in terms of binding to Rab4 compared with the full-length l-PGDS protein (blots not shown). We then investigated the l-PGDS domain comprising residues 75–98 (Fig. 8B). The l-PGDS structure revealed that the 75–98 and 85–92 amino acid sequences are part of two β strands folded to form an antiparallel β loop protruding from the core of the structure. Because these strands appeared to be accessible for protein interactions, it seemed plausible that they could serve as a Rab4-binding site. Interestingly, our results showed that Rab4 does not interact with the l-PGDS Δ75–98 and Δ85–92 deletion mutants (Fig. 8C).
      Figure thumbnail gr8
      Figure 8Identification of the L-PGDS region that interacts with Rab4. A, schematic representation of the different GST-tagged l-PGDS mutants. The Rab4-binding properties of the l-PGDS constructs are indicated on the right. B, the illustration of the complete l-PGDS structure (shown in green) was prepared with PyMOL (Schrödinger, LLC, New York) using the known crystal structure of l-PGDS (Protein Data Bank entry 2WWP). The two potential Rab4-binding sites are shown in yellow (residues 75–98) and blue (residues 85–92), respectively. C, binding assays were carried out using purified GSH-Sepharose–bound GST-l-PGDS WT or its mutants incubated with His6-Rab4. The binding of Rab4 to l-PGDS was detected by immunoblotting (IB) using an anti-His antibody, and GST-l-PGDS was detected using an anti-GST antibody. D, binding assays were carried out using purified GSH-Sepharose–bound GST-l-PGDS WT or GST-l-PGDS 75–98 incubated with His6-Rab4. The binding of Rab4 to l-PGDS was detected by immunoblotting using an anti-His antibody, and GST-l-PGDS was detected using an anti-GST antibody. The graph shows densitometry analyses performed on three different experiments. Rab4 pixels were normalized on l-PGDS pixels (means ± S.D. (error bars)).
      To further corroborate these results, we produced a construct consisting of amino acids 75–98 of l-PGDS fused with GST. In vitro binding assays were carried out using the GST-tagged construct and purified His6-Rab4. As can be seen in Fig. 8D, Rab4 bound to the GST-l-PGDS 75–98 construct but not to GST alone. Moreover, a peptide corresponding to amino acids 78–98 of l-PGDS was synthesized together with its scrambled control peptide. The 75GGK77 amino acids were not included in the peptide because of solubility issues. The peptides were preincubated individually with purified His6-Rab4 prior to performing GST-l-PGDS pulldown assays as described above to determine whether they would compete in the l-PGDS–Rab4 interaction. Remarkably, the l-PGDS 78–98 peptide completely abrogated the binding of Rab4 to l-PGDS, whereas the scrambled peptide had no significant effect (Fig. 9A).
      Figure thumbnail gr9
      Figure 9The interaction with Rab4 is necessary for L-PGDS to regulate DP1 recycling. A, peptides were incubated for 1 h with His6-Rab4 prior to binding assays. Assays were carried out using purified GSH-Sepharose–bound GST-l-PGDS WT incubated with peptides and His6-Rab4. The binding of Rab4 to l-PGDS was detected by immunoblotting using an anti-His antibody, and GST-l-PGDS was detected using an anti-GST antibody. The graph shows densitometry analyses performed on three different experiments. Rab4 pixels were normalized on l-PGDS pixels, and results are presented as -fold of these values (means ± S.D. (error bars)). ***, p < 0.001; ****, p < 0.0001; ns, not significant. B, HEK293 cells were co-transfected with pcDNA3-FLAG-DP1 and pcDNA3-l-PGDS, pcDNA3-l-PGDS Δ75–98, or pcDNA3-l-PGDS Δ85–92. Cells were treated with 1 μm PGD2 for 30 min at 37 °C and then incubated in DMEM for the indicated time periods to prevent further internalization and to allow receptor recycling. Cell-surface expression of the receptor was detected by ELISA, and the percentage of receptor recycling was calculated. Results are means ± S.E. (error bars) of three separate experiments. ***, p < 0.001, ****, p < 0.0001.
      Finally, we tested the ability of the Rab4 binding–deficient l-PGDS Δ75–98 and Δ85–92 deletion mutants to promote DP1 recycling. Unlike the WT l-PGDS, the l-PGDS Δ75–98 and Δ85–92 deletion mutants failed to enhance DP1 recycling after agonist-induced internalization in HEK293 cells (Fig. 9B). Altogether, our results indicate that amino acids 78–98 of l-PGDS are involved in Rab4 binding and that the l-PGDS–Rab4 interaction is required for l-PGDS to participate in the recycling of DP1.

      Discussion

      To maintain the sensitivity of cells to their environment, it is crucial for receptors to be able to recycle back to the cell surface. We and others have shown that the spatial and temporal vesicular transport of many GPCRs is regulated by direct interactions with various members of the Rab subfamily of small GTPases (
      • Hamelin E.
      • Thériault C.
      • Laroche G.
      • Parent J.-L.
      The intracellular trafficking of the G protein-coupled receptor TPβ depends on a direct interaction with Rab11.
      ,
      • Parent A.
      • Hamelin E.
      • Germain P.
      • Parent J.-L.
      Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction.
      • Dateyama I.
      • Sugihara Y.
      • Chiba S.
      • Ota R.
      • Nakagawa R.
      • Kobayashi T.
      • Itoh H.
      RABL2 positively controls localization of GPCRs in mammalian primary cilia.
      ,
      • Dong C.
      • Yang L.
      • Zhang X.
      • Gu H.
      • Lam M.L.
      • Claycomb W.C.
      • Xia H.
      • Wu G.
      Rab8 interacts with distinct motifs in α2B- and β2-adrenergic receptors and differentially modulates their transport.
      ,
      • Li C.
      • Wei Z.
      • Fan Y.
      • Huang W.
      • Su Y.
      • Li H.
      • Dong Z.
      • Fukuda M.
      • Khater M.
      • Wu G.
      The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.
      ,
      • Esseltine J.L.
      • Ribeiro F.M.
      • Ferguson S.S.G.
      Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.
      • O'Keeffe M.B.
      • Reid H.M.
      • Kinsella B.T.
      Agonist-dependent internalization and trafficking of the human prostacyclin receptor: a direct role for Rab5a GTPase.
      ,
      • Seachrist J.L.
      • Anborgh P.H.
      • Ferguson S.S.
      β2-Adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by Rab GTPases.
      ,
      • Smythe E.
      Direct interactions between rab GTPases and cargo.
      • Wikström K.
      • Reid H.M.
      • Hill M.
      • English K.A.
      • O'Keeffe M.B.
      • Kimbembe C.C.
      • Kinsella B.T.
      Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase.
      ). The central aspect of Rab GTPase function is their specific localization in distinct subcellular compartments, which makes it possible to precisely control trafficking (
      • Seabra M.C.
      • Wasmeier C.
      Controlling the location and activation of Rab GTPases.
      ). The mechanisms by which Rabs are recruited to GPCRs in particular membrane compartments are poorly understood.
      Like other small GTPases, the spatiotemporal activation and inactivation of Rab GTPases are tightly regulated by GEFs and GAPs (
      • Blümer J.
      • Rey J.
      • Dehmelt L.
      • Mazel T.
      • Wu Y.-W.
      • Bastiaens P.
      • Goody R.S.
      • Itzen A.
      RabGEFs are a major determinant for specific Rab membrane targeting.
      ,
      • Barr F.
      • Lambright D.G.
      Rab GEFs and GAPs.
      ). Rab4 is a small GTPase that is critical for the recycling of many key cargo proteins. Surprisingly, there is no identified GEF for Rab4, and how it is activated and recruited to cargo proteins is still an open question. In addition to being involved in normal cell physiology, Rab4 may be associated with disease. For example, Rab4 expression is elevated in Alzheimer's disease (
      • Ginsberg S.D.
      • Mufson E.J.
      • Alldred M.J.
      • Counts S.E.
      • Wuu J.
      • Nixon R.A.
      • Che S.
      Upregulation of select rab GTPases in cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease.
      ,
      • Soejima N.
      • Ohyagi Y.
      • Nakamura N.
      • Himeno E.
      • Iinuma K.M.
      • Sakae N.
      • Yamasaki R.
      • Tabira T.
      • Murakami K.
      • Irie K.
      • Kinoshita N.
      • LaFerla F.M.
      • Kiyohara Y.
      • Iwaki T.
      • Kira J.
      Intracellular accumulation of toxic turn amyloid-β is associated with endoplasmic reticulum stress in Alzheimer's disease.
      ). It has been proposed that Rab4 could be a target for the treatment of systemic lupus erythematosus (
      • Caza T.N.
      • Fernandez D.R.
      • Talaber G.
      • Oaks Z.
      • Haas M.
      • Madaio M.P.
      • Lai Z.-W.
      • Miklossy G.
      • Singh R.R.
      • Chudakov D.M.
      • Malorni W.
      • Middleton F.
      • Banki K.
      • Perl A.
      HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE.
      ). Accumulating evidence also indicates that Rab4 is involved in tumor growth and metastasis by regulating plasma membrane levels of receptor tyrosine kinases (
      • Hu C.-T.
      • Wu J.-R.
      • Wu W.-S.
      The role of endosomal signaling triggered by metastatic growth factors in tumor progression.
      ), integrins (
      • Do M.T.
      • Chai T.F.
      • Casey P.J.
      • Wang M.
      Isoprenylcysteine carboxylmethyltransferase function is essential for RAB4A-mediated integrin β3 recycling, cell migration and cancer metastasis.
      ), P-glycoprotein in multidrug resistance (
      • Ferrándiz-Huertas C.
      • Fernández-Carvajal A.
      • Ferrer-Montiel A.
      Rab4 interacts with the human P-glycoprotein and modulates its surface expression in multidrug resistant K562 cells.
      ), and proteases (
      • Arsenault D.
      • Lucien F.
      • Dubois C.M.
      Hypoxia enhances cancer cell invasion through relocalization of the proprotein convertase furin from the trans-Golgi network to the cell surface.
      ,
      • Frittoli E.
      • Palamidessi A.
      • Marighetti P.
      • Confalonieri S.
      • Bianchi F.
      • Malinverno C.
      • Mazzarol G.
      • Viale G.
      • Martin-Padura I.
      • Garré M.
      • Parazzoli D.
      • Mattei V.
      • Cortellino S.
      • Bertalot G.
      • Di Fiore P.P.
      • Scita G.
      A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination.
      ). It is thus essential to identify Rab4-interacting proteins to better understand the biology of this important small GTPase.
      Additional regulatory mechanisms were reported to be involved in the regulation of Rab GTPases activity. For example, Rab4 is regulated by protein kinase A following activation of the β2AR (
      • Yudowski G.A.
      • Puthenveedu M.A.
      • Henry A.G.
      • von Zastrow M.
      Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.
      ). We described previously (
      • Lachance V.
      • Degrandmaison J.
      • Marois S.
      • Robitaille M.
      • Génier S.
      • Nadeau S.
      • Angers S.
      • Parent J.-L.
      Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
      ) how a complex between the β2AR and the ubiquitin ligase HACE1 results in Rab11a ubiquitination on Lys-145. This ubiquitination is involved in the activation of Rab11a and in the regulation of β2AR recycling to the plasma membrane (
      • Lachance V.
      • Degrandmaison J.
      • Marois S.
      • Robitaille M.
      • Génier S.
      • Nadeau S.
      • Angers S.
      • Parent J.-L.
      Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
      ). Research efforts are also targeted at understanding whether GPCRs interact with other components of the Rab-associated machinery. In this regard, we observed that the interaction between the β2AR and Rab geranylgeranyltransferase modulates the trafficking of the receptor and the geranylgeranylation of Rab6a, Rab8a, and Rab11a (
      • Lachance V.
      • Cartier A.
      • Génier S.
      • Munger S.
      • Germain P.
      • Labrecque P.
      • Parent J.-L.
      Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.
      ).
      Our earlier work showed that DP1 recycles to the cell surface through Rab4 after agonist-induced internalization (
      • Gallant M.A.
      • Slipetz D.
      • Hamelin E.
      • Rochdi M.D.
      • Talbot S.
      • de Brum-Fernandes A.J.
      • Parent J.-L.
      Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
      ). In a separate study, we demonstrated that l-PGDS participates in the anterograde transport of DP1 through an interaction with the receptor and the Hsp90 chaperone. Furthermore, l-PGDS promoted the formation of a DP1-ERK1/2 complex and increased DP1-mediated ERK1/2 signaling. Interestingly, DP1 augmented PGD2 synthesis by l-PGDS, revealing an intracrine signaling loop between DP1 and l-PGDS (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). l-PGDS thus appears as a multifunctional protein capable not only of PGD2 synthesis and transport of lipophilic molecules, but also of regulating protein complex formation involved in DP1 trafficking and signaling. We were thus interested in studying whether l-PGDS is involved in recycling of the DP1 receptor and if this would involve Rab4.
      Experiments using overexpression or depletion of endogenous l-PGDS revealed that it regulates DP1 recycling after agonist-induced internalization. Inhibiting endogenous Rab4 expression inhibited the promotion of DP1 recycling by l-PGDS, whereas conversely, knocking down endogenous l-PGDS prevented the Rab4-mediated recycling of DP1. l-PGDS and Rab4 thus appear to work in conjunction with each other in regulating DP1 recycling.
      Agonist-induced internalization of DP1 is rather slow compared with other GPCRs. To detect an effect of recycling on receptor internalization after the addition of the agonist, the receptor has to first internalize and then recycle. This may explain why the l-PGDS–mediated recycling effect on DP1 internalization can be observed starting after 30 min of agonist treatment and becomes statistically significant after 90 min of DP1 stimulation. On the other hand, the effect of l-PGDS appears faster in the recycling assays (apparent after 15 min of agonist removal). This may be because in the latter context, the receptor was stimulated for 30 min prior to the recycling measurements, so the system was already “turned on” for 30 min when the recycling measurements began. Furthermore, the agonist is removed in the recycling assays, so the effect of l-PGDS on recycling does not compete with concomitant DP1 internalization, facilitating its detection more rapidly. We cannot totally exclude the possibility that the role of l-PGDS in anterograde transport (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ) is involved, at least in part, in regulating replenishment of DP1 to the plasma membrane after agonist-induced internalization. However, the l-PGDS W43A/G47A mutant, defective in the interaction with Hsp90 necessary for l-PGDS to promote anterograde transport (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ), had effects similar to those of WT l-PGDS on agonist-induced internalization and recycling of DP1. In contrast, the l-PGDS W43A/G47A mutant had a 70% reduced capacity to promote DP1 anterograde transport compared with WT l-PGDS (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). Altogether, our data support the idea that l-PGDS promotes recycling of DP1 and that its interaction with Hsp90 as well as its role in anterograde trafficking do not play a significant role, if any, in the recycling of DP1.
      How Rab GTPases are recruited to particular cargo proteins is poorly characterized. We and others have shown that a number of Rabs interact directly with the C-terminal ends of GPCRs (
      • Esseltine J.L.
      • Dale L.B.
      • Ferguson S.S.G.
      Rab GTPases bind at a common site within the angiotensin II type I receptor carboxyl-terminal tail: evidence that Rab4 regulates receptor phosphorylation, desensitization, and resensitization.
      • Hamelin E.
      • Thériault C.
      • Laroche G.
      • Parent J.-L.
      The intracellular trafficking of the G protein-coupled receptor TPβ depends on a direct interaction with Rab11.
      ,
      • Parent A.
      • Hamelin E.
      • Germain P.
      • Parent J.-L.
      Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction.
      • Dateyama I.
      • Sugihara Y.
      • Chiba S.
      • Ota R.
      • Nakagawa R.
      • Kobayashi T.
      • Itoh H.
      RABL2 positively controls localization of GPCRs in mammalian primary cilia.
      ,
      • Seachrist J.L.
      • Anborgh P.H.
      • Ferguson S.S.
      β2-Adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by Rab GTPases.
      ). The C terminus of DP1 was identified as the interaction site with Rab4, and Rab4 binding was enhanced by agonist stimulation of the receptor and by the presence of l-PGDS. The l-PGDS–Rab4 co-localization and interaction was also promoted by DP1 activation. We confirmed that Rab4 directly binds to l-PGDS using in vitro binding assays with purified recombinant proteins. Our data showed that l-PGDS did not bind to the other Rabs tested (Rab1, -5, -8, and -11), suggesting that l-PGDS displays at least a certain degree of specificity toward Rab4. Further experiments will be needed to determine the full extent of the l-PGDS interaction spectrum within the family of Rab GTPases. l-PGDS did not modulate the internalization of the β2AR, indicating that it does not play a general role in GPCR trafficking.
      Like all members of the Ras superfamily, Rab4 cycles between a GDP-bound and a GTP-bound form. Whereas the GDP-bound form is considered inactive, the GTP-bound form binds to effectors, switching on downstream cellular responses (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ). Interestingly, our binding assays revealed that l-PGDS preferentially interacts with the GDP-bound form of Rab4. Moreover, l-PGDS increased the levels of activated Rab4 in cells following DP1 stimulation, as well as Rab4 GTPγS loading in vitro using purified proteins, as reflected by binding to its effector rabaptin. This indicated that l-PGDS may be involved in regulating the activation of Rab4. This was further supported by the stimulation of Rab4 GDP-GTP exchange by l-PGDS in vitro using a TR-FRET assay. In a nutshell, we observed that: 1) l-PGDS associates with Rab4 in a time-dependent manner after DP1 stimulation; 2) l-PGDS only binds significantly to the GDP-bound inactive state of Rab4 but is constitutively associated with DP1; 3) l-PGDS favors Rab4 recruitment and activation by DP1; and 4) l-PGDS is not detected in the rabaptin pulldowns. Because the rabaptin pulldowns would only capture active GTP-bound Rab4, this would be consistent with a model in which l-PGDS binds inactive Rab4 and recruits it to the receptor, participates in its activation, and then dissociates. However, additional work will be necessary to characterize the nature of the mechanism involved in the increased levels of activated Rab4 in the presence of l-PGDS.
      Deletion mutagenesis studies led to the identification of two constructs (the l-PGDS Δ75–98 and Δ85–92 mutants) that could no longer bind Rab4. These l-PGDS mutants failed to regulate DP1 recycling, indicating that the l-PGDS–Rab4 interaction is required for this function. The l-PGDS–Rab4–binding domain was further confirmed by the ability of the l-PGDS 75–98 amino acid sequence fused to GST to interact with Rab4 and by the in vitro inhibition of the l-PGDS-Rab4 interaction by the l-PGDS 78–98 synthetic peptide.
      We cannot state at the moment whether l-PGDS acts, for example, as a GEF, a holdase, or other conformational change–inducing binding partner on Rab4. l-PGDS does not display a Vps9 domain, a DENN domain, a multisubunit TRAPP complex, or a Sec2 domain that can be found in Rab GEFs (
      • Cherfils J.
      • Zeghouf M.
      Regulation of small GTPases by GEFs, GAPs, and GDIs.
      ,
      • Ishida M.
      • Oguchi M.E.
      • Fukuda M.
      Multiple types of guanine nucleotide exchange factors (GEFs) for Rab small GTPases.
      ,
      • Müller M.P.
      • Goody R.S.
      Molecular control of Rab activity by GEFs, GAPs and GDI.
      ,
      • Yoshimura S.
      • Gerondopoulos A.
      • Linford A.
      • Rigden D.J.
      • Barr F.A.
      Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.
      ,
      • Barr F.
      • Lambright D.G.
      Rab GEFs and GAPs.
      ,
      • Delprato A.
      • Lambright D.G.
      Structural basis for Rab GTPase activation by VPS9 domain exchange factors.
      ,
      • Marat A.L.
      • Dokainish H.
      • McPherson P.S.
      DENN domain proteins: regulators of Rab GTPases.
      • Zhang X.
      • He X.
      • Fu X.-Y.
      • Chang Z.
      Varp is a Rab21 guanine nucleotide exchange factor and regulates endosome dynamics.
      ). Detection of GDP-GTP exchange or GTP-loading promotion by a protein is not necessarily indicative of a GEF protein per se. Indeed, as elegantly demonstrated by Gulbranson et al. (
      • Gulbranson D.R.
      • Davis E.M.
      • Demmitt B.A.
      • Ouyang Y.
      • Ye Y.
      • Yu H.
      • Shen J.
      RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis.
      ), instead of functioning as a Rab GEF as previously postulated, RABIF/MSS4 is a holdase chaperone that is crucial for the expression of its cognate Rab GTPases. Solution NMR studies are under way in our laboratory to determine how l-PGDS binds to Rab4 and favors its activation.
      In summary, we have discovered an original mechanism for the regulation of DP1 recycling by the synthase of its agonist, l-PGDS, which recruits Rab4 to the receptor and participates in the activation of the small GTPase.

      Experimental procedures

      Reagents

      The polyclonal anti-FLAG antibody, the FLAG-specific monoclonal antibodies (M1 and M2), the anti-mouse and anti-rabbit IgG peroxidase–linked species-specific whole antibodies, the alkaline phosphatase-conjugated goat anti-mouse antibody, the isoproterenol hydrochloride, the alkaline phosphatase substrate kit, and GDP (G7127) and GTPγS (G8634) were purchased from Sigma-Aldrich. The monoclonal anti-HA (16B12) was from Covance. The monoclonal anti-HA-peroxidase high-affinity antibody (3F10) was bought from Roche Applied Science. The monoclonal c-MYC antibody was from Biolegend. The anti-MYC-peroxidase high-affinity polyclonal antibody was from Abcam, whereas the anti-GST polyclonal antibody was from Bethyl Laboratories. The monoclonal anti-His was from Cell Signaling Technology. The polyclonal anti-HA, normal mouse, and rabbit IgG isotypic control antibodies, anti-GAPDH, anti-Rab4 (FL-213), and the protein G-agarose and A-agarose beads were purchased from Santa Cruz Biotechnology, Inc. The polyclonal and monoclonal anti-l-PGDS antibodies and PGD2 were from Cayman Chemical Co. The Antarctic Phosphatase was from New England BioLabs, Inc. (M0289S). Alexa Fluor 633 goat anti-rabbit and ProLong® Gold antifade reagent were bought from Invitrogen.

      Plasmid constructs

      The cDNA fragment coding for human Rab4 was amplified by PCR from a human HeLa MATCH-MAKER cDNA library (Clontech) using the high-fidelity DNA polymerase (Phusion, New England Biolabs, Inc.) and the following primers: Rab4 forward (5′-GAG GAA TTC ATG TCC GAA ACC TAC GAT TTT TTG-3′) and Rab4 reverse (5′-GAG CTC GAG CTA ACA ACC ACA CTC CTG AGC-3′). The full-length fragment was digested with EcoRI and XhoI and ligated into pcDNA3-HA vector digested likewise. Site-directed mutagenesis was carried out by PCR. The Rab4S22N and Rab4Q67L mutants were prepared from the pcDNA3-HA-Rab4 construct by using these primers: Rab4S22N forward (5′-GGA AAT GCA GGA ACT GGC AAA AAT TGC TTA CTT CAT CAG-3′), Rab4S22N reverse (5′-CTG ATG AAC TAA GCA ATT TTT GCC AGT TCC TGC ATT TCC-3′), Rab4Q67L forward (5′-ACA GCA GGA CTA GAA CGA TTC AGG-3′), Rab4Q67L reverse (5′-CCT GAA TCG TTC TAG TCC TGC TGT-3′), and Rab4 forward and Rab4 reverse as mentioned previously. The fragments were ligated by the PCR extension method. The full-length mutant fragments were digested with EcoRI and XhoI and ligated into pcDNA3-HA digested with the same enzymes. The His6-Rab4 construct was prepared from pcDNA3-HA-Rab4 by using the following primers: pRSETA Rab4 forward (5′-CTAG GGA TCC ATG TCC GAA ACC TAC GAT TTT TTG TTT AAG TTC-3′) and pRSETA Rab4 reverse (5′-CTAG GAA TTC CTA ACA ACC ACA CTC CTG AGC GTT CGG GGC CTG GGT GCG CCG CGG TGA CCT CAG-3′). The PCR fragment was digested with BamHI and EcoRI and inserted into pRSETA previously digested the same way. The GST-rabaptin construct was prepared by PCR from the human RABEP1 sequence-verified cDNA clone template purchased from GE Dharmacon (Clone ID: 6046320) with the use of the following primers: rabaptin forward (5′-CTAG GTC GAC ATG GCG CAG CCG GGC CCG GCT TCC CAG CCT-3′) and rabaptin reverse (5′-CTAG GTC GAC TCA TGT CTC AGG AAG CTG GTT AAT GTC TGT CAG TTT AGT ATC ATT CAG-3′). The PCR fragment was digested with SalI and inserted into pGEX-4T1 previously digested with SalI and treated with the Antarctic Phosphatase according to the manufacturer’s instruction to prevent self-ligation of the vector. The pcDNA3-l-PGDS-HA, pcDNA3-l-PGDS-MYC, pGEX-4T1-l-PGDS, and pRSETA-LPGDS were produced as described previously (
      • Mathurin K.
      • Gallant M.A.
      • Germain P.
      • Allard-Chamard H.
      • Brisson J.
      • Iorio-Morin C.
      • de Brum Fernandes A.
      • Caron M.G.
      • Laporte S.A.
      • Parent J.-L.
      An interaction between L-prostaglandin D synthase and arrestin increases PGD2 production.
      ). The pcDNA3-FLAG-DP1 construct was generated as described earlier (
      • Gallant M.A.
      • Slipetz D.
      • Hamelin E.
      • Rochdi M.D.
      • Talbot S.
      • de Brum-Fernandes A.J.
      • Parent J.-L.
      Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
      ). The pcDNA3-l-PGDS-W43A/G47A-HA, pGEX4T1-DP1-CT, and pGEX-4T1-DP1-ICL1 were generated as described previously (
      • Binda C.
      • Génier S.
      • Cartier A.
      • Larrivée J.-F.
      • Stankova J.
      • Young J.C.
      • Parent J.-L.
      A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
      ). The other ICLs of DP1 were subcloned as described previously (
      • Parent A.
      • Roy S.J.
      • Iorio-Morin C.
      • Lépine M.-C.
      • Labrecque P.
      • Gallant M.A.
      • Slipetz D.
      • Parent J.-L.
      ANKRD13C acts as a molecular chaperone for G protein-coupled receptors.
      ).

      Cell culture and transfections

      HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. Transient transfection of HEK293 and HeLa cells grown to 50–70% confluence was performed using TransIT®-LT1 reagent (Mirus) and Lipofectamine 2000 (Invitrogen), respectively, and according to the manufacturer's instructions. The total amount of DNA was kept constant by adding empty pcDNA3 vector per plate.

      Immunoprecipitation

      HEK293 cells were transiently transfected with the indicated constructs and were maintained as described above for 48 h. When stimulation was needed, cells were incubated in the presence of 1 μm PGD2 for the desired times in serum-free DMEM containing 20 mm HEPES and 0.5% BSA before harvesting. The cells were then washed with ice-cold PBS and harvested in 400 μl of lysis buffer (150 mm NaCl, 50 mm Tris (pH 8.0), 0.5% deoxycholate, 0.1% SDS, 10 mm Na4P2O7, 1% IGEPAL, and 5 mm EDTA or 1 mm CaCl2, depending on the antibody used for the assay) supplemented with protease inhibitors (10 μm chymostatin, 10 μm leupeptin, 9 μm antipain, and 9 μm pepstatin) (Roche Applied Science). After a 1-h incubation at 4 °C, the lysates were centrifuged for 20 min at 13,500 × g at 4 °C. Proteins were immunoprecipitated using 1 μg of specific antibodies overnight. 40 μl of 50% protein G- or A-agarose beads were added to the lysates for 1 h the next morning. Samples were then centrifuged for 2 min in a microcentrifuge and washed three times with 1 ml of lysis buffer supplemented with protease inhibitors as mentioned above. 40 μl of SDS sample buffer was added to elute the immunoprecipitated proteins, followed by a 60-min incubation at room temperature. Initial lysates and immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting. Endogenous immunoprecipitations were performed in HeLa cells. Cells were harvested and processed as described above, except proteins were immunoprecipitated using 5 μg of l-PGDS–specific or isotypic control IgG antibody, and 40 μl of 50% protein G-agarose beads overnight.

      Recombinant protein production and pulldown analysis

      All of the constructs in pGEX-4T1 vector (Amersham Biosciences) listed previously were used to produce GST-tagged fusion proteins in the OverexpressTM C41 (DE3) Escherichia coli strain (Avidis) as indicated by the manufacturer. Glutathione-Sepharose 4B (Amersham Biosciences) was used for protein purification, and the purified recombinant proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. The pRSETA constructs were used to produce His-tagged fusion proteins using the OverexpressTM C41 system as mentioned above. The fusion proteins were purified using nickel-nitrilotriacetic acid-agarose resin (Qiagen) by following the manufacturer's instructions. 5 μg (0.5 μm final concentration) of glutathione-Sepharose–bound GST-tagged fusion proteins were incubated with 10 μg (1.5 μm final concentration) of the purified His-tagged proteins in binding buffer (10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 10% glycerol, 0.5% IGEPAL, and 2 mm DTT) supplemented with protease inhibitors (10 μm chymostatin, 10 μm leupeptin, 9 μm antipain, and 9 μm pepstatin). The binding reactions were then washed three times with binding buffer. SDS sample buffer was added to each reaction before boiling the tubes for 5 min. All reactions were analyzed by Western blotting using specific antibodies as indicated. Where indicated, 5 μg of glutathione-Sepharose–bound GST-tagged fusion proteins were incubated with 300 μl of cell lysates. The cells were transfected with the indicated constructs, cultured, harvested, and lysed in the absence of EDTA as mentioned earlier, and the binding reactions were processed as mentioned above.

      Immunofluorescence staining and confocal microscopy

      For co-localization experiments, HeLa cells were plated directly onto coverslips previously coated with 0.1 mg/ml poly-l-lysine (Sigma-Aldrich) at a density of 7.5 × 104 cells/well in 6-well plates. The cells were then transiently transfected with the indicated constructs using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacturer's instructions. After 48 h, the cells were fixed with 4% paraformaldehyde in PBS, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 0.1% Triton X-100 in PBS containing 2% BSA. They were then incubated with primary antibodies diluted in blocking solution for 60 min, washed twice with 0.1% Triton X-100 in PBS, blocked with 0.1% Triton X-100 in PBS containing 2% BSA, and incubated with the appropriate secondary antibodies diluted in blocking solution for 60 min. The cells were then washed twice with 0.1% Triton X-100 in PBS followed by three washes with PBS. The coverslips were mounted using ProLong® Gold antifade reagent. Confocal microscopy was performed using a scanning confocal system (TCS SP8, Leica) coupled to an inverted microscope with a ×60 oil immersion objective (DMI8, Leica), and the images were processed using LAS X software (Leica).

      Measurement of DP1 internalization and recycling

      For quantification of receptor internalization, HEK293 cells were plated at 5 × 105 cells in 24-well plates pretreated with 0.1 mg/ml poly-l-lysine (Sigma). Cells were transfected the next day with the indicated constructs, and 48 h post-transfection, cells were stimulated with 1 μm PGD2 or 10 μm isoproterenol for the desired times in serum-free DMEM containing 20 mm HEPES and 0.5% BSA, fixed in 3.7% formaldehyde/Tris-buffered saline (TBS) (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm CaCl2) for 10 min, and then washed twice with TBS. Cells were blocked with TBS containing 1% BSA for 45 min to avoid nonspecific binding. A FLAGM1-specific mAb was then added at a dilution of 1:2000 in 1% TBS-BSA for 60 min. Cells were then washed three times and blocked again with 1% TBS-BSA for 15 min. Cells were incubated with an alkaline phosphatase–conjugated goat anti-mouse antibody at a 1:10,000 dilution in 1% TBS-BSA for 60 min. The cells were then washed three times before adding 250 μl of a colorimetric alkaline phosphatase substrate. The plates were incubated at 37 °C for 30 min, followed by the addition of 250 μl of 0.4 m NaOH to stop the reaction. 100 μl of the colorimetric reaction was collected, and the absorbance was measured at 405 nm using a spectrophotometer (Titertek Multiskan MCC/340, Labsystem). For quantification of receptor internalization using siRNAs, HEK293 cells or HeLa cells stably expressing the FLAG-DP1 receptor were plated at 5 × 105 and 3 × 105 cells, respectively, in 24-well plates and transfected the same day with the desired siRNAs. ELISAs were carried out as mentioned above 72 h post-transfection. For quantification of receptor recycling, cells were plated and transfected as described earlier and maintained for 48 h. Cells were then stimulated with 1 μm PGD2 for 30 min at 37 °C to allow receptor internalization. Cells were washed once with PBS before adding DMEM containing 0.5% BSA and 20 mm HEPES to allow receptor recycling. Recycling was then stopped at the desired times, and cell-surface receptor expression was assessed as described above.

      GTPγS-loading assays

      Purified Rab4 protein concentration was estimated by comparing Coomassie staining with known BSA control concentrations on a 10% SDS-polyacrylamide gel. GTPγS loading was performed essentially as described by Jean et al. (
      • Jean S.
      • Cox S.
      • Schmidt E.J.
      • Robinson F.L.
      • Kiger A.
      Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling.
      ). Purified His6-Rab4 (estimated 10 μm) was incubated in GTPase-loading buffer (40 μm GDP, 20 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm EDTA) at 30 °C for 10 min to allow loading of GDP. 10 mm MgCl2 was then added to stabilize Rab4 GTPase in the GDP-loaded form. GTPγS exchange reactions were performed at room temperature by adding exchange buffer (0.5 mg/ml BSA, 5 μm GTPγS, 0.5 mm DTT, 5 mm MgCl2, 100 mm NaCl, 20 mm Tris-HCl, pH 7.4, with or without 3 μm His6-l-PGDS or l-PGDS peptides) to 3 μm Rab4-GDP in a total volume of 130 μl for the indicated time intervals. After the allotted GTPγS-loading time, 70 μl of ice-cold wash buffer (20 mm Tris-HCl, pH 7.4, 100 mm NaCl, 20 mm MgCl2) was added to the mix. Pulldown assays using purified GST-rabaptin were carried out for 2 h as mentioned earlier. The binding reactions were then washed three times with ice-cold wash buffer and further processed as mentioned above.

      Solid-phase peptide synthesis

      Unless otherwise noted, all reactions were performed under nitrogen pressure. All solvents used were HPLC grade and were used without further purification. Water-sensitive reactions were performed in anhydrous solvents. TentaGel S RAM resin (0.22 mmol g−1) was purchased from Rapp Polymere (Tübingen, Germany). All of the amino acid derivatives and coupling reagents were purchased from ChemImpex International (Wood Dale, IL). Piperidine and N-methylpyrrolidinone were obtained from A&C American Chemicals Ltd. (Saint-Laurent, Quebec, Canada). All other reagents were purchased from Sigma-Aldrich. The UPLC-MS analysis was performed on a Waters (Milford, MA) AQUITY H-Class separation module coupled with a Waters SQD2 mass spectrometer equipped with an analytical column BEH C18 (1.7 μm, 2.1 × 50 mm). Preparative HPLC was carried out using a Waters 2535 module with an ACE C18 column (5 μm, 250 × 21.2 mm) (Canadian Life Science, Peterborough, Canada). The peptide syntheses were performed on an automated system using Tentagel S RAM resin. The resin was first loaded in reaction vessels on the Symphony-X peptide synthesizer (Gyros Protein Technologies, Tucson, AZ). The deprotection step was performed using 20% piperidine in N,N-dimethylformamide, and Fmoc amino acids were added in a 5-fold excess using HATU in the presence of DIPEA. Once all amino acids were coupled and the terminal Fmoc was removed, the peptides were cleaved from the polymer solid support using a mixture of TFA/H2O/TIPS/EDT (92.5:2.5:2.5:2.5, v/v/v/v) with stirring for 3 h. The mixture was filtered and then precipitated in diethyl ether. The precipitated crude peptides were centrifuged, and the ether layer was removed by decantation. The crude peptides were dissolved in a mixture of water and acetonitrile, filtered, diluted with water, lyophilized, and then purified with a preparative HPLC. The fractions containing the pure peptide were pooled and lyophilized to yield the final peptides as white powders. The identity of the peptides was confirmed by MS.

      siRNAs

      The control nontargeting DsiRNA duplex (DS NC1) and the DsiRNA duplexes targeting the human Rab4 gene (HSC.RNAI.N004578.12.9 and 13.3), the negative control siRNA (Silencer Negative control 1, catalogue no. 4390843) and the siRNA targeting the human l-PGDS (PTGDS) gene (siRNA ID s11446 and CDS4/5) were purchased from IDT. HeLa cells stably expressing the FLAG-DP1 receptor were transfected with 200 nm oligonucleotide using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Cells were harvested as mentioned above, and protein expression was assessed by Western blotting 72 h post-transfection.

      Transcreener GDP TR-FRET red assays

      As indicated in the Transcreener GDP TR-FRET red assay's technical manual (BellBrook Labs LLC), the enzyme reactions were performed in a 10-μl volume containing purified His6-Rab4 in the presence or absence of His6-l-PGDS, both diluted in FRET assay buffer (25 mm Tris (pH 7.5), 2.5 mm MgCl2, 0.5 mm EDTA, 0.5% DMSO, 0.01% SDS). The reactions were started by the addition of 10 μm GTP and carried out at room temperature for the indicated period of time. The reactions were then stopped by the addition of 10 μl of a 1× GDP detection mixture (8 nm GDP Antibody-Tb, 1× Stop and Detect Buffer C, 26.8 nm GDP HiLyte647 Tracer) according to the technical manual, bringing the final volume to 20 μl. The plate was rocked at room temperature for 90 min, and TR-FRET was measured. White 384-well microplates (AlphaPlateTM-384 SW, PerkinElmer Life Sciences) were used, and FRET signals were recorded on an Infinite M1000 plate reader (TECAN). The terbium conjugate was excited at 320 nm, and emission was measured at 615 and 665 nm after a delay time of 150 μs and total integration time of 500 μs. All TR-FRET signals were expressed as TR-FRET ratios (665/615), and values in graphs are means of data of three separate experiments. The range of enzyme concentrations and time of assay were determined based on a previous publication from BellBrook Labs (
      • Reichman M.
      • Schabdach A.
      • Kumar M.
      • Zielinski T.
      • Donover P.S.
      • Laury-Kleintop L.D.
      • Lowery R.G.
      A high-throughput assay for Rho guanine nucleotide exchange factors based on the transcreener GDP assay.
      ). Titration assays were done in 2-fold serial dilutions starting at a 10 μm enzyme concentration. Reactions were started as described above and mixed at room temperature for 60 min before adding the GDP detection mixture. IC50 values calculated following titrations (Rab4, 80 nm; l-PGDS, 320 nm) were used as optimized enzyme concentrations in the time-course TR-FRET assays. All data were analyzed using GraphPad Prism using a four-parameter nonlinear regression curve fitting.

      Statistical analysis

      Statistical analysis was performed using Prism version 5.0 (GraphPad Software) using a two-tailed Student’s t test or two-way analysis of variance with multiple comparisons. Data were considered significant when p values were <0.05 (*), <0.01 (**), <0.001 (***), or <0.0001 (****).

      Author contributions

      C. B., S. G., J. D., S. P., L. F., S. J., E. M., and J.-L. P. conceptualization; C. B., S. G., J. D., S. P., L. F., and J.-L. P. data curation; C. B., S. G., J. D., S. P., L. F., S. J., E. M., and J.-L. P. formal analysis; C. B., S. G., J. D., L. F., S. J., E. M., and J.-L. P. investigation; C. B., S. G., J. D., S. P., L. F., S. J., E. M., and J.-L. P. methodology; C. B. and J.-L. P. writing-original draft; C. B., S. G., J. D., S. P., L. F., S. J., E. M., and J.-L. P. writing-review and editing; S. G., J. D., S. P., L. F., E. M., and J.-L. P. validation; S. G., J. D., S. P., L. F., and J.-L. P. visualization; E. M. and J.-L. P. supervision; J.-L. P. resources; J.-L. P. funding acquisition; J.-L. P. project administration.

      Acknowledgments

      We thank Alexandre Desroches for help in setting up the TR-FRET assays.

      References

        • Hardy C.C.
        • Robinson C.
        • Tattersfield A.E.
        • Holgate S.T.
        The bronchoconstrictor effect of inhaled prostaglandin D2 in normal and asthmatic men.
        N. Engl. J. Med. 1984; 311 (6588293): 209-213
        • Ueno R.
        • Honda K.
        • Inoué S.
        • Hayaishi O.
        Prostaglandin D2, a cerebral sleep-inducing substance in rats.
        Proc. Natl. Acad. Sci. U.S.A. 1983; 80 (6572936): 1735-1737
        • Eguchi N.
        • Minami T.
        • Shirafuji N.
        • Kanaoka Y.
        • Tanaka T.
        • Nagata A.
        • Yoshida N.
        • Urade Y.
        • Ito S.
        • Hayaishi O.
        Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96 (9892701): 726-730
        • Matsuoka T.
        • Hirata M.
        • Tanaka H.
        • Takahashi Y.
        • Murata T.
        • Kabashima K.
        • Sugimoto Y.
        • Kobayashi T.
        • Ushikubi F.
        • Aze Y.
        • Eguchi N.
        • Urade Y.
        • Yoshida N.
        • Kimura K.
        • Mizoguchi A.
        • et al.
        Prostaglandin D2 as a mediator of allergic asthma.
        Science. 2000; 287 (10720327): 2013-2017
        • Ishizuka T.
        • Matsui T.
        • Okamoto Y.
        • Ohta A.
        • Shichijo M.
        Ramatroban (BAY u 3405): a novel dual antagonist of TXA2 receptor and CRTh2, a newly identified prostaglandin D2 receptor.
        Cardiovasc. Drug Rev. 2004; 22 (15179446): 71-90
        • Gilroy D.W.
        • Colville-Nash P.R.
        • Willis D.
        • Chivers J.
        • Paul-Clark M.J.
        • Willoughby D.A.
        Inducible cyclooxygenase may have anti-inflammatory properties.
        Nat. Med. 1999; 5 (10371510): 698-701
        • Ianaro A.
        • Ialenti A.
        • Maffia P.
        • Pisano B.
        • Di Rosa M.
        Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.
        FEBS Lett. 2001; 508 (11707269): 61-66
        • Vong L.
        • Ferraz J.G.P.
        • Panaccione R.
        • Beck P.L.
        • Wallace J.L.
        A pro-resolution mediator, prostaglandin D2, is specifically up-regulated in individuals in long-term remission from ulcerative colitis.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20547854): 12023-12027
        • Gallant M.A.
        • Chamoux E.
        • Bisson M.
        • Wolsen C.
        • Parent J.-L.
        • Roux S.
        • de Brum-Fernandes A.J.
        Increased concentrations of prostaglandin D2 during post-fracture bone remodeling.
        J. Rheumatol. 2010; 37 (20080921): 644-649
        • Gallant M.A.
        • Samadfam R.
        • Hackett J.A.
        • Antoniou J.
        • Parent J.-L.
        • de Brum-Fernandes A.J.
        Production of prostaglandin D2 by human osteoblasts and modulation of osteoprotegerin, RANKL, and cellular migration by DP and CRTH2 receptors.
        J. Bone Miner. Res. 2005; 20 (15765187): 672-681
        • Urade Y.
        • Eguchi N.
        Lipocalin-type and hematopoietic prostaglandin D synthases as a novel example of functional convergence.
        Prostaglandins Other Lipid Mediat. 2002; 68 (12432930): 375-382
        • Urade Y.
        • Ujihara M.
        • Horiguchi Y.
        • Igarashi M.
        • Nagata A.
        • Ikai K.
        • Hayaishi O.
        Mast cells contain spleen-type prostaglandin D synthetase.
        J. Biol. Chem. 1990; 265 (2403560): 371-375
        • Fujimori K.
        • Kanaoka Y.
        • Sakaguchi Y.
        • Urade Y.
        Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells: roles of the oct-1 element in the 5′-flanking region and the AP-2 element in the untranslated exon 1.
        J. Biol. Chem. 2000; 275 (10998423): 40511-40516
        • Tanaka K.
        • Ogawa K.
        • Sugamura K.
        • Nakamura M.
        • Takano S.
        • Nagata K.
        Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets.
        J. Immunol. 2000; 164 (10679060): 2277-2280
        • Urade Y.
        • Fujimoto N.
        • Hayaishi O.
        Purification and characterization of rat brain prostaglandin D synthetase.
        J. Biol. Chem. 1985; 260 (3930495): 12410-12415
        • Blödorn B.
        • Mäder M.
        • Urade Y.
        • Hayaishi O.
        • Felgenhauer K.
        • Brück W.
        Choroid plexus: the major site of mRNA expression for the β-trace protein (prostaglandin D synthase) in human brain.
        Neurosci. Lett. 1996; 209 (8761996): 117-120
        • Eguchi Y.
        • Eguchi N.
        • Oda H.
        • Seiki K.
        • Kijima Y.
        • Matsu-ura Y.
        • Urade Y.
        • Hayaishi O.
        Expression of lipocalin-type prostaglandin D synthase (β-trace) in human heart and its accumulation in the coronary circulation of angina patients.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94 (9405674): 14689-14694
        • Beuckmann C.T.
        • Gordon W.C.
        • Kanaoka Y.
        • Eguchi N.
        • Marcheselli V.L.
        • Gerashchenko D.Y.
        • Urade Y.
        • Hayaishi O.
        • Bazan N.G.
        Lipocalin-type prostaglandin D synthase (β-trace) is located in pigment epithelial cells of rat retina and accumulates within interphotoreceptor matrix.
        J. Neurosci. 1996; 16 (8815894): 6119-6124
        • Gerena R.L.
        • Eguchi N.
        • Urade Y.
        • Killian G.J.
        Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis.
        J. Androl. 2000; 21 (11105911): 848-854
        • Tanaka T.
        • Urade Y.
        • Kimura H.
        • Eguchi N.
        • Nishikawa A.
        • Hayaishi O.
        Lipocalin-type prostaglandin D synthase (β-trace) is a newly recognized type of retinoid transporter.
        J. Biol. Chem. 1997; 272 (9188476): 15789-15795
        • Beuckmann C.T.
        • Aoyagi M.
        • Okazaki I.
        • Hiroike T.
        • Toh H.
        • Hayaishi O.
        • Urade Y.
        Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.
        Biochemistry. 1999; 38 (10387044): 8006-8013
        • Mohri I.
        • Taniike M.
        • Okazaki I.
        • Kagitani-Shimono K.
        • Aritake K.
        • Kanekiyo T.
        • Yagi T.
        • Takikita S.
        • Kim H.-S.
        • Urade Y.
        • Suzuki K.
        Lipocalin-type prostaglandin D synthase is up-regulated in oligodendrocytes in lysosomal storage diseases and binds gangliosides.
        J. Neurochem. 2006; 97 (16515539): 641-651
        • Boie Y.
        • Sawyer N.
        • Slipetz D.M.
        • Metters K.M.
        • Abramovitz M.
        Molecular cloning and characterization of the human prostanoid DP receptor.
        J. Biol. Chem. 1995; 270 (7642548): 18910-18916
        • Hirai H.
        • Tanaka K.
        • Yoshie O.
        • Ogawa K.
        • Kenmotsu K.
        • Takamori Y.
        • Ichimasa M.
        • Sugamura K.
        • Nakamura M.
        • Takano S.
        • Nagata K.
        Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2.
        J. Exp. Med. 2001; 193 (11208866): 255-261
        • Lebon G.
        • Tate C.G.
        [G protein-coupled receptors in the spotlight].
        Med. Sci. (Paris). 2012; 28 (23067420): 876-882
        • Lachance V.
        • Degrandmaison J.
        • Marois S.
        • Robitaille M.
        • Génier S.
        • Nadeau S.
        • Angers S.
        • Parent J.-L.
        Ubiquitylation and activation of a Rab GTPase is promoted by a β2AR-HACE1 complex.
        J. Cell Sci. 2014; 127 (24190883): 111-123
        • Costanzi S.
        • Tikhonova I.G.
        • Harden T.K.
        • Jacobson K.A.
        Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands.
        J. Comput. Aided Mol. Des. 2009; 23 (18483766): 747-754
        • Pierce K.L.
        • Premont R.T.
        • Lefkowitz R.J.
        Seven-transmembrane receptors.
        Nat. Rev. Mol. Cell Biol. 2002; 3 (12209124): 639-650
        • Ritter S.L.
        • Hall R.A.
        Fine-tuning of GPCR activity by receptor-interacting proteins.
        Nat. Rev. Mol. Cell Biol. 2009; 10 (19935667): 819-830
        • Magalhaes A.C.
        • Dunn H.
        • Ferguson S.S.
        Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins.
        Br. J. Pharmacol. 2012; 165 (21699508): 1717-1736
        • Esseltine J.L.
        • Dale L.B.
        • Ferguson S.S.G.
        Rab GTPases bind at a common site within the angiotensin II type I receptor carboxyl-terminal tail: evidence that Rab4 regulates receptor phosphorylation, desensitization, and resensitization.
        Mol. Pharmacol. 2011; 79 (20943774): 175-184
        • Hamelin E.
        • Thériault C.
        • Laroche G.
        • Parent J.-L.
        The intracellular trafficking of the G protein-coupled receptor TPβ depends on a direct interaction with Rab11.
        J. Biol. Chem. 2005; 280 (16126723): 36195-36205
        • Parent A.
        • Hamelin E.
        • Germain P.
        • Parent J.-L.
        Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction.
        Biochem. J. 2009; 418 (18983266): 163-172
        • Dateyama I.
        • Sugihara Y.
        • Chiba S.
        • Ota R.
        • Nakagawa R.
        • Kobayashi T.
        • Itoh H.
        RABL2 positively controls localization of GPCRs in mammalian primary cilia.
        J Cell Sci. 2018; 132 (30578315): jcs224428
        • Seachrist J.L.
        • Ferguson S.S.G.
        Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases.
        Life Sci. 2003; 74 (14607250): 225-235
        • Dong C.
        • Yang L.
        • Zhang X.
        • Gu H.
        • Lam M.L.
        • Claycomb W.C.
        • Xia H.
        • Wu G.
        Rab8 interacts with distinct motifs in α2B- and β2-adrenergic receptors and differentially modulates their transport.
        J. Biol. Chem. 2010; 285 (20424170): 20369-20380
        • Li C.
        • Wei Z.
        • Fan Y.
        • Huang W.
        • Su Y.
        • Li H.
        • Dong Z.
        • Fukuda M.
        • Khater M.
        • Wu G.
        The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.
        Cell Rep. 2017; 21 (29069590): 1089-1101
        • Cherfils J.
        • Zeghouf M.
        Regulation of small GTPases by GEFs, GAPs, and GDIs.
        Physiol. Rev. 2013; 93 (23303910): 269-309
        • Hutagalung A.H.
        • Novick P.J.
        Role of Rab GTPases in membrane traffic and cell physiology.
        Physiol. Rev. 2011; 91 (21248164): 119-149
        • Stenmark H.
        Rab GTPases as coordinators of vesicle traffic.
        Nat. Rev. Mol. Cell Biol. 2009; 10 (19603039): 513-525
        • Wandinger-Ness A.
        • Zerial M.
        Rab proteins and the compartmentalization of the endosomal system.
        Cold Spring Harb. Perspect. Biol. 2014; 6 (25341920): a022616
        • Bhuin T.
        • Roy J.K.
        Rab proteins: the key regulators of intracellular vesicle transport.
        Exp. Cell Res. 2014; 328 (25088255): 1-19
        • Wang G.
        • Wu G.
        Small GTPase regulation of GPCR anterograde trafficking.
        Trends Pharmacol. Sci. 2012; 33 (22015208): 28-34
        • Pfeffer S.R.
        Rab GTPases: specifying and deciphering organelle identity and function.
        Trends Cell Biol. 2001; 11 (11719054): 487-491
        • Christoforidis S.
        • Zerial M.
        Purification and identification of novel Rab effectors using affinity chromatography.
        Methods. 2000; 20 (10720461): 403-410
        • Zhen Y.
        • Stenmark H.
        Cellular functions of Rab GTPases at a glance.
        J. Cell Sci. 2015; 128 (26272922): 3171-3176
        • Blümer J.
        • Rey J.
        • Dehmelt L.
        • Mazel T.
        • Wu Y.-W.
        • Bastiaens P.
        • Goody R.S.
        • Itzen A.
        RabGEFs are a major determinant for specific Rab membrane targeting.
        J. Cell Biol. 2013; 200 (23382462): 287-300
        • Pfeffer S.R.
        Rab GTPase regulation of membrane identity.
        Curr. Opin. Cell Biol. 2013; 25 (23639309): 414-419
        • Segev N.
        Ypt/rab gtpases: regulators of protein trafficking.
        Sci. STKE. 2001; 2001 (11579231): re11
        • Bos J.L.
        • Rehmann H.
        • Wittinghofer A.
        GEFs and GAPs: critical elements in the control of small G proteins.
        Cell. 2007; 129 (17540168): 865-877
        • Ishida M.
        • Oguchi M.E.
        • Fukuda M.
        Multiple types of guanine nucleotide exchange factors (GEFs) for Rab small GTPases.
        Cell Struct. Funct. 2016; 41 (27246931): 61-79
        • Yudowski G.A.
        • Puthenveedu M.A.
        • Henry A.G.
        • von Zastrow M.
        Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.
        Mol. Biol. Cell. 2009; 20 (19369423): 2774-2784
        • Gallant M.A.
        • Slipetz D.
        • Hamelin E.
        • Rochdi M.D.
        • Talbot S.
        • de Brum-Fernandes A.J.
        • Parent J.-L.
        Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2.
        Eur. J. Pharmacol. 2007; 557 (17207480): 115-123
        • Koch D.
        • Rai A.
        • Ali I.
        • Bleimling N.
        • Friese T.
        • Brockmeyer A.
        • Janning P.
        • Goud B.
        • Itzen A.
        • Müller M.P.
        • Goody R.S.
        A pull-down procedure for the identification of unknown GEFs for small GTPases.
        Small GTPases. 2016; 7 (26918858): 93-106
        • Müller M.P.
        • Goody R.S.
        Molecular control of Rab activity by GEFs, GAPs and GDI.
        Small GTPases. 2018; 9 (28055292): 5-21
        • Yoshimura S.
        • Gerondopoulos A.
        • Linford A.
        • Rigden D.J.
        • Barr F.A.
        Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.
        J. Cell Biol. 2010; 191 (20937701): 367-381
        • Do M.T.
        • Chai T.F.
        • Casey P.J.
        • Wang M.
        Isoprenylcysteine carboxylmethyltransferase function is essential for RAB4A-mediated integrin β3 recycling, cell migration and cancer metastasis.
        Oncogene. 2017; 36 (28604748): 5757-5767
        • Barbarin A.
        • Frade R.
        Procathepsin L secretion, which triggers tumour progression, is regulated by Rab4a in human melanoma cells.
        Biochem. J. 2011; 437 (21501115): 97-107
        • Arsenault D.
        • Lucien F.
        • Dubois C.M.
        Hypoxia enhances cancer cell invasion through relocalization of the proprotein convertase furin from the trans-Golgi network to the cell surface.
        J. Cell Physiol. 2012; 227 (21503879): 789-800
        • Frittoli E.
        • Palamidessi A.
        • Marighetti P.
        • Confalonieri S.
        • Bianchi F.
        • Malinverno C.
        • Mazzarol G.
        • Viale G.
        • Martin-Padura I.
        • Garré M.
        • Parazzoli D.
        • Mattei V.
        • Cortellino S.
        • Bertalot G.
        • Di Fiore P.P.
        • Scita G.
        A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination.
        J. Cell Biol. 2014; 206 (25049275): 307-328
        • Binda C.
        • Génier S.
        • Cartier A.
        • Larrivée J.-F.
        • Stankova J.
        • Young J.C.
        • Parent J.-L.
        A G protein-coupled receptor and the intracellular synthase of its agonist functionally cooperate.
        J. Cell Biol. 2014; 204 (24493589): 377-393
        • Génier S.
        • Degrandmaison J.
        • Moreau P.
        • Labrecque P.
        • Hébert T.E.
        • Parent J.-L.
        Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.
        Mol. Biol. Cell. 2016; 27 (27708139): 3800-3812
        • Roy S.J.
        • Glazkova I.
        • Fréchette L.
        • Iorio-Morin C.
        • Binda C.
        • Pétrin D.
        • Trieu P.
        • Robitaille M.
        • Angers S.
        • Hébert T.E.
        • Parent J.-L.
        Novel, gel-free proteomics approach identifies RNF5 and JAMP as modulators of GPCR stability.
        Mol. Endocrinol. 2013; 27 (23798571): 1245-1266
        • Lachance V.
        • Cartier A.
        • Génier S.
        • Munger S.
        • Germain P.
        • Labrecque P.
        • Parent J.-L.
        Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.
        J. Biol. Chem. 2011; 286 (21990357): 40802-40813
        • Dale L.B.
        • Seachrist J.L.
        • Babwah A.V.
        • Ferguson S.S.G.
        Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases.
        J. Biol. Chem. 2004; 279 (14711821): 13110-13118
        • Hammad M.M.
        • Kuang Y.-Q.
        • Morse A.
        • Dupré D.J.
        Rab1 interacts directly with the β2-adrenergic receptor to regulate receptor anterograde trafficking.
        Biol. Chem. 2012; 393 (22628317): 541-546
        • Mulvaney E.P.
        • O'Meara F.
        • Khan A.R.
        • O'Connell D.J.
        • Kinsella B.T.
        Identification of α-helix 4 (α4) of Rab11a as a novel Rab11-binding domain (RBD): interaction of Rab11a with the prostacyclin receptor.
        Biochim. Biophys. Acta Mol. Cell Res. 2017; 1864 (28739266): 1819-1832
        • Langemeyer L.
        • Nunes Bastos R.
        • Cai Y.
        • Itzen A.
        • Reinisch K.M.
        • Barr F.A.
        Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation.
        Elife. 2014; 3 (24520163): e01623
        • Kanie T.
        • Jackson P.K.
        Guanine nucleotide exchange assay using fluorescent MANT-GDP.
        Bio Protoc. 2018; 8 (29951569): e2795
        • Esseltine J.L.
        • Ribeiro F.M.
        • Ferguson S.S.G.
        Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.
        J. Neurosci. 2012; 32 (23175844): 16933-16942a
        • O'Keeffe M.B.
        • Reid H.M.
        • Kinsella B.T.
        Agonist-dependent internalization and trafficking of the human prostacyclin receptor: a direct role for Rab5a GTPase.
        Biochim. Biophys. Acta. 2008; 1783 (18498773): 1914-1928
        • Seachrist J.L.
        • Anborgh P.H.
        • Ferguson S.S.
        β2-Adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by Rab GTPases.
        J. Biol. Chem. 2000; 275 (10854436): 27221-27228
        • Smythe E.
        Direct interactions between rab GTPases and cargo.
        Mol. Cell. 2002; 9 (11864591): 205-206
        • Wikström K.
        • Reid H.M.
        • Hill M.
        • English K.A.
        • O'Keeffe M.B.
        • Kimbembe C.C.
        • Kinsella B.T.
        Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase.
        Cell. Signal. 2008; 20 (18832025): 2332-2346
        • Seabra M.C.
        • Wasmeier C.
        Controlling the location and activation of Rab GTPases.
        Curr. Opin. Cell Biol. 2004; 16 (15261679): 451-457
        • Barr F.
        • Lambright D.G.
        Rab GEFs and GAPs.
        Curr. Opin. Cell Biol. 2010; 22 (20466531): 461-470
        • Ginsberg S.D.
        • Mufson E.J.
        • Alldred M.J.
        • Counts S.E.
        • Wuu J.
        • Nixon R.A.
        • Che S.
        Upregulation of select rab GTPases in cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease.
        J. Chem. Neuroanat. 2011; 42 (21669283): 102-110
        • Soejima N.
        • Ohyagi Y.
        • Nakamura N.
        • Himeno E.
        • Iinuma K.M.
        • Sakae N.
        • Yamasaki R.
        • Tabira T.
        • Murakami K.
        • Irie K.
        • Kinoshita N.
        • LaFerla F.M.
        • Kiyohara Y.
        • Iwaki T.
        • Kira J.
        Intracellular accumulation of toxic turn amyloid-β is associated with endoplasmic reticulum stress in Alzheimer's disease.
        Curr. Alzheimer. Res. 2013; 10 (22950910): 11-20
        • Caza T.N.
        • Fernandez D.R.
        • Talaber G.
        • Oaks Z.
        • Haas M.
        • Madaio M.P.
        • Lai Z.-W.
        • Miklossy G.
        • Singh R.R.
        • Chudakov D.M.
        • Malorni W.
        • Middleton F.
        • Banki K.
        • Perl A.
        HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE.
        Ann. Rheum. Dis. 2014; 73 (23897774): 1888-1897
        • Hu C.-T.
        • Wu J.-R.
        • Wu W.-S.
        The role of endosomal signaling triggered by metastatic growth factors in tumor progression.
        Cell. Signal. 2013; 25 (23571269): 1539-1545
        • Ferrándiz-Huertas C.
        • Fernández-Carvajal A.
        • Ferrer-Montiel A.
        Rab4 interacts with the human P-glycoprotein and modulates its surface expression in multidrug resistant K562 cells.
        Int. J. Cancer. 2011; 128 (20209493): 192-205
        • Delprato A.
        • Lambright D.G.
        Structural basis for Rab GTPase activation by VPS9 domain exchange factors.
        Nat. Struct. Mol. Biol. 2007; 14 (17450153): 406-412
        • Marat A.L.
        • Dokainish H.
        • McPherson P.S.
        DENN domain proteins: regulators of Rab GTPases.
        J. Biol. Chem. 2011; 286 (21330364): 13791-13800
        • Zhang X.
        • He X.
        • Fu X.-Y.
        • Chang Z.
        Varp is a Rab21 guanine nucleotide exchange factor and regulates endosome dynamics.
        J. Cell Sci. 2006; 119 (16525121): 1053-1062
        • Gulbranson D.R.
        • Davis E.M.
        • Demmitt B.A.
        • Ouyang Y.
        • Ye Y.
        • Yu H.
        • Shen J.
        RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis.
        Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28894007): E8224-E8233
        • Mathurin K.
        • Gallant M.A.
        • Germain P.
        • Allard-Chamard H.
        • Brisson J.
        • Iorio-Morin C.
        • de Brum Fernandes A.
        • Caron M.G.
        • Laporte S.A.
        • Parent J.-L.
        An interaction between L-prostaglandin D synthase and arrestin increases PGD2 production.
        J. Biol. Chem. 2011; 286 (21112970): 2696-2706
        • Parent A.
        • Roy S.J.
        • Iorio-Morin C.
        • Lépine M.-C.
        • Labrecque P.
        • Gallant M.A.
        • Slipetz D.
        • Parent J.-L.
        ANKRD13C acts as a molecular chaperone for G protein-coupled receptors.
        J. Biol. Chem. 2010; 285 (20959461): 40838-40851
        • Jean S.
        • Cox S.
        • Schmidt E.J.
        • Robinson F.L.
        • Kiger A.
        Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling.
        Mol. Biol. Cell. 2012; 23 (22648168): 2723-2740
        • Reichman M.
        • Schabdach A.
        • Kumar M.
        • Zielinski T.
        • Donover P.S.
        • Laury-Kleintop L.D.
        • Lowery R.G.
        A high-throughput assay for Rho guanine nucleotide exchange factors based on the transcreener GDP assay.
        J. Biomol. Screen. 2015; 20 (26195453): 1294-1299