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

ABSTRACT: 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, GTP?S loading and time-resolved FRET assays with purified proteins suggested that l-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how l-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation.

SUBMITTER: Binda C

PROVIDER: S-EPMC6851311 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Binda Chantal C Génier Samuel S Degrandmaison Jade J Picard Samuel S Fréchette Louis L Jean Steve S Marsault Eric E Parent Jean-Luc JL

The Journal of biological chemistry 20191001 45

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. Dep ...[more]

PMID: 31575663

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Project description:BackgroundProstaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S (LbrPGF2S) expression and pathogenicity in mice.MethodsLbrPGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate LbrPGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry-LbrPGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle.ResultsLeishmania braziliensis promastigotes synthesize prostaglandin F2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. LbrPGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of LbrPGF2S leads to an increase of infectivity in vitro. The data suggest that LbrPGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites.ConclusionsLbrPGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of LbrPGF2S in L. braziliensis. Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis.

Project description:Eicosanoids, oxygenated metabolites of C20 polyunsaturated fatty acids (PUFAs), mediate fundamental physiological processes, including immune reactions and reproduction, in insects. Prostaglandins (PGs) make up one group of eicosanoids, of which PGE2 is a relatively well-known mediator in various insect taxa. While PG biosynthesis has been reported, the specific biosynthetic pathway for PGE2 is not known in insects. Here, we posed the hypothesis that Se-mPGES2 mediates biosynthesis of physiologically active PGE2 through its cognate protein. To test this hypothesis, we interrogated a transcriptome of the lepidopteran insect, Spodoptera exigua, to identify a candidate PGE2 synthase (Se-mPGES2) and analyzed its sequence and expression. Its predicted amino acid sequence contains a consensus thioredoxin homology sequence (Cys-x-x-Cys) responsible for catalytic activity along with an N-terminal membrane-associated hydrophobic domain and C-terminal cytosolic domain. It also shares sequence homology (36.5%) and shares almost overlapping three dimensional structures with a membrane-bound human PGES2 (mPGES2). Se-mPGES2 was expressed in all developmental stages with high peaks during the late larval instar and adult stages. Immune challenge significantly up-regulated its expression levels in hemocytes and fat body. Injecting double-stranded RNA (dsRNA) specific to Se-mPGES2 significantly impaired two cellular immune responses, hemocyte-spreading behavior and nodule formation following bacterial challenge. Humoral immunity was also significantly suppressed, registered as reduced phenoloxidase activity and antimicrobial peptide expression levels. The suppressed immune responses were reversed following PGE2, but not arachidonic acid (AA), treatments. RNAi treatments also reduced the egg-laying behavior of females. Control females mated with the RNAi-treated males led to substantially reduced egg-laying behavior, which was also reversed following PGE2 injections into females. These results strongly bolster our hypothesis that Se-mPGES2 acts in the biosynthesis of PGE2, a crucial biochemical signal mediating immune and reproductive physiology of S. exigua.

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

Publications

L-type prostaglandin D synthase regulates the trafficking of the PGD<sub>2</sub> DP1 receptor by interacting with the GTPase Rab4.

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