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Peroxiredoxin-4 interacts with and regulates the thromboxane A2 receptor
FEBS Letters 581 (2007) 3863–3868
Peroxiredoxin-4 interacts with and regulates the thromboxane A2 receptor Patrick Gigue`rea,1, Marie-Eve Turcottea,1, Emilie Hamelina, Audrey Parenta, Jessy Brissona, Genevie`ve Larochea, Pascale Labrecquea, Gilles Dupuisb, Jean-Luc Parenta,* a
Service de Rhumatologie, De´partement de Me´decine, Faculte´ de Me´decine and Centre de Recherche Clinique-E´tienne Lebel, Que´bec, Canada b Programme d’Immunologie, Universite´ de Sherbrooke, Que´bec, Canada Received 21 February 2007; revised 6 June 2007; accepted 3 July 2007 Available online 17 July 2007 Edited by Christian Griesinger
Abstract We identified peroxiredoxin-4 (Prx-4) as a protein interacting with the b isoform of the thromboxane A2 receptor (TPb) by yeast two-hybrid analysis. Prx-4 co-immunoprecipitated constitutively with TPb in HEK293 cells. The second and third intracellular loops as well as the C-terminus of TPb interacted directly with Prx-4. Co-expression of Prx-4 caused a 60% decrease in cell surface expression of TPb. Prx-4 and TPb predominantly co-localized in the endoplasmic reticulum. Coexpression of Prx-4 in cells treated with H2O2 targeted TPb for degradation. We show for the first time an interaction between a receptor involved in oxidative stress and Prx-4, an anti-oxidative enzyme. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: GPCR; G protein-coupled receptor; Peroxiredoxin; Oxidative stress; Trafficking; Signalling
1. Introduction Thromboxane A2 (TXA2) is the sequential metabolic product of arachidonic acid by the cyclooxygenases and thromboxane synthase. The TXA2 receptor (TP) is a G protein-coupled receptor (GPCR) that can couple to the G proteins Gaq, Gas, Gai2, Ga12, Ga13, and Ga16[1]. The TP receptor is expressed as two alternatively spliced isoforms, a (343 residues) and b (407 residues) that share the first 328 residues[2,3]. Oxidative stress can be induced by diverse mechanisms, including activation of TP [4,5]. A major product of peroxidation is a group of compounds termed isoprostanes. Isoprostanes are mainly derived by free radical-mediated oxidation of arachidonic acid [6]. The isoprostanes exert significant biological activity and there is evidence that they interact with TP [7–10].
* Corresponding author. Fax: +1 819 564 5265. E-mail address: [email protected] (J.-L. Parent). 1
These authors contributed equally to this work.
Abbreviations: GPCR, G protein-coupled receptor; TP, thromboxane A2 receptor; TXA2, thromboxane A2; TPbCT, C-terminal tail of TPb; GST, glutathione-S-transferase; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; HA, hemagglutinin; ER, endoplasmic reticulum; ICL, intracellular loop of the TPb receptor
Peroxiredoxins (Prxs) have recently been the subject of growing interest as a new family of thiol-specific antioxidant proteins [11,12]. Prxs perform their protective antioxidant role in cells through their peroxidase activity whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides are reduced and detoxified [13]. At least six Prxs were identified in mammalian cells (Prx1–Prx6). Prxs are ubiquitously and abundantly expressed in the various tissues of the human body [14] and were reported to be not redundant functionally [15]. The data that we present here show that an anti-oxidant enzyme, Prx-4, can regulate the biology of TPb, a GPCR mediating oxidative stress.
2. Materials and methods 2.1. Materials The HA- and Myc-specific monoclonal antibodies were from Covance. The rabbit anti-calnexin A polyclonal antibody was acquired from Sigma. The Cascade Blue goat anti-mouse, goat anti-rabbit Texas Red, and fluorescein isothiocyanate (FITC)-conjugated goat antimouse antibodies were purchased from Molecular Probes. The goat anti-mouse alkaline-phosphatase-conjugated antibody and the alkaline phosphatase substrate kit were obtained from Bio-Rad. U46619 was from Cayman. 2.2. Two-hybrid screening assay Yeast two-hybrid screening was performed with the C-terminus of TPb as a bait on a Human HeLa MATCHMAKER cDNA Library (Clontech) as we described previously [16]. 2.3. Cell culture and transfection HEK293 cells were maintained and transfected as detailed before [16]. 2.4. Immunoprecipitations 6-Well plates of HEK293 cells were transfected with the indicated combinations of constructs. Transfected cells were maintained for 48 h and incubated for 0–30 min at 37 C in the presence of 1 lM U46619 prior to harvesting. Thereafter, the cells were processed for immunoprecipitations as we reported previously [16]. 2.5. Recombinant protein production and binding assays A pRSET A-myc-Prx-4 construct was used to produce a His-tagged myc-Prx-4 fusion protein in OverExpress C41(DE3) Escherichia coli strain (Avidis, SA) by following the manufacturer’s instructions. Amino acids 53–66 (ICL1), 129–150 (ICL2), 217–244 (ICL3), and 312–407 (C-tail) were produced as fusion proteins with glutathione-S-transferase (GST) and the pulldown assays performed as we described before [17].
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.011
3864 2.6. Inositol phosphates measurements Inositol phosphates measurements were carried out as we detailed previously [18]. 2.7. Endocytosis and cell surface expression assays Endocytosis and cell surface expression of receptors was measured by enzyme-linked immunosorbent assay (ELISA) using transiently transfected HEK293 cells as we previously reported [17]. 2.8. Immunofluorescence microscopy 1.0 · 106 HEK293 cells were transiently co-transfected with the pcDNA3-HA-TPb green fluorescent protein (GFP) [19] and pcDNA3-myc-Prx-4 constructs and maintained overnight.The cells were then processed for immunofluorescence microscopy as we detailed before on a Nikon Eclipse TE2000-U microscope using a 60· objective, and images were collected using SimplePCI Camera software and processed with Adobe Photoshop software [17].
3. Results and discussion 3.1. Prx-4 interacts with TPb We recently showed that the C-terminus conferred specific regulatory mechanisms to TPb [20,21]. In our efforts to elucidate the mechanisms involved in the regulation of TPb, we performed yeast two-hybrid screening experiments using the yeast strain pJ69-4a transformed with pAS2.1-TPbCT and a human HeLa cell cDNA library. A total of 1.5 · 106 independent clones were screened yielding over 300 positives. One hundred clones demonstrating a strong growth on selective yeast medium (TRP , LEU , HIS , and ADE ) were then isolated and characterized by dideoxy sequencing, and then aligned using the NCBI blast alignment search tool. Four clones contained a cDNA fragment coding for the Prx-4 protein. Other members of the Prx protein family were not isolated in the clones that were sequenced.
Fig. 1. Prx-4 co-immunoprecipitates with TPb. HEK293 cells were transiently transfected with the indicated constructs. Forty-eight hours post-transfection, the cells were stimulated or not with 1 lM U46619, lysed and processed for immunoprecipitation of the receptor with a HA-specific polyclonal antibody. Immunoblotting was performed with HA-specific polyclonal or Myc-specific monoclonal antibodies as described in Section 2. Data presented are representative of three different experiments. IP, immunoprecipitation; IB, immunoblotting.
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868
3.2. Co-immunoprecipitations and in vitro binding assay To investigate the interaction of Prx-4 with TPb in a cellular context, we performed immunoprecipitation experiments in HEK293 cells transfected with pcDNA3-Myc-Prx-4 and pcDNA3-HA-TPb in the presence or absence of stimulation with the stable TP receptor agonist, U46619. Our results demonstrate that Myc-Prx-4 was co-immunoprecipitated with TPb in absence of agonist (Fig. 1). Agonist treatment did not modulate the interaction between the two proteins (Fig. 1). To further characterize the interaction of Prx-4 with the TPb receptor, we carried out an in vitro binding assay using the purified recombinant intracellular loop 1 (ICL1), ICL2, ICL3, or C-tail (CT) of TPb fused to GST along with purified His6-Myc-Prx-4 protein. A schematic representation of the TPb topology is illustrated in Fig. 2A. The results showed that Prx-4 interacted specifically with glutathione sepharose-bound GST-TPb-ICL2, -ICL3, and -CT but not with glutathione sepharose-bound GST or GST-TPb-ICL1 (Fig. 2B). Taken together, our data show that Prx-4 interacts directly with intracellular domains of TPb, predominantly with the C-tail, in an agonist-independent manner. Interestingly, this consti-
Fig. 2. Prx-4 directly interacts with intracellular domains of TPb. (A) Schematic representation of the topology of TPb. The receptor is composed of seven transmembrane domains connected by three extracellular and three intracellular loops. The C-terminus completes the intracellular domains of the receptor. The numbers indicate the first and the last of the amino acids for each intracellular domain that were used to produce fusion proteins with GST. (B) The intracellular loops 1–3 (ICL1, ICL2, and ICL3) and the C-tail of the receptor were produced as GST-fusion proteins and used to perform GST-pulldown assays on purified His6-Prx-4myc protein. The upper panel shows the Prx-4 binding to the receptor domains, while the GST-fusion proteins present in the reaction can be seen in the middle panel. The bottom panel shows the level of Prx-4 protein present in the initial reaction. Data presented are representative of three different experiments. IB, immunoblotting.
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868
tutes the first study whereby a direct interaction of a Prx protein with a G protein-coupled receptor, and to our knowledge to any transmembrane receptor, is shown. 3.3. Regulation of TPb receptor signaling by Prx-4 Members of the Prx protein family have been shown to regulate the signaling of various classes of receptors, including the receptors for EGF, PDGF, and TNFa (reviewed in Ref. [23]). We thus assessed if Prx-4 could modulate the generation of inositol phosphates by TPb following agonist stimulation. Fig. 3A illustrates that co-expression of Prx-4 with the receptor in HEK293 cells resulted in a 70% inhibition of receptormediated production of inositol phosphates following agonist treatment when compared to cells expressing the receptor alone. Production of inositol phosphates by a Gaq-coupled receptor involves the receptor-Gaq interaction and activation of the G protein, with subsequent stimulation of phospholipase Cb by the activated Gaq. Interestingly, Prx-4 overexpression did not affect the generation of inositol phosphates by a constitutive mutant of Gaq, Gaq-R183C (Fig. 3B). This indicated that Prx-4 was not acting at the level of the phospholipase Cb activation by Gaq, but rather at the level of the receptor itself. Co-immunoprecipitation experiments demonstrated that the TPb receptor-Gaq interaction was not pre-
Fig. 3. Regulation of TPb signaling by Prx-4. HEK293 cells were transiently co-transfected with the indicated constructs and labelled with 3H-myo-inositol. Total inositol phosphates were measured after treatment of the cells with 500 nM U46619 for 30 min (A) or after 30 min of accumulation in presence of the constitutive Gq-R183C mutant (B) as described in Section 2. Results are means ± S.E. from five different experiments.
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vented by co-expression of Prx-4 (data not shown). Thus, this suggested that Prx-4 was acting on the receptor to suppress its signaling but not by interfering with its interaction with the Gaq protein. 3.4. Regulation of TPb cell surface expression by Prx-4 Since the response of a cell surface receptor to an agonist is controlled in part by the number of receptors present at the plasma membrane, we next studied whether Prx-4 could alter the internalization and/or the cell surface expression of TPb. HEK293 cells expressing HA-tagged TPb alone or in combination with Prx-4 were stimulated with 100 nM U46619 or ethanol (vehicle) for 120 min and receptors present at the cell surface measured by ELISA, as we did before [16,19–21]. Prx-4 expression had no effect on internalization of the receptor (Fig. 4A). On the other hand, the expression of TPb at the cell surface was reduced by roughly 60% when Prx-4 was present (Fig. 4B), which is most likely the cause of the reduced receptor-mediated signaling after agonist treatment. 3.5. TPb and Prx-4 co-localize intracellularly Given that our results suggested that Prx-4 inhibited the cell surface expression of TPb, we were interested in determining if and where the two proteins co-localized in the cell. Fluorescence microscopy experiments were performed on HEK293 cells co-expressing GFP-tagged TPb [19] and Myc-Prx-4. Initial experiments showed that the two proteins co-localized in an intracellular compartment that could be the endoplasmic reticulum (ER). Three-color fluorescence microscopy,
Fig. 4. Prx-4 overexpression inhibits TPb cell surface expression. HEK293 cells were transiently co-transfected with the HA-tagged TPb and pcDNA3 or pcDNA3-Prx-4myc. The percentage of cell surface receptor loss following a 2 h stimulation with 100 nM U46619 (A) or cell surface expression (B) was measured by ELISA as described in Section 2. Results are means ± S.E. from six different experiments.
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including labelling of calnexin A as an ER-marker, was carried out. The receptor and Prx-4 co-localized intracellularly in absence of agonist stimulation, as shown in Fig. 5. Agonist treatment failed to significantly change the intracellular colocalization pattern of both proteins. We observed that colocalization of TPb (green) with Prx-4 (purple) occurred for the major part in the ER (red), as indicated by the white-colored region (Fig. 5). Co-localization of the receptor with Prx-4 also appeared in some cells in a compartment reminiscent of the Golgi (data not shown). We have previously demonstrated that the trafficking of TPb present at the cell surface can be followed by immunofluorescence microscopy by labelling the receptor with an anti-HA antibody at 16 C prior to constitutive or ago-
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868
nist-induced internalization of the receptor at 37 C [20,21]. TPb trafficking from the cell surface was never found to colocalize with Prx-4 (Fig. 6). Interestingly, these results indicate that TPb and Prx-4 interact intracellularly, predominantly at the ER, during synthesis/maturation of the receptor protein, and not during the internalization and recycling of the receptor from and back to the cell surface, respectively. 3.6. Prx-4 targets TPb for degradation during oxidative stress Proteins accumulating at the ER can be targeted to the ERAD (endoplasmic reticulum-associated degradation), which employs a series of processes leading to protein degradation by the proteasome pathway. Of relevance to our study, the
Fig. 5. Prx-4 and TPb co-localize in the endoplasmic reticulum. HEK293 cells transiently co-expressing GFP-tagged TPb and Prx-4myc were processed for immunofluorescence microscopy on an inverted Nikon Eclipse TE-2000 microscope as described in Section 2. Prx-4 and calnexin A were visualized with mouse monoclonal anti-Myc and rabbit polyclonal anti-calnexin A antibodies, followed by goat anti-mouse Cascade Blue- and goat anti-rabbit Texas Red-conjugated antibodies, respectively. The receptor is thus seen in green, Prx-4 in purple, and calnexin A in red. Colocalization of the three proteins is detected by the presence of white color. Results are representative of three independent experiments.
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868
Fig. 6. Cell surface TPb do not co-localize with Prx-4 after agonistinduced endocytosis. HEK293 cells were co-transfected with pcDNA3HA-tagged TPb and pcDNA3-Prx-4myc. Cell surface receptors were labelled with a monoclonal anti-HA antibody prior to stimulating the cells with 100 nM U46619 for 60 min, as we detailed before [20,21]. The cells were then processed for immunofluorescence microscopy with a polyclonal anti-myc antibody, followed by incubation with goat anti-mouse fluorescein- and goat anti-rabbit Texas-Red-conjugated antibodies. The receptors are seen in green while Prx-4 is visualized in red. Results are representative of three separate experiments.
ERAD response is regulated by the redox potential of a cell [22]. Because Prx-4 co-localized with TPb at the ER and inhib-
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ited its cell surface expression, we went on to verify if Prx-4 could modulate the level of receptor protein during oxidative stress. HEK293 cells expressing the HA-tagged receptor alone or in combination with Myc-Prx4 were treated with 10 lM H2O2 or with H2O (vehicle) for different time intervals. The cells were then lysed and TPb protein levels determined by Western blots. Stimulation of the cells with H2O2 did not affect the TPb-Prx-4 co-immunoprecipitation (data not shown). It can be seen in Fig. 7 that treatment of the cells with H2O (vehicle) had no significant effect on levels of receptor protein. The addition of H2O2 to cells expressing the receptor alone caused a small reduction in the amount of receptor protein detected in the cell lysates. Surprisingly, co-expression of Prx-4 largely reduced the total amount of detectable TPb protein in the cell lysates after 180 min of H2O2 treatment, indicating that Prx-4 targets the receptor to degradation during oxidative stress. Prxs are ubiquitously and abundantly expressed in the various tissues of the human body [14]. These enzymes are produced at high levels in cells: they are the second or third most abundant protein in erythrocytes and compose 0.1– 0.8% of the soluble protein in other mammalian cells [11]. Many organisms produce more than one isoform of Prx, including at least six Prxs identified in mammalian cells (Prx1 to Prx-6). Although located primarily in the cytosol, Prxs are also found within mitochondria and peroxisomes, and associated with nuclei and membranes [13]. Prxs contain a reactive Cys in a conserved region near the N-terminus, and this catalytic Cys residue forms cysteine-sulfenic acid as a reaction intermediate during the reduction of peroxide. All the Prxs exhibit peroxidase activity dependent on reduced forms of thioredoxin and/or glutathione. Prx-1–Prx-4 have an additional Cys at a conserved position near the C-terminus. Prx1–Prx-4 are cross members of the same family and form a
Fig. 7. Co-expression of Prx-4 targets TPb for degradation during oxidative stress. HEK293 cells co-transfected with the indicated constructs were treated with 1 lM H2O2 or H2O (vehicle) for different time intervals. Levels of receptor proteins were detected in cell lysates by Western blotting with a monoclonal anti-HA Ab. H2O2 triggers receptor degradation of the receptor only when Prx-4 is co-expressed. Treatment with the vehicle had no effect on receptor protein levels in presence of Prx-4. Results are representative of four independent experiments.
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homodimer with head-to-tail type association. Prx5 and Prx6 are monomeric and presumed to be far from the other Prxs from an evolutionary point of view [24]. Prxs are not redundant functionally [15]. In this context, it is interesting to note that only Prx-4 was pulled out as a specific interacting partner of TPb by the yeast two-hybrid screen. Further experiments will substantiate whether TPb interacts with the other isoforms of the Prxs in a cellular context. Oxidative stress in blood vessels and the kidney can be induced by diverse mechanisms, including activation of the TP receptor [4,5]. The study of the involvement of Prxs in receptor signaling has received considerable attention in the recent years. It was shown that Prx-1 and Prx-2 inhibit EGF- and PDGF-induced H2O2 generation [reviewed in 23]. These and other studies showed that Prxs also suppress downstream signaling responses of receptors, such as apoptosis, NF-kB transcriptional and JNK activities [23]. However, there is a lack of information regarding a direct mechanism by which Prxs influence receptor-mediated signaling. Here we report for the first time that an anti-oxidant enzyme, Prx-4, interacts with and regulates a pro-oxidant receptor, TPb. This interaction may correspond to one mechanism by which a cell could cope with oxidative stress. By interacting intracellularly with a receptor, Prx-4 could limit the responsiveness of the receptor involved in generating oxidative stress by regulating its cell surface expression. Moreover, Prx-4 could put a stop to an amplification loop by targeting the receptor for degradation during oxidative stress. Acknowledgements: This work was supported by a grant from the Kidney Foundation of Canada to J.L.P. The authors also thank the NSERC and the Heart & Stroke Foundation of Canada for the studentships to A. Parent and G. Laroche, respectively. J.L.P benefited from a CIHR New Investigator Award and a Chercheur BoursierJunior 2 from FRSQ during the completion of this work.
References [1] Wise, H., Wong, Y.H. and Jones, R.L. (2002) Prostanoid signal integration and cross talk. Neurosignals 11, 20–28. [2] Hirata, M., Hayashi, Y., Ushikubi, F., Yokota, Y., Kageyama, R., Nakanishi, S. and Narumiya, S. (1991) Cloning and expression of cDNA for a human thromboxane A2 receptor. Nature 349, 617–620. [3] Raychowdhury, M.K., Yukawa, M., Collins, L.J., McGrail, S.H., Kent, K.C. and Ware, J.A. (1994) Alternative splicing produces a divergent cytoplasmic tail in the human endothelial thromboxane A2 receptor. J. Biol. Chem. 269, 19256–19261, Correction (1995) J. Biol. Chem. 270, 7011. [4] Wilcox, C.S. (2002) Reactive oxygen species: roles in blood pressure and kidney function. Curr. Hypertension Rep. 4, 160– 166. [5] Ganafa, A.A., Socci, R.R., Eatman, D., Silvestora, N., Abukhalaf, I.K. and Bayorh, M.A. (2002) Acute inhibition of glutathione biosynthesis alters endothelial function and blood pressure in rats. Eur. J. Pharmacol. 454, 217–223. [6] Morrow, J.D. and Roberts II, L.J. (1996) The isoprostanes. Current knowledge and directions for future research. Biochem. Pharmacol. 51, 1–9. [7] Habib, A. and Badr, K.F. (2004) Molecular pharmacology of isoprostanes in vascular smooth muscle. Chem. Phys. Lipids 128, 69–73.
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868 [8] Weber, T.J. and Markillie, L.M. (2003) Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner. Mol. Pharmacol. 63, 1075–1081. [9] Kromer, B.M. and Tippins, J.R. (1996) Coronary artery constriction by the isoprostane 8-epi prostaglandin F2 alpha. Br. J. Pharmacol. 122, 899–905. [10] Audoly, L.P., Rocca, B., Fabre, J.E., Koller, B.H., Thomas, D., Loeb, A.L., Coffman, T.M. and FitzGerald, G.A. (2000) Cardiovascular responses to the isoprostanes iPF(2alpha)-III and iPE(2)III are mediated via the thromboxane A(2) receptor in vivo. Circulation 101, 2833–2840. [11] Chae, H.Z., Robison, K., Poole, L.B., Church, G., Storz, G. and Rhee, S.G. (1994) Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc. Natl Acad. Sci. USA 91, 7017– 7021. [12] Chae, H.Z., Chung, S.J. and Rhee, S.G. (1994) Thioredoxindependent peroxide reductase from yeast. J. Biol. Chem. 269, 27670–27678. [13] Hofmann, B., Hecht, H.J. and Flohe´, L. (2002) Peroxiredoxins. Biol. Chem. 383, 347–364. [14] Veal, E.A., Findlay, V.J., Day, A.M., Bozonet, S.M., Evans, J.M., Quinn, J. and Morgan, B.A. (2004) A 2-cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stressactivated MAP kinase mol. Cell 15, 129–139. [15] Shen, C. and Nathan, C. (2002) Nonredundant antioxidant defense by multiple two-cysteine peroxiredoxins in human prostate cancer cells. Mol. Med. 8, 95–102. [16] Rochdi, M.D., Laroche, G., Dupre´, E., Gigue`re, P., Lebel, A., Watier, V., Hamelin, E., Le´pine, M.-C., Dupuis, G. and Parent, J.-L. (2004) Nm23-H2 interacts with a G protein-coupled receptor to regulate its endocytosis through a Rac1-dependent mechanism. J. Biol. Chem. 279, 18981–18989. [17] Hamelin, E., Theriault, C., Laroche, G. and Parent, J.L. (2005) The intracellular trafficking of the G protein-coupled receptor TPbeta depends on a direct interaction with Rab11. J. Biol. Chem. 280, 36195–36205. [18] Rochdi, M.D., Watier, V., La Madeleine, C., Nakata, H., Kozasa, T. and Parent, J.-L. (2002) Regulation of GTP-binding protein aq (Gaq) signaling by the ezrin–radixin–moesin-binding phosphoprotein-50 (EBP50). J. Biol. Chem. 277, 40751– 40759. [19] Laroche, G., Rochdi, M.D., Laporte, S.A. and Parent, J.L. (2005) Involvement of actin in agonist-induced endocytosis of the G protein-coupled receptor for thromboxane A2: overcoming of actin disruption by arrestin-3 but not arrestin-2. J. Biol. Chem. 280, 23215–23224. [20] Parent, J.-L., Labrecque, P., Rochdi, M.D. and Benovic, J.L. (2001) Role of the differentially spliced carboxyl terminus in thromboxane A2 receptor trafficking: identification of a distinct motif for tonic internalization. J. Biol. Chem. 276, 7079– 7085. [21] Parent, J.L., Labrecque, P., Orsini, M.J. and Benovic, J.L. (1999) Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced COOH terminus in agonistpromoted receptor internalization. J. Biol. Chem. 274, 8941– 8948. [22] Molinari, M., Galli, C., Piccaluga, V., Pieren, M. and Paganetti, P. (2002) Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER. J. Cell Biol. 158, 247–257. [23] Kang, S.W., Rhee, S.G., Chang, T.S., Jeong, W. and Choi, M.H. (2005) 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends Mol. Med. 11, 571–578. [24] Fujii, J. and Ikeda, Y. (2002) Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep. 7, 123–130.
Peroxiredoxin-4 interacts with and regulates the thromboxane A2 receptor Patrick Gigue`rea,1, Marie-Eve Turcottea,1, Emilie Hamelina, Audrey Parenta, Jessy Brissona, Genevie`ve Larochea, Pascale Labrecquea, Gilles Dupuisb, Jean-Luc Parenta,* a
Service de Rhumatologie, De´partement de Me´decine, Faculte´ de Me´decine and Centre de Recherche Clinique-E´tienne Lebel, Que´bec, Canada b Programme d’Immunologie, Universite´ de Sherbrooke, Que´bec, Canada Received 21 February 2007; revised 6 June 2007; accepted 3 July 2007 Available online 17 July 2007 Edited by Christian Griesinger
Abstract We identified peroxiredoxin-4 (Prx-4) as a protein interacting with the b isoform of the thromboxane A2 receptor (TPb) by yeast two-hybrid analysis. Prx-4 co-immunoprecipitated constitutively with TPb in HEK293 cells. The second and third intracellular loops as well as the C-terminus of TPb interacted directly with Prx-4. Co-expression of Prx-4 caused a 60% decrease in cell surface expression of TPb. Prx-4 and TPb predominantly co-localized in the endoplasmic reticulum. Coexpression of Prx-4 in cells treated with H2O2 targeted TPb for degradation. We show for the first time an interaction between a receptor involved in oxidative stress and Prx-4, an anti-oxidative enzyme. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: GPCR; G protein-coupled receptor; Peroxiredoxin; Oxidative stress; Trafficking; Signalling
1. Introduction Thromboxane A2 (TXA2) is the sequential metabolic product of arachidonic acid by the cyclooxygenases and thromboxane synthase. The TXA2 receptor (TP) is a G protein-coupled receptor (GPCR) that can couple to the G proteins Gaq, Gas, Gai2, Ga12, Ga13, and Ga16[1]. The TP receptor is expressed as two alternatively spliced isoforms, a (343 residues) and b (407 residues) that share the first 328 residues[2,3]. Oxidative stress can be induced by diverse mechanisms, including activation of TP [4,5]. A major product of peroxidation is a group of compounds termed isoprostanes. Isoprostanes are mainly derived by free radical-mediated oxidation of arachidonic acid [6]. The isoprostanes exert significant biological activity and there is evidence that they interact with TP [7–10].
* Corresponding author. Fax: +1 819 564 5265. E-mail address: [email protected] (J.-L. Parent). 1
These authors contributed equally to this work.
Abbreviations: GPCR, G protein-coupled receptor; TP, thromboxane A2 receptor; TXA2, thromboxane A2; TPbCT, C-terminal tail of TPb; GST, glutathione-S-transferase; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; HA, hemagglutinin; ER, endoplasmic reticulum; ICL, intracellular loop of the TPb receptor
Peroxiredoxins (Prxs) have recently been the subject of growing interest as a new family of thiol-specific antioxidant proteins [11,12]. Prxs perform their protective antioxidant role in cells through their peroxidase activity whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides are reduced and detoxified [13]. At least six Prxs were identified in mammalian cells (Prx1–Prx6). Prxs are ubiquitously and abundantly expressed in the various tissues of the human body [14] and were reported to be not redundant functionally [15]. The data that we present here show that an anti-oxidant enzyme, Prx-4, can regulate the biology of TPb, a GPCR mediating oxidative stress.
2. Materials and methods 2.1. Materials The HA- and Myc-specific monoclonal antibodies were from Covance. The rabbit anti-calnexin A polyclonal antibody was acquired from Sigma. The Cascade Blue goat anti-mouse, goat anti-rabbit Texas Red, and fluorescein isothiocyanate (FITC)-conjugated goat antimouse antibodies were purchased from Molecular Probes. The goat anti-mouse alkaline-phosphatase-conjugated antibody and the alkaline phosphatase substrate kit were obtained from Bio-Rad. U46619 was from Cayman. 2.2. Two-hybrid screening assay Yeast two-hybrid screening was performed with the C-terminus of TPb as a bait on a Human HeLa MATCHMAKER cDNA Library (Clontech) as we described previously [16]. 2.3. Cell culture and transfection HEK293 cells were maintained and transfected as detailed before [16]. 2.4. Immunoprecipitations 6-Well plates of HEK293 cells were transfected with the indicated combinations of constructs. Transfected cells were maintained for 48 h and incubated for 0–30 min at 37 C in the presence of 1 lM U46619 prior to harvesting. Thereafter, the cells were processed for immunoprecipitations as we reported previously [16]. 2.5. Recombinant protein production and binding assays A pRSET A-myc-Prx-4 construct was used to produce a His-tagged myc-Prx-4 fusion protein in OverExpress C41(DE3) Escherichia coli strain (Avidis, SA) by following the manufacturer’s instructions. Amino acids 53–66 (ICL1), 129–150 (ICL2), 217–244 (ICL3), and 312–407 (C-tail) were produced as fusion proteins with glutathione-S-transferase (GST) and the pulldown assays performed as we described before [17].
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.011
3864 2.6. Inositol phosphates measurements Inositol phosphates measurements were carried out as we detailed previously [18]. 2.7. Endocytosis and cell surface expression assays Endocytosis and cell surface expression of receptors was measured by enzyme-linked immunosorbent assay (ELISA) using transiently transfected HEK293 cells as we previously reported [17]. 2.8. Immunofluorescence microscopy 1.0 · 106 HEK293 cells were transiently co-transfected with the pcDNA3-HA-TPb green fluorescent protein (GFP) [19] and pcDNA3-myc-Prx-4 constructs and maintained overnight.The cells were then processed for immunofluorescence microscopy as we detailed before on a Nikon Eclipse TE2000-U microscope using a 60· objective, and images were collected using SimplePCI Camera software and processed with Adobe Photoshop software [17].
3. Results and discussion 3.1. Prx-4 interacts with TPb We recently showed that the C-terminus conferred specific regulatory mechanisms to TPb [20,21]. In our efforts to elucidate the mechanisms involved in the regulation of TPb, we performed yeast two-hybrid screening experiments using the yeast strain pJ69-4a transformed with pAS2.1-TPbCT and a human HeLa cell cDNA library. A total of 1.5 · 106 independent clones were screened yielding over 300 positives. One hundred clones demonstrating a strong growth on selective yeast medium (TRP , LEU , HIS , and ADE ) were then isolated and characterized by dideoxy sequencing, and then aligned using the NCBI blast alignment search tool. Four clones contained a cDNA fragment coding for the Prx-4 protein. Other members of the Prx protein family were not isolated in the clones that were sequenced.
Fig. 1. Prx-4 co-immunoprecipitates with TPb. HEK293 cells were transiently transfected with the indicated constructs. Forty-eight hours post-transfection, the cells were stimulated or not with 1 lM U46619, lysed and processed for immunoprecipitation of the receptor with a HA-specific polyclonal antibody. Immunoblotting was performed with HA-specific polyclonal or Myc-specific monoclonal antibodies as described in Section 2. Data presented are representative of three different experiments. IP, immunoprecipitation; IB, immunoblotting.
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3.2. Co-immunoprecipitations and in vitro binding assay To investigate the interaction of Prx-4 with TPb in a cellular context, we performed immunoprecipitation experiments in HEK293 cells transfected with pcDNA3-Myc-Prx-4 and pcDNA3-HA-TPb in the presence or absence of stimulation with the stable TP receptor agonist, U46619. Our results demonstrate that Myc-Prx-4 was co-immunoprecipitated with TPb in absence of agonist (Fig. 1). Agonist treatment did not modulate the interaction between the two proteins (Fig. 1). To further characterize the interaction of Prx-4 with the TPb receptor, we carried out an in vitro binding assay using the purified recombinant intracellular loop 1 (ICL1), ICL2, ICL3, or C-tail (CT) of TPb fused to GST along with purified His6-Myc-Prx-4 protein. A schematic representation of the TPb topology is illustrated in Fig. 2A. The results showed that Prx-4 interacted specifically with glutathione sepharose-bound GST-TPb-ICL2, -ICL3, and -CT but not with glutathione sepharose-bound GST or GST-TPb-ICL1 (Fig. 2B). Taken together, our data show that Prx-4 interacts directly with intracellular domains of TPb, predominantly with the C-tail, in an agonist-independent manner. Interestingly, this consti-
Fig. 2. Prx-4 directly interacts with intracellular domains of TPb. (A) Schematic representation of the topology of TPb. The receptor is composed of seven transmembrane domains connected by three extracellular and three intracellular loops. The C-terminus completes the intracellular domains of the receptor. The numbers indicate the first and the last of the amino acids for each intracellular domain that were used to produce fusion proteins with GST. (B) The intracellular loops 1–3 (ICL1, ICL2, and ICL3) and the C-tail of the receptor were produced as GST-fusion proteins and used to perform GST-pulldown assays on purified His6-Prx-4myc protein. The upper panel shows the Prx-4 binding to the receptor domains, while the GST-fusion proteins present in the reaction can be seen in the middle panel. The bottom panel shows the level of Prx-4 protein present in the initial reaction. Data presented are representative of three different experiments. IB, immunoblotting.
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tutes the first study whereby a direct interaction of a Prx protein with a G protein-coupled receptor, and to our knowledge to any transmembrane receptor, is shown. 3.3. Regulation of TPb receptor signaling by Prx-4 Members of the Prx protein family have been shown to regulate the signaling of various classes of receptors, including the receptors for EGF, PDGF, and TNFa (reviewed in Ref. [23]). We thus assessed if Prx-4 could modulate the generation of inositol phosphates by TPb following agonist stimulation. Fig. 3A illustrates that co-expression of Prx-4 with the receptor in HEK293 cells resulted in a 70% inhibition of receptormediated production of inositol phosphates following agonist treatment when compared to cells expressing the receptor alone. Production of inositol phosphates by a Gaq-coupled receptor involves the receptor-Gaq interaction and activation of the G protein, with subsequent stimulation of phospholipase Cb by the activated Gaq. Interestingly, Prx-4 overexpression did not affect the generation of inositol phosphates by a constitutive mutant of Gaq, Gaq-R183C (Fig. 3B). This indicated that Prx-4 was not acting at the level of the phospholipase Cb activation by Gaq, but rather at the level of the receptor itself. Co-immunoprecipitation experiments demonstrated that the TPb receptor-Gaq interaction was not pre-
Fig. 3. Regulation of TPb signaling by Prx-4. HEK293 cells were transiently co-transfected with the indicated constructs and labelled with 3H-myo-inositol. Total inositol phosphates were measured after treatment of the cells with 500 nM U46619 for 30 min (A) or after 30 min of accumulation in presence of the constitutive Gq-R183C mutant (B) as described in Section 2. Results are means ± S.E. from five different experiments.
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vented by co-expression of Prx-4 (data not shown). Thus, this suggested that Prx-4 was acting on the receptor to suppress its signaling but not by interfering with its interaction with the Gaq protein. 3.4. Regulation of TPb cell surface expression by Prx-4 Since the response of a cell surface receptor to an agonist is controlled in part by the number of receptors present at the plasma membrane, we next studied whether Prx-4 could alter the internalization and/or the cell surface expression of TPb. HEK293 cells expressing HA-tagged TPb alone or in combination with Prx-4 were stimulated with 100 nM U46619 or ethanol (vehicle) for 120 min and receptors present at the cell surface measured by ELISA, as we did before [16,19–21]. Prx-4 expression had no effect on internalization of the receptor (Fig. 4A). On the other hand, the expression of TPb at the cell surface was reduced by roughly 60% when Prx-4 was present (Fig. 4B), which is most likely the cause of the reduced receptor-mediated signaling after agonist treatment. 3.5. TPb and Prx-4 co-localize intracellularly Given that our results suggested that Prx-4 inhibited the cell surface expression of TPb, we were interested in determining if and where the two proteins co-localized in the cell. Fluorescence microscopy experiments were performed on HEK293 cells co-expressing GFP-tagged TPb [19] and Myc-Prx-4. Initial experiments showed that the two proteins co-localized in an intracellular compartment that could be the endoplasmic reticulum (ER). Three-color fluorescence microscopy,
Fig. 4. Prx-4 overexpression inhibits TPb cell surface expression. HEK293 cells were transiently co-transfected with the HA-tagged TPb and pcDNA3 or pcDNA3-Prx-4myc. The percentage of cell surface receptor loss following a 2 h stimulation with 100 nM U46619 (A) or cell surface expression (B) was measured by ELISA as described in Section 2. Results are means ± S.E. from six different experiments.
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including labelling of calnexin A as an ER-marker, was carried out. The receptor and Prx-4 co-localized intracellularly in absence of agonist stimulation, as shown in Fig. 5. Agonist treatment failed to significantly change the intracellular colocalization pattern of both proteins. We observed that colocalization of TPb (green) with Prx-4 (purple) occurred for the major part in the ER (red), as indicated by the white-colored region (Fig. 5). Co-localization of the receptor with Prx-4 also appeared in some cells in a compartment reminiscent of the Golgi (data not shown). We have previously demonstrated that the trafficking of TPb present at the cell surface can be followed by immunofluorescence microscopy by labelling the receptor with an anti-HA antibody at 16 C prior to constitutive or ago-
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868
nist-induced internalization of the receptor at 37 C [20,21]. TPb trafficking from the cell surface was never found to colocalize with Prx-4 (Fig. 6). Interestingly, these results indicate that TPb and Prx-4 interact intracellularly, predominantly at the ER, during synthesis/maturation of the receptor protein, and not during the internalization and recycling of the receptor from and back to the cell surface, respectively. 3.6. Prx-4 targets TPb for degradation during oxidative stress Proteins accumulating at the ER can be targeted to the ERAD (endoplasmic reticulum-associated degradation), which employs a series of processes leading to protein degradation by the proteasome pathway. Of relevance to our study, the
Fig. 5. Prx-4 and TPb co-localize in the endoplasmic reticulum. HEK293 cells transiently co-expressing GFP-tagged TPb and Prx-4myc were processed for immunofluorescence microscopy on an inverted Nikon Eclipse TE-2000 microscope as described in Section 2. Prx-4 and calnexin A were visualized with mouse monoclonal anti-Myc and rabbit polyclonal anti-calnexin A antibodies, followed by goat anti-mouse Cascade Blue- and goat anti-rabbit Texas Red-conjugated antibodies, respectively. The receptor is thus seen in green, Prx-4 in purple, and calnexin A in red. Colocalization of the three proteins is detected by the presence of white color. Results are representative of three independent experiments.
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Fig. 6. Cell surface TPb do not co-localize with Prx-4 after agonistinduced endocytosis. HEK293 cells were co-transfected with pcDNA3HA-tagged TPb and pcDNA3-Prx-4myc. Cell surface receptors were labelled with a monoclonal anti-HA antibody prior to stimulating the cells with 100 nM U46619 for 60 min, as we detailed before [20,21]. The cells were then processed for immunofluorescence microscopy with a polyclonal anti-myc antibody, followed by incubation with goat anti-mouse fluorescein- and goat anti-rabbit Texas-Red-conjugated antibodies. The receptors are seen in green while Prx-4 is visualized in red. Results are representative of three separate experiments.
ERAD response is regulated by the redox potential of a cell [22]. Because Prx-4 co-localized with TPb at the ER and inhib-
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ited its cell surface expression, we went on to verify if Prx-4 could modulate the level of receptor protein during oxidative stress. HEK293 cells expressing the HA-tagged receptor alone or in combination with Myc-Prx4 were treated with 10 lM H2O2 or with H2O (vehicle) for different time intervals. The cells were then lysed and TPb protein levels determined by Western blots. Stimulation of the cells with H2O2 did not affect the TPb-Prx-4 co-immunoprecipitation (data not shown). It can be seen in Fig. 7 that treatment of the cells with H2O (vehicle) had no significant effect on levels of receptor protein. The addition of H2O2 to cells expressing the receptor alone caused a small reduction in the amount of receptor protein detected in the cell lysates. Surprisingly, co-expression of Prx-4 largely reduced the total amount of detectable TPb protein in the cell lysates after 180 min of H2O2 treatment, indicating that Prx-4 targets the receptor to degradation during oxidative stress. Prxs are ubiquitously and abundantly expressed in the various tissues of the human body [14]. These enzymes are produced at high levels in cells: they are the second or third most abundant protein in erythrocytes and compose 0.1– 0.8% of the soluble protein in other mammalian cells [11]. Many organisms produce more than one isoform of Prx, including at least six Prxs identified in mammalian cells (Prx1 to Prx-6). Although located primarily in the cytosol, Prxs are also found within mitochondria and peroxisomes, and associated with nuclei and membranes [13]. Prxs contain a reactive Cys in a conserved region near the N-terminus, and this catalytic Cys residue forms cysteine-sulfenic acid as a reaction intermediate during the reduction of peroxide. All the Prxs exhibit peroxidase activity dependent on reduced forms of thioredoxin and/or glutathione. Prx-1–Prx-4 have an additional Cys at a conserved position near the C-terminus. Prx1–Prx-4 are cross members of the same family and form a
Fig. 7. Co-expression of Prx-4 targets TPb for degradation during oxidative stress. HEK293 cells co-transfected with the indicated constructs were treated with 1 lM H2O2 or H2O (vehicle) for different time intervals. Levels of receptor proteins were detected in cell lysates by Western blotting with a monoclonal anti-HA Ab. H2O2 triggers receptor degradation of the receptor only when Prx-4 is co-expressed. Treatment with the vehicle had no effect on receptor protein levels in presence of Prx-4. Results are representative of four independent experiments.
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homodimer with head-to-tail type association. Prx5 and Prx6 are monomeric and presumed to be far from the other Prxs from an evolutionary point of view [24]. Prxs are not redundant functionally [15]. In this context, it is interesting to note that only Prx-4 was pulled out as a specific interacting partner of TPb by the yeast two-hybrid screen. Further experiments will substantiate whether TPb interacts with the other isoforms of the Prxs in a cellular context. Oxidative stress in blood vessels and the kidney can be induced by diverse mechanisms, including activation of the TP receptor [4,5]. The study of the involvement of Prxs in receptor signaling has received considerable attention in the recent years. It was shown that Prx-1 and Prx-2 inhibit EGF- and PDGF-induced H2O2 generation [reviewed in 23]. These and other studies showed that Prxs also suppress downstream signaling responses of receptors, such as apoptosis, NF-kB transcriptional and JNK activities [23]. However, there is a lack of information regarding a direct mechanism by which Prxs influence receptor-mediated signaling. Here we report for the first time that an anti-oxidant enzyme, Prx-4, interacts with and regulates a pro-oxidant receptor, TPb. This interaction may correspond to one mechanism by which a cell could cope with oxidative stress. By interacting intracellularly with a receptor, Prx-4 could limit the responsiveness of the receptor involved in generating oxidative stress by regulating its cell surface expression. Moreover, Prx-4 could put a stop to an amplification loop by targeting the receptor for degradation during oxidative stress. Acknowledgements: This work was supported by a grant from the Kidney Foundation of Canada to J.L.P. The authors also thank the NSERC and the Heart & Stroke Foundation of Canada for the studentships to A. Parent and G. Laroche, respectively. J.L.P benefited from a CIHR New Investigator Award and a Chercheur BoursierJunior 2 from FRSQ during the completion of this work.
References [1] Wise, H., Wong, Y.H. and Jones, R.L. (2002) Prostanoid signal integration and cross talk. Neurosignals 11, 20–28. [2] Hirata, M., Hayashi, Y., Ushikubi, F., Yokota, Y., Kageyama, R., Nakanishi, S. and Narumiya, S. (1991) Cloning and expression of cDNA for a human thromboxane A2 receptor. Nature 349, 617–620. [3] Raychowdhury, M.K., Yukawa, M., Collins, L.J., McGrail, S.H., Kent, K.C. and Ware, J.A. (1994) Alternative splicing produces a divergent cytoplasmic tail in the human endothelial thromboxane A2 receptor. J. Biol. Chem. 269, 19256–19261, Correction (1995) J. Biol. Chem. 270, 7011. [4] Wilcox, C.S. (2002) Reactive oxygen species: roles in blood pressure and kidney function. Curr. Hypertension Rep. 4, 160– 166. [5] Ganafa, A.A., Socci, R.R., Eatman, D., Silvestora, N., Abukhalaf, I.K. and Bayorh, M.A. (2002) Acute inhibition of glutathione biosynthesis alters endothelial function and blood pressure in rats. Eur. J. Pharmacol. 454, 217–223. [6] Morrow, J.D. and Roberts II, L.J. (1996) The isoprostanes. Current knowledge and directions for future research. Biochem. Pharmacol. 51, 1–9. [7] Habib, A. and Badr, K.F. (2004) Molecular pharmacology of isoprostanes in vascular smooth muscle. Chem. Phys. Lipids 128, 69–73.
P. Gigue`re et al. / FEBS Letters 581 (2007) 3863–3868 [8] Weber, T.J. and Markillie, L.M. (2003) Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner. Mol. Pharmacol. 63, 1075–1081. [9] Kromer, B.M. and Tippins, J.R. (1996) Coronary artery constriction by the isoprostane 8-epi prostaglandin F2 alpha. Br. J. Pharmacol. 122, 899–905. [10] Audoly, L.P., Rocca, B., Fabre, J.E., Koller, B.H., Thomas, D., Loeb, A.L., Coffman, T.M. and FitzGerald, G.A. (2000) Cardiovascular responses to the isoprostanes iPF(2alpha)-III and iPE(2)III are mediated via the thromboxane A(2) receptor in vivo. Circulation 101, 2833–2840. [11] Chae, H.Z., Robison, K., Poole, L.B., Church, G., Storz, G. and Rhee, S.G. (1994) Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc. Natl Acad. Sci. USA 91, 7017– 7021. [12] Chae, H.Z., Chung, S.J. and Rhee, S.G. (1994) Thioredoxindependent peroxide reductase from yeast. J. Biol. Chem. 269, 27670–27678. [13] Hofmann, B., Hecht, H.J. and Flohe´, L. (2002) Peroxiredoxins. Biol. Chem. 383, 347–364. [14] Veal, E.A., Findlay, V.J., Day, A.M., Bozonet, S.M., Evans, J.M., Quinn, J. and Morgan, B.A. (2004) A 2-cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stressactivated MAP kinase mol. Cell 15, 129–139. [15] Shen, C. and Nathan, C. (2002) Nonredundant antioxidant defense by multiple two-cysteine peroxiredoxins in human prostate cancer cells. Mol. Med. 8, 95–102. [16] Rochdi, M.D., Laroche, G., Dupre´, E., Gigue`re, P., Lebel, A., Watier, V., Hamelin, E., Le´pine, M.-C., Dupuis, G. and Parent, J.-L. (2004) Nm23-H2 interacts with a G protein-coupled receptor to regulate its endocytosis through a Rac1-dependent mechanism. J. Biol. Chem. 279, 18981–18989. [17] Hamelin, E., Theriault, C., Laroche, G. and Parent, J.L. (2005) The intracellular trafficking of the G protein-coupled receptor TPbeta depends on a direct interaction with Rab11. J. Biol. Chem. 280, 36195–36205. [18] Rochdi, M.D., Watier, V., La Madeleine, C., Nakata, H., Kozasa, T. and Parent, J.-L. (2002) Regulation of GTP-binding protein aq (Gaq) signaling by the ezrin–radixin–moesin-binding phosphoprotein-50 (EBP50). J. Biol. Chem. 277, 40751– 40759. [19] Laroche, G., Rochdi, M.D., Laporte, S.A. and Parent, J.L. (2005) Involvement of actin in agonist-induced endocytosis of the G protein-coupled receptor for thromboxane A2: overcoming of actin disruption by arrestin-3 but not arrestin-2. J. Biol. Chem. 280, 23215–23224. [20] Parent, J.-L., Labrecque, P., Rochdi, M.D. and Benovic, J.L. (2001) Role of the differentially spliced carboxyl terminus in thromboxane A2 receptor trafficking: identification of a distinct motif for tonic internalization. J. Biol. Chem. 276, 7079– 7085. [21] Parent, J.L., Labrecque, P., Orsini, M.J. and Benovic, J.L. (1999) Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced COOH terminus in agonistpromoted receptor internalization. J. Biol. Chem. 274, 8941– 8948. [22] Molinari, M., Galli, C., Piccaluga, V., Pieren, M. and Paganetti, P. (2002) Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER. J. Cell Biol. 158, 247–257. [23] Kang, S.W., Rhee, S.G., Chang, T.S., Jeong, W. and Choi, M.H. (2005) 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends Mol. Med. 11, 571–578. [24] Fujii, J. and Ikeda, Y. (2002) Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep. 7, 123–130.