ER‐anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
2021; Springer Nature; Volume: 40; Issue: 16 Linguagem: Inglês
10.15252/embj.2020107403
ISSN1460-2075
AutoresShengkai Zuo, Bei Wang, Jiao Liu, Deping Kong, Hui Cui, Yao-Nan Jia, Chenyao Wang, Xin Xu, Guilin Chen, Yuanyang Wang, Linlin Yang, Kai Zhang, Ding Ai, Jie Du, Yujun Shen, Ying Yu,
Tópico(s)Occupational and environmental lung diseases
ResumoArticle5 July 2021free access Source DataTransparent process ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6 Shengkai Zuo Shengkai Zuo Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Bei Wang Bei Wang Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Jiao Liu Jiao Liu Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Deping Kong Deping Kong Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Hui Cui Hui Cui School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Yaonan Jia Yaonan Jia School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China Search for more papers by this author Chenyao Wang Chenyao Wang Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Xin Xu Xin Xu orcid.org/0000-0002-8840-4813 Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Guilin Chen Guilin Chen Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Yuanyang Wang Yuanyang Wang Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Linlin Yang Linlin Yang Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China Search for more papers by this author Kai Zhang Kai Zhang Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Ding Ai Ding Ai Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Jie Du Jie Du Beijing Anzhen Hospital of Capital Medical University and Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China Search for more papers by this author Yujun Shen Corresponding Author Yujun Shen [email protected] orcid.org/0000-0002-9266-9064 Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Ying Yu Corresponding Author Ying Yu [email protected] orcid.org/0000-0002-6476-1752 Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Shengkai Zuo Shengkai Zuo Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Bei Wang Bei Wang Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Jiao Liu Jiao Liu Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Deping Kong Deping Kong Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Hui Cui Hui Cui School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Yaonan Jia Yaonan Jia School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China Search for more papers by this author Chenyao Wang Chenyao Wang Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Xin Xu Xin Xu orcid.org/0000-0002-8840-4813 Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Guilin Chen Guilin Chen Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Yuanyang Wang Yuanyang Wang Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Linlin Yang Linlin Yang Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China Search for more papers by this author Kai Zhang Kai Zhang Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Ding Ai Ding Ai Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Jie Du Jie Du Beijing Anzhen Hospital of Capital Medical University and Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China Search for more papers by this author Yujun Shen Corresponding Author Yujun Shen [email protected] orcid.org/0000-0002-9266-9064 Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Ying Yu Corresponding Author Ying Yu [email protected] orcid.org/0000-0002-6476-1752 Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China Search for more papers by this author Author Information Shengkai Zuo1, Bei Wang1, Jiao Liu1, Deping Kong1, Hui Cui2, Yaonan Jia3, Chenyao Wang4, Xin Xu1, Guilin Chen1, Yuanyang Wang1, Linlin Yang5, Kai Zhang6, Ding Ai7, Jie Du8, Yujun Shen *,1 and Ying Yu *,1 1Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Pharmacology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China 2School of Life Science and Technology, Shanghai Tech University, Shanghai, China 3School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China 4Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 5Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China 6Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China 7Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China 8Beijing Anzhen Hospital of Capital Medical University and Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China **Corresponding author. Tel: +86 22 83336642; E-mail: [email protected] ***Corresponding author. Tel: +86 22 83336627; E-mail: [email protected] The EMBO Journal (2021)40:e107403https://doi.org/10.15252/embj.2020107403 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Excessive deposition of extracellular matrix, mainly collagen protein, is the hallmark of organ fibrosis. The molecular mechanisms regulating fibrotic protein biosynthesis are unclear. Here, we find that chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2), a plasma membrane receptor for prostaglandin D2, is trafficked to the endoplasmic reticulum (ER) membrane in fibroblasts in a caveolin-1-dependent manner. ER-anchored CRTH2 binds the collagen mRNA recognition motif of La ribonucleoprotein domain family member 6 (LARP6) and promotes the degradation of collagen mRNA in these cells. In line, CRTH2 deficiency increases collagen biosynthesis in fibroblasts and exacerbates injury-induced organ fibrosis in mice, which can be rescued by LARP6 depletion. Administration of CRTH2 N-terminal peptide reduces collagen production by binding to LARP6. Similar to CRTH2, bumetanide binds the LARP6 mRNA recognition motif, suppresses collagen biosynthesis, and alleviates bleomycin-triggered pulmonary fibrosis in vivo. These findings reveal a novel anti-fibrotic function of CRTH2 in the ER membrane via the interaction with LARP6, which may represent a therapeutic target for fibrotic diseases. Synopsis The molecular mechanisms underlying excessive collagen biosynthesis and tissue fibrosis remain unclear. This study reports a role for ER-localized prostaglandin D2 receptor CRTH2 in promoting collagen degradation and suppressing tissue fibrosis via blocking the ribonucleoprotein LARP6. Caveolin-1 mediates plasma membrane-to-ER trafficking of CRTH2 in fibroblasts. ER-anchored CRTH2 binds the collagen mRNA recognition motif of LARP6, decreasing collagen biogenesis. CRTH2 depletion enhances injury-induced lung fibrosis in mice in a LARP6-dependent manner. A small molecule screen identifies Bumetanide as anti-fibrotic agent, mimicking CRTH2 binding to LARP6. Introduction Fibrosis is the common pathological feature of many chronic inflammatory diseases. It affects virtually all tissues and organs in the body. Histologically, fibrosis is characterized by the accumulation of excess extracellular matrix (ECM) materials, such as collagen and fibronectin, in the inflamed or damaged tissues. Although this fibrotic response is an indispensable and reversible process for wound healing, if it persists as chronic inflammation or if tissue is severely injured, fibrosis can progressively become irreversible and ultimately lead to organ malfunction and death. Despite considerable progress in the understanding of the molecular signals and cellular mechanisms underlying fibrosis, there are still few effective clinical therapies targeting fibrogenesis (Weiskirchen et al, 2019). In parenchymal organs, tissue damage that includes toxicity or infection leads to an inflammatory reaction by recruiting a variety of immune cells and releasing different biologically active mediators (cytokines and chemokines). The infiltrated immune cells elicit the activation of effector cells, which prime the fibrogenic process. Fibroblasts and differentiated myofibroblasts are the main fibrotic effector cell response for ECM synthesis in many organs (Wynn & Ramalingam, 2012; Rockey et al, 2015). Various precursor cell populations can transit into myofibroblasts or matrix-producing cells. However, resident fibroblasts appear to be the predominant contributing source of myofibroblasts and the resulting tissue fibrosis in diseases, such as systemic sclerosis and idiopathic pulmonary fibrosis (Kendall & Feghali-Bostwick, 2014). Thus, targeting fibroblasts may be a promising option for the treatment of organ fibrosis. Chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2) is the cell membrane receptor for prostaglandin D2 (PGD2). CRTH2 is highly expressed in type 2 helper T cells (Th2), innate lymphoid cells, eosinophils, and basophils (Hirai et al, 2001). The PGD2/CRTH2 axis is involved in type 2 inflammation reactions that occur in asthma and allergic rhinitis (Kupczyk & Kuna, 2017; Marone et al, 2019). Inhibition of CRTH2-mediated Th2 activation ameliorates unilateral ureteral obstruction (UUO)-induced renal fibrosis (Ito et al, 2012). In contrast, genetic deficiency of CRTH2 aggravates bleomycin-induced pulmonary fibrosis in mice, accompanied by reduced infiltration of γδT cells in the lungs (Ito et al, 2012; Ueda et al, 2019). These observations indicate that CRTH2 in different inflammatory cells may function differently in organ fibrogenesis. We and others have found that CRTH2 is also highly expressed in fibroblasts (Maruyama et al, 2008; Zuo et al, 2018a). However, the exact role of CRTH2 in fibroblasts remains unknown in organ fibrosis. In this study, we observed CRTH2 located unexpectedly in endoplasmic reticulum (ER) membrane in fibroblasts, and the ER-anchored CRTH2 suppressed collagen biosynthesis in fibroblasts through binding La ribonucleoprotein domain family member 6 (LARP6) at the RNA recognition motif (RRM). Bumetanide inhibited TGF-β1-induced collagen expression in fibroblasts and reduced bleomycin-induced pulmonary fibrosis in mice by mimicking the structurally binding of CRTH2 with LARP6. These observations indicate that CRTH2 regulates collagen biogenesis in fibroblasts by targeting LARP6 in the ER. Results CRTH2 is sustained on the ER membrane in fibroblasts by caveolin-1 As a G protein-coupled receptor (GPCR), CRTH2 was located in the cell membrane in all cell lines tested (Fig 1A). Interestingly, CRTH2 colocalized with calnexin in the ER membrane in HEK293T, human fibroblasts MRC-5, NIH 3T3, and primary mouse organ fibroblasts (Fig 1A). Western blot analysis verified CRTH2 protein expression in both cytoplasmic and ER membranes in HEK293T and NIH3T3 cells (Fig 1B). We envision ER CRTH2 may be trafficked originally from cell membrane in fibroblasts. Figure 1. CRTH2 is located in the endoplasmic reticulum (ER) and cell membrane in fibroblasts CRTH2 subcellular localization in GFP-fused CRTH2-expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER, and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; and red, calnexin. Scale bar, 20 μm. Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2-expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole-cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively. The effect of TGF-β1 (10 ng/ml) on the expression of caveolin-1 in NIH 3T3 cells. Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/ml) treatment. Red arrows indicate CRTH2 localized in the ER, and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; and red, calnexin. Scale bar, 20 μm. Quantification of CRTH2 colocalization with endoplasmic reticulum after TGF-β1 or vehicle treatment. *P < 0.05 vs vehicle (Mann–Whitney U-test); n = 4 for all groups. Data are expressed as the mean ± standard error of the mean. Source data are available online for this figure. Source Data for Figure 1 [embj2020107403-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Scaffold proteins, such as β-arrestins (β-arrestin-1 and β-arrestin-2) and caveolins (caveolin-1 and caveolin-2), are involved in the mediation of the trafficking of GPCRs (Shenoy & Lefkowitz, 2011; Busija et al, 2017). We observed that caveolin-1 and caveolin-2 were highly expressed, while expressions of β-arrestin-1 and β-arrestin-2 were relatively low in NIH 3T3 fibroblasts (Fig EV1A). Knockdown of β-arrestin-1 or β-arrestin-2 (Fig EV1B and C) did not markedly influence CRTH2 expression on the ER and fibroblast membranes (Fig EV1D and E). However, caveolin-1 knockdown reduced the ER distribution, but increased plasma membrane distribution, of CRTH2 (Fig EV1F–H), while overexpression of caveolin-1 increased CRTH2 accumulation on ER in fibroblasts (Fig EV1I). Density gradient ultracentrifugation further demonstrated colocalization of CRTH2 and caveolin-1 in lipid rafts (Fig EV1J). TGF-β1 treatment suppressed caveolin-1 protein in fibroblasts (Fig 1C), consistent with a previous report (Wang et al, 2006a). TGF-β1 stimulation reduced ER trafficking of CRTH2 protein from cell membrane in fibroblasts (Fig 1D and E). Using HA-tagged CRTH2 mice (Appendix Fig S1A and B), we found that TGF-β1 also inhibited the endogenous CRTH2 translocation in fibrocytes (collagen-1+CD45+CXCR4+) (Appendix Fig S1C and D)–bone marrow-derived fibroblast precursors (Reilkoff et al, 2011). Indeed, pro-fibrotic growth factor PDGF-BB (Leask, 2010) reduced, while anti-fibrotic cytokine IL-10 (Nakagome et al, 2006) promoted the ER trafficking of CRTH2 in fibroblasts (Appendix Fig S2A and B). The collective results indicate that caveolin-1 mediates CRTH2 trafficking from the cytoplasmic membrane to the ER membrane in fibroblasts. Click here to expand this figure. Figure EV1. CRTH2 trafficking to the ER membrane in fibroblasts through caveolin-1 A. Relative mRNA levels of β-arrestin-1 and β-arrestin-2 and caveolin-1 and caveolin-2 in NIH 3T3 cells (n = 4 for all groups). B, C. Knockdown efficiency of β-arrestin-1 (B) and β-arrestin-2(C) siRNA (Si-β-arrestin-1 and Si-β-arrestin-2) detected by qRT–PCR in NIH 3T3 cells. *P < 0.05 vs scramble (two-tailed Student’s t-test); n = 6 for all groups. D, E. Effect of knocking down of β-arrestin-1 and β-arrestin-2 on CRTH2 subcellular distribution in NIH 3T3 cells. F. Knockdown efficiency of caveolin-1 siRNA (Si-caveolin-1) detected by qRT–PCR in NIH 3T3 cells. *P < 0.05 vs scramble (two-tailed Student’s t-test); n = 6 for all groups. G. Knockdown of caveolin-1 reduces CRTH2 expression in the ER fraction in NIH 3T3 cells. H. Quantification of myc-CRTH2 protein densitometry after caveolin-1 knockdown. Calnexin and ATP1B were used as a loading control for densitometry analysis. *P < 0.05 vs scramble (Mann–Whitney U-test); n = 4 for all groups. I. Effect of caveolin-1 overexpression on CRTH2 subcellular distribution in NIH 3T3 cells. J. Western blot analysis of CRTH2 and caveolin-1 cellular distribution in NIH 3T3 cells by fractionation. Myc-tagged CRTH2 was transfected into NIH 3T3 cells. At 48 h after the transfection, cell fractionations were collected from cell lysates by sucrose gradient centrifugation. Data information: All data are expressed as the mean ± standard error of the mean. Source data are available online for this figure. Download figure Download PowerPoint Activation of CRTH2 on plasma membrane triggers intracellular Ca2+ mobilization into the cytosol (Zuo et al, 2018b). However, inhibition of caveolin-1 by genistein did not influence CRTH2 agonist DK-PGD2-boosted cytosolic Ca2+ signals in fibroblasts (Appendix Fig S3A), but did reduce the localization of CRTH2 on ER (Appendix Fig S3B). Moreover, CRTH2 receptor antagonist CAY10595 attenuated DK-PGD2-induced Ca2+ flux (Appendix Fig S3A), but had no effect on ER distribution of CRTH2 in fibroblasts (Appendix Fig S3B). Thus, CRTH2 on ER mediates different cellular signaling from that on plasma membrane in fibroblasts. CRTH2 interacts with LARP6 RRM in the ER membrane To investigate the physiological role of ER CRTH2 in fibroblasts, we tried to identify CRTH2-associated proteins in the ER membrane using co-immunoprecipitation (co-IP) and mass spectrometry (MS). Co-IP revealed the CRTH2 formed a complex with LARP6 and the 78-kDa glucose-regulated protein (GRP78) (Fig 2A–D). Western blot assay followed by co-IP and immune staining confirmed the interaction of CRTH2 with LARP6 in the ER membrane (Fig 2E–G). GPR78 is a chaperone protein located in the ER organelle (Wang et al, 2009). Binding of CRTH2 to GPR78 at the ER was evident (Fig 2H–J). Thus, CRTH2, LARP6, and GRP78 formed a protein complex in the ER organelle. To determine the structural basis for the interaction of the three proteins, we constructed a series of truncated fragments fused with different tags that included glutathione S-transferase (GST), 3×Flag-streptavidin-binding peptide (Flag-SBP), and Myc (Fig EV2A). Protein pulldown assays revealed that an N-terminal region of CRTH2 comprising amino acids 1-36 interacted with the LARP6 RNA RRM in 293T cells (Fig EV2B and C). The interaction was verified by an in vitro binding assay with purified recombinant proteins (Fig EV2D and E). Similarly, we observed that the C-terminal region of CRTH2 directly bound to the substrate binding domain (SBD) of GPR78 (Fig EV2F–I). Despite the association of GRP78 and LARP6 that was evident using the pulldown assay (Appendix Fig S4A and B), direct binding of these two proteins was not detected (Appendix Fig S4C and D). These observations suggest CRTH2 is anchored in the ER by GPR78 and interacts with cytoplasmic LARP6 (Fig 2K). Figure 2. CRTH2 interacts with LARP6 and GRP78 in the ER A. Immunopurification and mass spectrometric analysis of CRTH2-containing protein complex in NIH 3T3 cells. Cellular extracts from NIH 3T3 cells stably expressing myc-CRTH2 protein were immunopurified with anti-myc affinity beads and eluted with myc peptide. The eluates were used for SDS–PAGE, and the gels were silver-stained and analyzed using mass spectrometry. B–D. Representative mass spectrogram of CRTH2 (B), LARP6 (C), and GRP78 (D) from CRTH2-containing protein complex in NIH 3T3 cells. E, F. Co-immunoprecipitation analysis of association of CRTH2 with LARP6. HEK293T cells were co-transfected with Myc-CRTH2- and Flag-LARP6-expressing plasmids. Whole-cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies. G. Subcellular localization of CRTH2 and LARP6 in NIH 3T3 cells. NIH 3T3 cells were transfected with GFP-fused CRTH2-expressing plasmid and fixed and immunostained with antibodies against the indicated proteins. Red arrow indicates colocalization of CRTH2 and LARP6 in the ER. Scale bar, 20 μm. H, I. Co-IP analysis of association of CRTH2 with GPR78. HEK293T cells were co-transfected with Flag-CRTH2- and Myc-GPR78-expressing plasmids. J. Colocalization of CRTH2, LARP6, and GRP78 in NIH 3T3 cells. White arrow indicates colocalization of CRTH2, LARP6, and GRP78. Scale bar, 20 μm. K. Schematic diagram depicting different domains of CRTH2 involved in the interaction with LARP6 and GRP78 in the endoplasmic reticulum membrane. Source data are available online for this figure. Source Data for Figure 2 [embj2020107403-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CRTH2 interacts with LARP6 and GRP78 via distinct structural motifs A. The strategy for GST-CRTH2, 3× Flag-SBP-LARP6, and Myc-GRP78 truncations. N, N terminus; C, C terminus; LA, La domain; RRM, RNA recognition motif; and SBD, substrate binding domain. B, C. Determination of LARP6 and CRTH2 domains required for their interaction with each other. Different LARP6-truncated constructs were co-transfected with GST-tagged CRTH2 (B) and different CRTH2-truncated constructs were co-transfected with 3×Flag-SBP-tagged LARP6 (C) into HEK293T cells. The cells were then subjected to co-immunoprecipitation and Western blot analyses with the indicated antibodies. D, E. GST and MBP pulldown assays to examine the direct interaction of purified LARP6 and CRTH2 fragments. GST agarose beads conjugated with the CRTH2-2 domain were incubated with purified MBP-LARP6-4 or MBP control protein for the GST pulldown analysis (D). The MBP agarose beads conjugated with the LARP6-4 domain were incubated with purified GST-CRTH2-2 or GST control protein for the MBP pulldown analysis (E). F, G. Determination of GRP78 and CRTH2 domains required for their mutual interaction. Different GRP78-truncated constructs were co-transfected with GST-tagged CRTH2 (F) and different CRTH2-truncated constructs were co-transfected with myc-tagged GRP78 (G) into HEK293T cells. The cells were then subjected to co-immunoprecipitation and Western blot analyses with the indicated antibodies. H, I. GST and MBP pulldown assays to examine the direct interactions of purified GRP78 and CRTH2 domains. The GST agarose beads conjugated with the CRTH2-2 domain were incubated with purified MBP-GRP78-4 or MBP control protein for the GST pulldown analysis (H). The MBP agarose beads conjugated with the GRP78-4 domain were incubated with purified GST-CRTH2-2 or GST control protein for the MBP pulldown analysis (I). Source data are available online for this figure. Download figure Download PowerPoint CRTH2 inhibits collagen synthesis in fibroblasts through LARP6 LARP6 regulates the stability of collagen mRNAs through the RRM binding of the 5’ untranslated region (UTR) of these mRNAs (Zhang & Stefanovic, 2016a). Presently, CRTH2 bound competitively to the RRM of LARP6 in the ER membrane (Fig 2). CRTH2 deletion promoted LARP6 binding with collagen mRNAs [collagen type 1, α1 (Col1a1), collagen type 1, α2 (Col1a2), and collagen type 3, α1 (Col3a1)] (Fig 3A) and subsequently suppressed cellular decay of collagen mRNAs and prolonged their half-lives in mouse primary lung fibroblasts (Fig 3B). The mRNA and protein expressions of collagen I and III were increased in mouse fibroblasts (Fig 3C and D). Knockdown of LARP6 (Fig 3E) marked
Referência(s)