DOCSLIB.ORG

TITLE Crystal Structure of Human Endothelin ETB Receptor in Complex

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
TITLE Crystal Structure of Human Endothelin ETB Receptor in Complex bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 1 TITLE 2 Crystal structure of human endothelin ETB receptor in complex 3 with peptide inverse agonist IRL2500. 4 Chisae Nagiri1*, Wataru Shihoya1*‡, Asuka Inoue2, Francois Marie Ngako Kadji2, Junken 5 Aoki2, Osamu Nureki1‡. 6 7 Affiliations: 8 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 9 Bunkyo, Tokyo 113-0033, Japan. 10 2 Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, 11 Aoba-ku, Sendai, Miyagi 980-8578, Japan. 12 13 *These authors contributed equally to this work. 14 ‡To whom correspondence should be addressed. E-mail: [email protected] (W.S.), 15 [email protected] (O.N.) – 1 – bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 16 Abstract 17 Endothelin receptors (ETA and ETB) are G-protein coupled receptors activated 18 by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide- 19 mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a 20 potent ETB-selective antagonist, which has preventive effects against brain edema. Here, 21 we report the crystal structure of the human ETB receptor in complex with IRL2500 at 22 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500 23 and ET-1, and provides structural insights into its ETB-selectivity. Notably, the biphenyl 24 group of IRL2500 penetrates into the transmembrane core proximal to D2.50, stabilizing 25 the inactive conformation. Using the newly-established constitutively active mutant, we 26 clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor. 27 The current findings will expand the chemical space of ETR antagonists and facilitate the 28 design of inverse agonists for other class A GPCRs. – 2 – bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 29 Main text 30 Introduction 31 Endothelin receptors (ETR) are G-protein coupled receptors activated by vaso 1 32 active peptide, endothelins . Two endothelin receptor subtypes (ETA and ETB) are widely 33 expressed in the vascular endothelium, brain, and other circulatory organs2,3. Endothelin- 34 1 (ET-1) activates the endothelin receptors (ETRs) with sub-nanomolar affinities. The 35 activation of the ETA receptor leads to potent and long-lasting vasoconstriction, whereas 36 that of the ETB receptor induces nitric oxide-mediated vasorelaxation. Therefore, the up- 37 regulation of ET-1 is significantly related to circulatory-system diseases, including 38 pulmonary arterial hypertension (PAH)4–7. Moreover, the autocrine and paracrine 39 signaling functions of ET-1 through the ETA receptor play a critical role in tumor growth 40 and survival8. Therefore, ETR antagonists have been developed for the treatment of 41 circulatory-system diseases and cancers6,7. Bosentan is the first orally-active ETR 9,10 42 antagonist , and is used to treat PAH. The ETB receptor is the prominent ET receptor 11 43 subtype in the brain, with high expression levels in astrocytes . Stimulation of the ETB 44 receptor modulates astrocytic responses, indicating its important roles in regulating 12 45 astrocytic functions . The up-regulation of the astrocytic ETB receptor by ET-1 increases 46 the vascular permeability and reduces the AQP4 levels, thereby aggravating vasogenic 11 47 brain edema . The application of ETB-selective antagonists may provide preventive 48 effects against brain edema in the acute phase of brain insults13–16. 49 To date, most endothelin receptor antagonists have been developed based on 50 bosentan17,18. The ETR antagonists that have been developed till now are mostly N- – 3 – bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 51 heterocyclic sulfonamides with similar structures and molecular weights, and non- 52 sulfonamide antagonists (atrasentan, ambrisentan, darusentan, and enrasentan) still retain 53 high similarities with each other and with the sulfonamides7. Therefore, the ETR agents 54 are chemically very similar, and expanded chemical space should be exploited. IRL2500 55 is a peptide ETR antagonist developed based on the partial structure of ET-119, rather than 56 bosentan. IRL2500 has been characterized as an ETB-selective antagonist with an IC50 57 value of 1.2 nM20, which shows higher affinity than that of bosentan. In an animal model, 58 the intracerebroventricular administration of IRL2500 attenuated cold injury-mediated 59 brain edema and disruption of the blood-brain barrier, indicating the neuroprotective 60 effect of IRL250014,15. An understanding of the IRL2500 binding mode would facilitate 61 the expansion of the chemical space of ET agents. 21 62 We previously reported the crystal structures of the ETB receptor bound to ET-1 22 63 and bosentan ; however, both the binding mode and ETB-selectivity of IRL2500 64 remained to be elucidated. Here, we present the crystal structure of the ETB receptor in 65 complex with IRL2500. This structure revealed the unique binding mode of IRL2500, 66 which differs from those of ET-1 and bosentan. Structure-guided functional analyses 67 clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor, 68 and thus will provide the basis for design of inverse agonists for other class A GPCRs. 69 70 – 4 – bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 71 Results 72 Overall structure 73 For crystallization, we used the previously established, thermostabilized ETB 22,23 74 receptor (ETB-Y4) . The IC50 value of IRL2500 for ETB-Y4 was similar to that for the 75 wild type receptor in the TGFα shedding assay24 (Fig. 1), suggesting that the 76 themostabilizing mutations minimally affect the IRL2500 binding. In contrast, the IC50 77 value of IRL2500 for the ETA receptor is over 3 M (Fig. 1), indicating that IRL2500 has 20 78 over 100-fold ETB-selectivity, consistent with the previous pharmacological analysis . 79 To facilitate crystallization, we replaced the third intracellular loop (ICL3) of the receptor 25 26 80 with minimal T4 Lysozyme (ETB-Y4-mT4L). Using in meso crystallization , we 81 obtained crystals of ETB-Y4-mT4L in complex with IRL2500 (Supplementary Fig. 1a, b). 82 In total, 58 datasets were collected and merged by the data processing system KAMO27. 83 Eventually, we determined the ETB structure in complex with IRL2500 at 2.7 Å resolution, 84 by molecular replacement using the antagonist-bound ETB structure (PDB code: 5X93) 85 (Table 1). 86 The overall structure consists of the canonical 7 transmembrane helices (TM), the 87 amphipathic helix 8 at the C-terminus (H8), and two antiparallel β-strands in the 88 extracellular loop 2 (ECL2), as in the previously determined ETB structures (Fig. 2a). The 89 IRL2500-bound structure is similar to the bosentan-bound structure, rather than the ET- 90 1-bound structure (R.M.S.D. values for Cα atoms=1.34 and 1.95 Ǻ, respectively), 91 reflecting the inactive conformation. We observed a remarkable difference in the 92 conformation of ECL2. The β strands are opened up by 9 Å, as compared with those in – 5 – bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 93 the ligand-free structure (Fig. 2b and Supplementary Fig. 2a), and are the widest among 94 the peptide-activated class A GPCRs (Supplementary Fig. 2b). This structural feature 95 indicates the innate flexibility of ECL2, to capture the large peptide ligand endothelin, in 96 the inactive conformation of the ETB receptor. 97 98 IRL2500 binding site 99 We first describe the IRL2500 binding mode. IRL2500 consists of a tryptophan, 100 a 3,5-dimethylbenzoyl group, and a biphenyl group19, which are connected by two peptide 101 bonds (Fig. 2c). IRL2500 binds to the transmembrane binding cleft exposed to the 102 extracellular side, with a clear electron density in the Fo − Fc omit map (Supplementary 103 Fig. 3a, b). The carboxylate group of the tryptophan moiety in IRL2500 forms salt bridges 104 with K1823.33 and R3436.55 (superscripts indicate Ballesteros–Weinstein numbers28) (Fig. 105 2c, d).
Load more

Recommended publications
  • Endothelin System and Therapeutic Application of Endothelin Receptor
    xperim ACCESS Freely available online & E en OPEN l ta a l ic P in h l a C r m f o a c l a o n l o r g u y o J Journal of ISSN: 2161-1459 Clinical & Experimental Pharmacology Research Article Endothelin System and Therapeutic Application of Endothelin Receptor Antagonists Abebe Basazn Mekuria, Zemene Demelash Kifle*, Mohammedbrhan Abdelwuhab Department of Pharmacology, School of Pharmacy, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia ABSTRACT Endothelin is a 21 amino acid molecule endogenous potent vasoconstrictor peptide. Endothelin is synthesized in vascular endothelial and smooth muscle cells, as well as in neural, renal, pulmonic, and inflammatory cells. It acts through a seven transmembrane endothelin receptor A (ETA) and endothelin receptor B (ETB) receptors belongs to G protein-coupled rhodopsin-type receptor superfamily. This peptide involved in pathogenesis of cardiovascular disorder like (heart failure, arterial hypertension, myocardial infraction and atherosclerosis), renal failure, pulmonary arterial hypertension and it also involved in pathogenesis of cancer. Potentially endothelin receptor antagonist helps the treatment of the above disorder. Currently, there are a lot of trails both per-clinical and clinical on endothelin antagonist for various cardiovascular, pulmonary and cancer disorder. Some are approved by FAD for the treatment. These agents are including both selective and non-selective endothelin receptor antagonist (ETA/B). Currently, Bosentan, Ambrisentan, and Macitentan approved
    [Show full text]
  • Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung’S Disease
    RESEARCH ARTICLE Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung’s Disease Ankush Gosain1,2*, Amanda J. Barlow-Anacker1, Chris S. Erickson1, Joseph F. Pierre1, Aaron F. Heneghan1, Miles L. Epstein2, Kenneth A. Kudsk1,3 a11111 1 Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, United States of America, 2 Department of Neuroscience, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, United States of America, 3 Veteran Administration Surgical Service, William S. Middleton Memorial Veterans Hospital, Madison, United States of America * [email protected] OPEN ACCESS Citation: Gosain A, Barlow-Anacker AJ, Erickson Abstract CS, Pierre JF, Heneghan AF, Epstein ML, et al. (2015) Impaired Cellular Immunity in the Murine Hirschsprung’s disease (HSCR) is characterized by aganglionosis from failure of neural crest Neural Crest Conditional Deletion of Endothelin cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung’s- Receptor-B ’ Model of Hirschsprung s Disease. PLoS associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative ONE 10(6): e0128822. doi:10.1371/journal. pone.0128822 to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hy- Academic Editor: Anatoly V. Grishin, Childrens Hospital Los Angeles, UNITED STATES pothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contrib- ute to enterocolitis. EdnrB was deleted from the neural crest (EdnrBNCC-/-) resulting in mutants Received: November 12, 2014 with defective NCC migration, distal colonic aganglionosis and the development of enterocoli- Accepted: May 1, 2015 tis.
    [Show full text]
  • University of Groningen Genetics Dissection and Functional
    University of Groningen Genetics dissection and functional studies in Hirschsprung disease Sribudiani, Yunia IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2011 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Sribudiani, Y. (2011). Genetics dissection and functional studies in Hirschsprung disease Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 11-02-2018 Genetic Dissection and Functional Studies in Hirschsprung Disease Yunia Sribudiani Genetic Dissection and Functional Studies in Hirschsprung Disease Y. Sribudiani PhD Thesis – Department of Genetics University Medical Center Groningen, University of Groningen Groningen, the Netherlands This study was financially supported by University of Groningen and Research Institute GUIDE. ISBN/EAN: 978-94-6182-039-6 Cover Ilustration: Ella Elviana, www.ellasworks.blogspot.com Cover & book layout: Briyan B Hendro, www.brianos.daportfolio.com Printed by: Off Page, Amsterdam, www.offpage.nl Printing of this thesis was financially supported by: University of Groningen, GUIDE and Department of Genetics - UMCG.
    [Show full text]
  • G-Protein Coupled Receptors in Lipid Rafts and Caveolae: How, When and Why Do They Go There?
    325 G-protein coupled receptors in lipid rafts and caveolae: how, when and why do they go there? B Chini and M Parenti1 CNR Institute of Neuroscience, Cellular and Molecular Pharmacology Section, Milan, Italy 1Department of Experimental and Environmental Medicine and Medical Biotechnologies, University of Milano-Bicocca, Monza, Italy (Requests for offprints should be addressed to B Chini; Email: [email protected]) Abstract This review describes the advances in our understanding of the role of G-protein coupled receptor (GPCR) localisation in membrane microdomains known as lipid rafts and caveolae. The growing interest in these specialised regions is due to the recognition that they are involved in the regulation of a number of cell functions, including the fine-tuning of various signalling molecules. As a number of GPCRs have been found to be enriched in lipid rafts and/or caveolae by means of different experimental approaches, we first discuss the pitfalls and uncertainties related to the use of these different procedures. We then analyse the addressing signals that drive and/or stabilise GPCRs in lipid rafts and caveolae, and explore the role of rafts/caveolae in regulating GPCR trafficking, particularly in receptor exo- and endocytosis. Finally, we review the growing evidence that lipid rafts and caveolae participate in the regulation of GPCR signalling by affecting both signalling selectivity and coupling efficacy. Journal of Molecular Endocrinology (2004) 32, 325–338 Introduction cascade (Dykstra et al. 2001, Sedwick & Altman 2002, Werlen & Palmer 2002), unravelling the role Lipid rafts and caveolae have been found to be of rafts and caveolae in the functional regulation of involved in the regulation of various cell functions, other signalling components is still at its very including the homeostasis of cholesterol (Ikonen & beginning.
    [Show full text]
  • Peripheral Regulation of Pain and Itch
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1596 Peripheral Regulation of Pain and Itch ELÍN INGIBJÖRG MAGNÚSDÓTTIR ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0746-6 UPPSALA urn:nbn:se:uu:diva-392709 2019 Dissertation presented at Uppsala University to be publicly examined in A1:107a, BMC, Husargatan 3, Uppsala, Friday, 25 October 2019 at 13:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor emeritus George H. Caughey (University of California, San Francisco). Abstract Magnúsdóttir, E. I. 2019. Peripheral Regulation of Pain and Itch. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1596. 71 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0746-6. Pain and itch are diverse sensory modalities, transmitted by the somatosensory nervous system. Stimuli such as heat, cold, mechanical pain and itch can be transmitted by different neuronal populations, which show considerable overlap with regards to sensory activation. Moreover, the immune and nervous systems can be involved in extensive crosstalk in the periphery when reacting to these stimuli. With recent advances in genetic engineering, we now have the possibility to study the contribution of distinct neuron types, neurotransmitters and other mediators in vivo by using gene knock-out mice. The neuropeptide calcitonin gene-related peptide (CGRP) and the ion channel transient receptor potential cation channel subfamily V member 1 (TRPV1) have both been implicated in pain and itch transmission. In Paper I, the Cre- LoxP system was used to specifically remove CGRPα from the primary afferent population that expresses TRPV1.
    [Show full text]
  • Glucagon-Like Peptide-1 and Its Class BG Protein–Coupled Receptors
    1521-0081/68/4/954–1013$25.00 http://dx.doi.org/10.1124/pr.115.011395 PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:954–1013, October 2016 Copyright © 2016 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license. ASSOCIATE EDITOR: RICHARD DEQUAN YE Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes Chris de Graaf, Dan Donnelly, Denise Wootten, Jesper Lau, Patrick M. Sexton, Laurence J. Miller, Jung-Mo Ahn, Jiayu Liao, Madeleine M. Fletcher, Dehua Yang, Alastair J. H. Brown, Caihong Zhou, Jiejie Deng, and Ming-Wei Wang Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (C.d.G.); School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom (D.D.); Drug Discovery Biology Theme and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia (D.W., P.M.S., M.M.F.); Protein and Peptide Chemistry, Global Research, Novo Nordisk A/S, Måløv, Denmark (J.La.); Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.); Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, Texas (J.-M.A.); Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, Riverside, California (J.Li.); National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (D.Y., C.Z., J.D., M.-W.W.); Heptares Therapeutics, BioPark, Welwyn Garden City, United Kingdom (A.J.H.B.); and School of Pharmacy, Fudan University, Zhangjiang High-Tech Park, Shanghai, China (M.-W.W.) Downloaded from Abstract.
    [Show full text]
  • NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer
    Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 11-7-2018 Decipher Mechanisms by which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer Jairo Ramos [email protected] Follow this and additional works at: https://digitalcommons.fiu.edu/etd Part of the Clinical Epidemiology Commons Recommended Citation Ramos, Jairo, "Decipher Mechanisms by which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer" (2018). FIU Electronic Theses and Dissertations. 3872. https://digitalcommons.fiu.edu/etd/3872 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida DECIPHER MECHANISMS BY WHICH NUCLEAR RESPIRATORY FACTOR ONE (NRF1) COORDINATES CHANGES IN THE TRANSCRIPTIONAL AND CHROMATIN LANDSCAPE AFFECTING DEVELOPMENT AND PROGRESSION OF INVASIVE BREAST CANCER A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in PUBLIC HEALTH by Jairo Ramos 2018 To: Dean Tomás R. Guilarte Robert Stempel College of Public Health and Social Work This dissertation, Written by Jairo Ramos, and entitled Decipher Mechanisms by Which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer, having been approved in respect to style and intellectual content, is referred to you for judgment.
    [Show full text]
  • A Unique Signal Sequence of the Chemokine Receptor CCR7 Promotes Package Into COPII Vesicles for Efficient Receptor Trafficking
    Erschienen in: Journal of leukocyte biology ; 104 (2018), 2. - S. 375-389 https://dx.doi.org/10.1002/JLB.2VMA1217-492R A unique signal sequence of the chemokine receptor CCR7 promotes package into COPII vesicles for efficient receptor trafficking Edith Uetz-von Allmen Alexandra V. Rippl Hesso Farhan∗ Daniel F. Legler Biotechnology Institute Thurgau (BITg), Univer- sity of Konstanz, Kreuzlingen, Switzerland Abstract Correspondence Chemokine receptors are considered to belong to the group of G protein-coupled receptors that Daniel F.Legler, BITg,University of Konstanz, use the first transmembrane domain as signal anchor sequence for membrane insertion instead of Unterseestrasse 47, CH-8280 Kreuzlingen, a cleavable N-terminal signal sequence. Chemokine recognition is determined by the N-termini Switzerland. Email: [email protected] of chemokine receptors. Here, we show that the chemokine receptor CCR7, which is essential ∗Present address: Institute of Basic Medical for directed migration of adaptive immune cells, possesses a 24 amino acids long N-terminal sig- Sciences, University of Oslo, Oslo, Norway nal sequence that is unique among chemokine receptors. This sequence is cleaved off the mature human and mouse protein. Introducing single point mutations in the hydrophobic core h-region or in the polar C-terminal segment (c-region) of the signal sequence to interfere with its cleav- age retained CCR7 in the ER and prevented its surface expression. Furthermore, we demonstrate the correct topology of the 35 amino acids short extracellular N-tail of CCR7 in a deletion mutant lacking the natural signal sequence. This signal sequence deletion mutant of CCR7 is fully func- tional as it efficiently binds its ligand, elicits chemokine-induced calcium mobilization, and directs cell migration.
    [Show full text]
  • Capturing Peptide–GPCR Interactions and Their Dynamics
    molecules Review Capturing Peptide–GPCR Interactions and Their Dynamics Anette Kaiser * and Irene Coin Faculty of Life Sciences, Institute of Biochemistry, Leipzig University, Brüderstr. 34, D-04103 Leipzig, Germany; [email protected] * Correspondence: [email protected] Academic Editor: Paolo Ruzza Received: 31 August 2020; Accepted: 9 October 2020; Published: 15 October 2020 Abstract: Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes. Keywords: GPCR activation; peptide–GPCR interactions; structural dynamics of GPCRs; peptide ligands; crosslinking; NMR; EPR 1.
    [Show full text]
  • The Clinical Significance of Endothelin Receptor Type B in Hepatocellular
    Experimental and Molecular Pathology 107 (2019) 141–157 Contents lists available at ScienceDirect Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp The clinical significance of endothelin receptor type B in hepatocellular T carcinoma and its potential molecular mechanism ⁎ Lu Zhanga,1, Bin Luob,1, Yi-wu Danga, Rong-quan Heb, Gang Chena, Zhi-gang Pengb, , ⁎ Zhen-bo Fenga, a Department of Pathology, First Affiliated Hospital of Guangxi Medical University, No. 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region530021,PR China b Department of Medical Oncology, First Affiliated Hospital of Guangxi Medical University, No. 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China ARTICLE INFO ABSTRACT Keywords: Objective: To explore the clinical significance and potential molecular mechanism of endothelin receptor typeB Endothelin receptor type B (EDNRB) in hepatocellular carcinoma (HCC). Hepatocellular carcinoma Methods: Immunohistochemistry was used to detect EDNRB protein expression level in 67 HCC paraffin em- Immunohistochemistry bedded tissues and adjacent tissues. Correlations between EDNRB expression level and clinicopathologic para- meters were analyzed in our study. The expression level and clinical significance of EDNRB in HCC were also evaluated from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. The cBioPortal for Cancer Genomics was employed to analyze the EDNRB related genes, and Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Protein-Protein Interaction (PPI) network were conducted for those EDNRB related genes. Results: Lower expression level of EDNRB in HCC was verified by immunohistochemistry than adjacent tissues (P < 0.0001). The expression level of EDNRB in HCC tissues was lower than normal control liver tissues based on TCGA and GEO data (standard mean difference [SMD] = −1.48, 95% [confidence interval] CI: 2 −1.63−(−1.33), P heterogeneity = 0.116, I = 32.4%).
    [Show full text]
  • New Drugs and Emerging Therapeutic Targets in the Endothelin Signaling Pathway and Prospects for Personalized Precision Medicine
    Physiol. Res. 67 (Suppl. 1): S37-S54, 2018 https://doi.org/10.33549/physiolres.933872 REVIEW New Drugs and Emerging Therapeutic Targets in the Endothelin Signaling Pathway and Prospects for Personalized Precision Medicine A. P. DAVENPORT1, R. E. KUC1, C. SOUTHAN2, J. J. MAGUIRE1 1Experimental Medicine and Immunotherapeutics, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom, 2Deanery of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom Received January 26, 2018 Accepted March 29, 2018 Summary Key words During the last thirty years since the discovery of endothelin-1, Allosteric modulators • Biased signaling • G-protein coupled the therapeutic strategy that has evolved in the clinic, mainly in receptors • Endothelin-1 • Monoclonal antibodies • Pepducins • the treatment of pulmonary arterial hypertension, is to block the Single nucleotide polymorphisms action of the peptide either at the ETA subtype or both receptors using orally active small molecule antagonists. Recently, there Corresponding author has been a rapid expansion in research targeting ET receptors A. P. Davenport, Experimental Medicine and Immunotherapeutics, using chemical entities other than small molecules, particularly University of Cambridge, Addenbrooke's Hospital, Cambridge, monoclonal antibody antagonists and selective peptide agonists CB2 0QQ, United Kingdom. Fax: 01223 762576. E-mail: and antagonists. While usually sacrificing oral bio-availability, [email protected] these compounds have other therapeutic advantages with the potential to considerably expand drug targets in the endothelin Introduction pathway and extend treatment to other pathophysiological conditions. Where the small molecule approach has been During the last thirty years since the discovery retained, a novel strategy to combine two vasoconstrictor of endothelin-1 (ET-1), the therapeutic strategy that has targets, the angiotensin AT1 receptor as well as the ETA receptor evolved in the clinic, mainly in the treatment of in the dual antagonist sparsentan has been developed.
    [Show full text]
  • Downloaded from Bioscientifica.Com at 10/02/2021 05:00:39PM Via Free Access
    58 1 G S Y ONG and M J YOUNG Mineralocorticoid cell signalling 58: 1 R33–R57 Review Mineralocorticoid regulation of cell function: the role of rapid signalling and gene transcription pathways Gregory S Y Ong1,2 and Morag J Young1,3 1 Cardiovascular Endocrinology Laboratory, Centre for Endocrinology and Metabolism, Hudson Institute Correspondence of Medical Research, Clayton, Victoria, Australia should be addressed 2 Department of Medicine, School of Clinical Sciences, Monash University, Clayton, Victoria, Australia to M J Young 3 Department of Physiology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia Email [email protected] Abstract The mineralocorticoid receptor (MR) and mineralocorticoids regulate epithelial handling Key Words of electrolytes, and induces diverse effects on other tissues. Traditionally, the effects f aldosterone of MR were ascribed to ligand–receptor binding and activation of gene transcription. f mineralocorticoid However, the MR also utilises a number of intracellular signalling cascades, often by receptor transactivating unrelated receptors, to change cell function more rapidly. Although f glucocorticoid aldosterone is the physiological mineralocorticoid, it is not the sole ligand for MR. f MAPK pathways Tissue-selective and mineralocorticoid-specific effects are conferred through the f GPER f EGFR enzyme 11β-hydroxysteroid dehydrogenase 2, cellular redox status and properties of the MR itself. Furthermore, not all aldosterone effects are mediated via MR, with implication of the involvement of other membrane-bound receptors such as GPER. Journal of Molecular Endocrinology This review will describe the ligands, receptors and intracellular mechanisms available for mineralocorticoid hormone and receptor signalling and illustrate their complex Journal of Molecular interactions in physiology and disease.
    [Show full text]