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The Involvement of Trace Amine-Associated Receptor 1 and Thyroid Hormone Transporters in Non-Classical Pathways of the Thyroid Gland Auto-Regulation

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The Involvement of Trace Amine-Associated Receptor 1 and Thyroid Hormone Transporters in Non-Classical Pathways of the Thyroid Gland Auto-Regulation The Involvement of Trace Amine-Associated Receptor 1 and Thyroid Hormone Transporters in Non-Classical Pathways of the Thyroid Gland Auto-Regulation by Maria Qatato a Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell Biology Approved Dissertation Committee Prof. Dr. Klaudia Brix Jacobs University Bremen Prof. Sebastian Springer, DPhil Jacobs University Bremen Dr. Georg Homuth Ernst-Moritz-Arndt-Universität Greifswald Date of Defence: 16 January 2018 Department of Life Sciences and Chemistry Statutory Declaration Family Name, Given/First Name Qatato, Maria Matriculation number 20330110 What kind of thesis are you submitting: PhD Thesis English: Declaration of Authorship I hereby declare that the thesis submitted was created and written solely by myself without any external support. Any sources, direct or indirect, are marked as such. I am aware of the fact that the contents of the thesis in digital form may be revised with regard to usage of unauthorized aid as well as whether the whole or parts of it may be identified as plagiarism. I do agree my work to be entered into a database for it to be compared with existing sources, where it will remain in order to enable further comparisons with future theses. This does not grant any rights of reproduction and usage, however. This document was neither presented to any other examination board nor has it been published. German: Erklärung der Autorenschaft (Urheberschaft) Ich erkläre hiermit, dass die vorliegende Arbeit ohne fremde Hilfe ausschließlich von mir erstellt und geschrieben worden ist. Jedwede verwendeten Quellen, direkter oder indirekter Art, sind als solche kenntlich gemacht worden. Mir ist die Tatsache bewusst, dass der Inhalt der Thesis in digitaler Form geprüft werden kann im Hinblick darauf, ob es sich ganz oder in Teilen um ein Plagiat handelt. Ich bin damit einverstanden, dass meine Arbeit in einer Datenbank eingegeben werden kann, um mit bereits bestehenden Quellen verglichen zu werden und dort auch verbleibt, um mit zukünftigen Arbeiten verglichen werden zu können. Dies berechtigt jedoch nicht zur Verwendung oder Vervielfältigung. Diese Arbeit wurde noch keiner anderen Prüfungsbehörde vorgelegt noch wurde sie bisher veröffentlicht. ………………………………………………………………………………………………………. Date, Signature III IV Table of Contents Statutory Declaration..................................................................................................................III List of Abbreviation .................................................................................................................. VII Publications, and Oral and Poster Presentations ...................................................................... XI 1. Abstract .............................................................................................................................. 1 2. Introduction ......................................................................................................................... 3 2.1 The Thyroid Gland ........................................................................................................ 3 2.1.1 Classical thyroid hormones, synthesis and release .................................................. 3 2.1.2 Thyroid phenotype of mice deficient in Tg-processing cathepsins ............................ 5 2.1.3 Thyroid hormone transport ....................................................................................... 6 2.1.4 Genomic action of thyroid hormone .......................................................................... 7 2.1.5 Non-genomic action of thyroid hormones ................................................................. 7 2.2 Trace amine-associated receptors............................................................................ 8 2.2.1 Trace amine-associated receptor 1 .........................................................................10 2.2.2 In vitro Taar1 expression .........................................................................................10 2.2.3 Thyronamines as Taar1 agonists ............................................................................11 2.2.4 Taar1-deficient mouse model ..................................................................................14 3. Aims of the Study ...............................................................................................................16 4. Results ...............................................................................................................................19 4.1 Thyroglobulin storage and degradation for thyroid hormone liberation ........................23 4.2 Interdependence of thyroglobulin processing and thyroid hormone export in the mouse thyroid ...................................................................................................................................25 4.3 Trace amine-associated receptor 1 localization at the apical plasma membrane domain of Fisher rat thyroid epithelial cells Is confined to cilia ............................................................27 4.4 Establishing an in vitro model to study trafficking of mouse Taar1 in thyroid epithelial cells ………………………………………………………………………………………………….29 4.5 Canonical TSH regulation in the thyroid resulting in regular cathepsin-mediated thyroglobulin processing requires Taar1 expression ..............................................................80 5. Discussion ....................................................................................................................... 111 5.1 Thyroid hormone transporters Mct8 and Mct10 serve as sensors of intrathyroidal TH levels and contribute to regulating Tg-processing cathepsins by TSH-independent means . 111 5.2 Taar1 localisation on the apical plasma membrane domain and ciliary appendages of thyrocytes in situ and in vitro ............................................................................................... 112 5.3 Altered TSHR localisation in situ in Taar1-deficient mouse thyroid epithelial cells ..... 114 V 5.4 The absence of functional Taar1 leads to a subtle disbalance in the proteolytic to anti- proteolytic activities in male mouse thyroid tissue ................................................................ 117 6. References ...................................................................................................................... 118 VI List of Abbreviation % Percent °C Degrees Celcius 3-T1AM 3-iodothyronamine β-PEA β-phenylalamine µg Microgram µl Microliter µm Micrometre AADC Aromatic amino acid decarboxylase Asn Asparagine ATP Adenosine triphosphate BLAST Basic Local Alignment Search Tool BRET Bioluminescence resonance energy transfer BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate cDNA Complementary deoxyribonucleic acid ConA Concanavalin A CMF-PBS Calcium and magnesium free phosphate buffered saline CNS Central nervous system -/- Ctsb Cathepsin B-deficient mouse Ctsk-/- Cathepsin K-deficient mouse Ctsl-/- Cathepsin L-deficient mouse Dio (1, 2 or 3) Iodothyronine deiodinase (type I, II or III) DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide DTT Dithiotreitol VII EDTA Ethylenediaminetetraacetic acid EGFP Enhanced green fluorescent protein EGTA Ethylene glycol tetraacetic acid ELISA Enzyme-linked immunosorbent assay EPPTB N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide ER Endoplasmic reticulum ERGIC ER-Golgi intermediate complex FACS Fluorescence activated cell sorting FRT Fisher rat thyroid cells FBS Foetal bovine serum g acceleration of gravity (9.81 m/sec) g gram Gα Guanine nucleotide-binding protein, alpha subunit GM130 Golgi matrix protein 130 GPCR G protein-coupled receptor HA Haemagglutinin tag HEK 293 Human embryonic kidney 293 cells HEK 293T Human embryonic kidney 293 transformed with large T antigen HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hr Hour HRP Horseradish peroxidise kDa Kilo Dalton KTC-1 Human thyroid papillary carcinoma cells KTC-Z KTC-1 cells stably expressing mTaar1-EGFP LacZ Lactose operon LAMP-2 Lysosomal-associated membrane protein 2 VIII Lat L-type amino acid transporter M Molar mM Millimolar MALDI Matrix assisted laser desorption ionisation MAP6 Microtubule associated protein 6 MAPK Mitogen-activated protein kinase MAO Monoamine oxidase MAO-B Monoamine oxidase type-B min Minutes ml Millilitre MCT8/Mct8 Monocarboxylate transporter 8 (human/rodent) Mct10 Monocarboxylate transporter 10 Mct8-/y Mct8-deficient (male) mouse Mct10-/- Mct10-deficient mouse NIS Sodium iodide symporter nM Nanomolar Nthy-ori 3-1 Human thyroid follicular epithelial cells Nthy-Z Nthy-ori 3-1 cells stably expressing mTaar1-EGFP OATP1C1 Organic anion-transporting polypeptide 1C1 PAX8 Paired box gene 8 PBS Phosphate buffered saline PBS-T Phosphate buffer saline + 0.3% Tween-20 PCR Polymerase chain reaction PFA Paraformaldehyde RLuc Renilla luciferase RXR Retinoic X receptor IX SDS Sodium dodecyl sulphate SDS-PAGE SDS polyacrylamide gel electrophoresis t1/2 Half-life T0AM Thyronamine T3 L-3,5,3’-triiodothyronine T4 L-3,5,3’,5’-tetraiodothyronine (L-thyroxine) TA Trace amines TAAR1/Taar1 Trace amine-associated receptor 1 (human/other) Taar5 Trace amine-associated receptor 5 Taar8b Trace amine-associated receptor 8b -/- Taar1 Taar1-deficient mouse TAM Thyronamines Tg Thyroglobulin TGN Trans-Golgi network TH Thyroid hormones THT Thyroid hormone transporter(s) TRα Thyroid hormone receptor α TRβ Thyroid hormone receptor β TPO Thyroid peroxidase TR Nuclear thyroid
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