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Functional and Structural Characterization of Olfactory Receptors in Human Heart and Eye

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Functional and Structural Characterization of Olfactory Receptors in Human Heart and Eye DISSERTATION to obtain the degree Doctor Rerum Naturalium (Dr.rer.nat.) at the Faculty of Biology and Biotechnology International Graduate School Biosciences Ruhr-University Bochum Functional and structural characterization of olfactory receptors in human heart and eye Department of Cellphysiology submitted by Nikolina Jovancevic from Zadar, Croatia Bochum February 2016 First Referee: Prof. Dr. Dr. Dr. Hanns Hatt Second Referee: Prof. Dr. Stefan Wiese DISSERTATION zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum Funktionale und strukturelle Charakterisierung olfaktorischer Rezeptoren im humanen Herzen und Auge Lehrstuhl für Zellphysiologie vorgelegt von Nikolina Jovancevic aus Zadar, Kroatien Bochum Februar 2016 Referent: Prof. Dr. Dr. Dr. Hanns Hatt Korreferent: Prof. Dr. Stefan Wiese To my family TABLE OF CONTENTS TABEL OF CONTENT 1 INTRODUCTION 1 1.1 G protein-coupled receptors 1 1.1.1 General 1 1.1.2 Structure and classification 2 1.1.3 Olfactory Receptors 4 1.2 Function of olfactory receptors 9 1.2.1 The olfactory system 9 1.2.2 Ectopic expression of olfactory receptors 11 1.3 Excursus: Anatomy and physiology of the heart 13 1.3.1 Anatomy of the heart and blood circuit 14 1.3.2 The cardiac conduction system 15 1.3.3 Excitation-contraction coupling 16 1.3.4 Cardiac GPCRs: Modulation of cardiac contraction 17 1.4 Excursus: Anatomy and physiology of the eye 18 1.4.1 Anatomy of the retina 19 1.4.2 Physiology of the retina: visual processing 20 1.4.3 The retinal pigment epithelium: Structure and function 21 1.5 Objectives 23 2 MATERIAL 38 2.1 Laboratory apparatus 38 2.2 Consumables 39 2.3 Chemicals 39 2.4 Solution and media 41 2.5 Odorants 43 2.6 Inhibitors 44 2.7 Transfection reagent 44 2.8 Kits and PCR mixes 44 2.9 Enzyme 45 2.10 Antibodies and blocking peptide 45 2.10.1 Primary antibodies and blocking peptide 45 2.10.2 Secondary antibodies 45 1 TABLE OF CONTENTS 2.11 DNA and protein standards 46 2.12 Primer sequences 46 2.13 siRNA 48 2.14 Plasmids 48 2.15 Cell culture 48 2.15.1 Cell culture supplies 48 2.15.2 Media and solution 49 2.15.3 Stem cell-derived cardiomyocytes 49 2.15.4 RPE cells 50 2.15.5 Hana3A cells 50 2.16 Human tissues 50 2.16.1 Heart tissue 50 2.16.2 Retina tissue 50 2.17 Competent bacterial strain 51 2.18 Databases 51 2.19 Software 51 3 METHODS 52 3.1 Cell culture 52 3.1.1 Culture and differentiation of human embryonic (hESC) and induced pluripotent stem cells (hiPSC) towards cardiomyocytes 52 3.1.2 Culture of RPE cells and Hana3A cells 53 3.2 Cell-based assays 53 3.2.1 Luciferase reporter assay 53 3.2.2 Cell proliferation assay 54 3.2.3 Cell migration assay: Scratch wound-healing assay 54 3.2.4 Matrigel inversion assay 54 3.3 Molecular biology 55 3.3.1 RNA isolation 55 3.3.2 Synthesis of complementary DNA 55 3.3.3 Reverse transcription polymerase chain reaction (RT-PCR) 55 3.3.4 Agarose gel electrophoresis 56 3.3.5 Purification of DNA fragments 56 3.3.6 DNA sequencing 56 3.3.7 Cloning of OR51E2-mutants 57 3.3.8 mRNA-Sequencing (RNAseq) 59 2 TABLE OF CONTENTS 3.4 siRNA transfection 61 3.5 Protein biochemistry 61 3.5.1 Protein isolation from cultured cells and human tissues 61 3.5.2 Sodium dodecylsulfate polyacrylamide gel electrophoresis 62 3.5.3 Western Blot 62 3.5.4 Detecting of protein phosphorylation 63 3.6 Immunofluorescence stainings 64 3.6.1 Immunohistochemistry of human retina 64 3.6.2 Immunohistochemistry of human heart tissue 64 3.6.3 Immunocytochemistry 65 3.7 Ca2+ imaging 65 3.8 Contractile force measurements of slice preparations of adult human ventricle 66 3.9 Determination of the fatty acid pattern in human serum epicardial adipose biopsies 67 4 RESULTS 68 4.1 Identification and functional characterization of olfactory receptors in the human heart 68 4.1.1 Olfactory receptor OR51E1 is expressed in the human heart and in stem cell-derived cardiomyocytes 68 4.1.2 Ligand screening on OR51E1 70 4.1.3 OR51E1-activation induces a negative chronotropic effect in human stem cell-derived cardiomyocytes 72 4.1.4 OR51E1 signaling involves Gβγ 76 4.1.5 OR51E1 agonists reduce contraction force of explanted heart preparations 78 4.1.6 OR51E1-agonists are present in human blood at receptor activating concentrations 80 4.2 Identification and functional characterization of olfactory receptors in the human eye 81 4.2.1 Identification of olfactory receptors in the neural retina 81 4.2.2 Identification and functional characterization olfactory receptors in the human retinal pigment epithelial cells 91 4.3 Structural characterization of OR51E2 100 5 DISCUSSION 105 5.1 Identification and functional characterization of olfactory receptors in the human heart 105 5.1.1 Expression of OR51E1 in the human heart 105 5.1.2 Activation of OR51E1 in cardiomyocytes 106 5.1.3 Possible role of OR51E1 in the heart 107 5.2 Identification and functional characterization of olfactory receptors in the human eye 110 3 TABLE OF CONTENTS 5.2.1 Detection of olfactory receptors in the neural retina 110 5.2.2 Identification and functional characterization of olfactory receptors in human retinal pigment epithelial cells 114 5.3 Structural characterization of OR51E2 119 6 SUMMARY 124 7 ZUSAMMENFASSUNG 127 8 REFERENCES 130 9 APPENDIX 157 9.1 List of abbreviations 157 9.2 List of figures 159 9.3 List of tables 160 9.4 Curriculum vitae 161 9.5 Publication list 162 9.6 Danksagung 163 9.7 Erklärung 165 4 INTRODUCTION 1 INTRODUCTION For humans is communication essential, it appears in various ways and is necessary for an intact society. Communication not only occurs between individuals but also within our bodies and is crucial for our survival. Cells are able to communicate via molecular antennas, so called receptors, on their cell surface. These receptors pick up signals from the extracellular environment, transfer them into the cells, and trigger signal-specific cellular responses. The superfamily of G protein-coupled receptors (GPCRs) represents with approximately 800 genes the largest and most diverse group of human membrane receptors (Pierce et al., 2002). Therefore, the following chapter focuses on this receptor family. 1.1 G protein-coupled receptors 1.1.1 General The superfamily of G protein-coupled receptors (GPCRs) is involved in the regulation of various physiological processes, such as the neuronal transduction, cellular metabolism, differentiation, proliferation, secretion and immunological reactions (Wu et al., 2012). They are activated by a broad spectrum of extracellular signals including neurotransmitters, hormones, cytokines, light energy, odorants, taste ligands and extracellular Ca2+ ions (Pierce et al., 2002). At the cellular level, these external signals are transmitted across the plasma membrane by GPCRs, which convert these extracellular cues into one or more intracellular responses. As their name implies, GPCRs interact intercellularly with heterotrimeric G proteins. Ligand- binding to a GPCR causes a conformational change, which in turn triggers the activation of the coupled G protein. The activated heterotrimeric G protein dissociates into the α-subunit and the -complex. The α-subunit inhibits or stimulates specific effector proteins, such as adenylyl cyclase, phosphodiesterase, phospholipase, phosphoinositol-3-kinase or ion channels (Strader et al., 1995). This results in a change of the concentration of second messengers within the cell, which regulate the activity of various proteins, thereby affecting e.g. the gene expression or secretion. Due to the participation of GPCRs in a variety of physiological 1 INTRODUCTION processes, it is not surprising that a dysfunction can lead to diseases such as retinitis pigmentosa, hypo- and hyperthyroidism, nephrogenic diabetes insipidus and even cancer (Schöneberg et al., 2004). Moreover, their key role in cellular communication makes them a favorable target for pharmacological research. Currently, more than 60% of the commercially available drugs achieve their effect through agonistic or antagonistic interaction with GPCRs, which include various psychotropic drugs, beta-blockers or antihistamines (Hopkins & Groom, 2002). 1.1.2 Structure and classification In the 1980s, a structure consisting of seven α-helical transmembrane (TM) domains was postulated for the β2 adrenoreceptor analogous to that of rhodopsin (Dixon et al., 1986). The hypothesis of the existence of a large receptor gene family developed and was rapidly confirmed by cloning other receptors (Dohlman et al., 1991). At the beginning, the crystal structure of bacteriorhodopsin served as a model for predicting the three-dimensional structure of the receptor family (Henderson & Unwin, 1975). However, in the year 2000 the first detailed structural analysis of GPCRs was performed with the determination of the X-ray structure of bovine rhodopsin (Palczewski et al., 2000). The results confirmed a heptahelical structure of the membrane protein with an extracellular amino-terminus (N-terminus) and an intracellular carboxyl terminus (C-terminus). The seven transmembrane domains are linked by alternating extra- and intracellular loops. To date, the structures of 24 GPCRs were determined by X-ray crystal structure analyses (Cherezov et al., 2007; Rasmussen et al., 2007; Jaakola et al., 2008; Warne et al., 2008; Chien et al., 2010; Wu et al., 2010; Shimamura et al., 2011; Granier et al., 2012; Haga et al., 2012; Hanson et al., 2012; Kruse et al., 2012; Liu et al., 2012; Manglik et al., 2012; Thompson et al., 2012; White et al., 2012; Wu et al., 2012a; Zhang et al., 2012a; Hollenstein et al., 2013; Siu et al., 2013; Tan et al., 2013, Wacker et al., 2013a; Wang et al., 2013a; Wang et al., 2013b; Wu et al., 2014; Zhang et al., 2014).
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