Characterization of allosteric mechanisms on the M2 und M4 mACh receptor using the FRET-technique Charakterisierung allosterischer Mechanismen am M2 und M4 mACh Rezeptor unter Anwendung der FRET-Technik Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Life Sciences submitted by Monika Maier-Peuschel from Nürnberg Würzburg, 2010 Submitted on: Members of the Promotionskomitee: Chairperson: Professor Paul Pauli Primary Supervisor: Professor Martin L. Lohse Supervisor (Second): Professor Caroline Kisker Supervisor (Third): Professor Arthur Christopoulos Date of Public Defence: Date of receipt of Certificates: Affidavit Affidavit I hereby declare that my thesis Characterization of allosteric mechanisms on the M2 und M4 mACh receptor using the FRET-technique is the result of my own work. I did not receive any help or support from commercial consultants. All sources and/or materials are listed and specified in the thesis. Furthermore I verify that this thesis has not yet been submitted as part of another examination process neither in identical nor similar form. Würzburg, 10.04.2010 Contents Contents ___________________________________________________________________________ 1 Introduction ........................................................................................................................1 1.1 Allostery ........................................................................................................................1 1.1.1 Allostery and receptors...........................................................................................1 1.1.2 Definition................................................................................................................4 1.1.3 Allosteric mechanisms on G-protein coupled receptors.........................................5 1.1.4 Allosteric ligands of GPCRs ................................................................................13 1.1.5 Atypical allosteric modulators..............................................................................15 1.1.6 Novel developments .............................................................................................15 1.2 Muscarinic receptors ...................................................................................................17 1.2.1 Receptor distribution ............................................................................................18 1.2.2 Signal transduction ...............................................................................................22 1.2.3 Desensitization and sequestration.........................................................................24 1.2.4 Muscarinic ligands................................................................................................25 1.2.5 Therapeutic opportunities.....................................................................................28 1.3 FRET Imaging.............................................................................................................34 1.3.1 Definition of FRET...............................................................................................34 1.3.2 Measuring ratiometric FRET................................................................................37 1.3.3 Potential problems using FRET technique...........................................................37 1.3.4 Fluorophores for FRET ........................................................................................38 1.3.5 Other FRET-applications......................................................................................45 2 Aims of the study ..............................................................................................................47 2.1 General considerations ................................................................................................47 2.1.1 Manufacturing of mACh-receptor constructs.......................................................47 2.1.2 Analysis of allosteric receptor modulation...........................................................48 3 Materials and Methods ....................................................................................................49 3.1 Materials......................................................................................................................49 3.1.1 Bacteria strains .....................................................................................................49 3.1.2 Plasmid vectors.....................................................................................................49 3.1.3 Template cDNA....................................................................................................49 3.1.4 Oligonucleotides...................................................................................................49 3.1.5 Cell lines...............................................................................................................49 3.1.6 Chemicals .............................................................................................................50 3.1.7 Kits .......................................................................................................................51 3.1.8 Enzymes and specified buffers.............................................................................52 3.1.9 Cell culture ...........................................................................................................52 3.1.10 Other materials ...................................................................................................52 3.2 Methods .......................................................................................................................54 3.2.1 Molecular biology ................................................................................................54 3.2.2 Cell culture and transfection methods ..................................................................57 3.2.3 Assays to determine receptor functionality ..........................................................60 3.2.4 Fluorescence measurements .................................................................................63 4 Results................................................................................................................................65 4.1 M2 mACh Receptor .....................................................................................................65 4.1.1 Cloning of the M2 mAChR FRET-sensor construct.............................................65 4.1.2 Confocal microscopy............................................................................................69 4.1.3 Receptor functionality ..........................................................................................70 4.1.4 FRET-measurements ............................................................................................74 Contents 4.2 M4 mAChR ..................................................................................................................89 4.2.1 Cloning of a functional M4-construct...................................................................90 5 Discussion ..........................................................................................................................93 5.1 Generation and optimization of a M2 mAChR FRET- sensor construct.....................93 5.2 Functionality of the M2 mAChR sensor ......................................................................94 5.3 FRET-measurements with orthosteric ligands ............................................................96 5.4 FRET-measurements with allosteric ligands...............................................................97 5.4.1 Allosteric ligands induce conformational changes in the receptor.......................97 5.4.2 Kinetics of the FRET responses for allosteric ligands .........................................98 5.5 Generation of the M4 mAChR sensor construct..........................................................99 6 Summary .........................................................................................................................100 7 Zusammenfassung ..........................................................................................................102 8 Appendix .........................................................................................................................104 8.1 Abbreviations ............................................................................................................104 8.2 List of Oligonucleotides ............................................................................................107 9 Bibliography....................................................................................................................110 10 Acknowledgment ..........................................................................................................132 11 Curriculum Vitae..........................................................................................................134 1 Introduction 1 Introduction ___________________________________________________________________________ 1.1 Allostery 1.1.1 Allostery and receptors Nature has found various ways, to transmit extracellular signalling into living cells. For example membrane proteins, like receptors or channels provide binding sites for different ligands such as neurotransmitters or hormones, which upon binding of the ligand, activate downstream