Characterization of allosteric mechanisms on the M_1tn2 and M_1tn4 mACh receptor using the FRET-technique [Elektronische Ressource] = Charakterisierung allosterischer Mechanismen am M_1tn2- und M_1tn4-mACh-Rezeptor unter Anwendung der FRET-Technik / submitted by Monika Maier-Peuschel

Monika Maier-Peuschel

Sujets

• Naturwissenschaften

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

Characterization of allosteric mechanisms
on the M und M mACh receptor 2 4
using the FRET-technique


Charakterisierung allosterischer Mechanismen
am M und M mACh Rezeptor 2 4
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 M und M 2 4
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 Allostery1
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 considerations47
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 biology54
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 M mACh Receptor .....................................65 2
4.1.1 Cloning of the M mAChR FRET-sensor construct.............65 2
4.1.2 Confocal microscopy............................................................69
4.1.3 Receptor functionality ..........................................................70
4.1.4 FRET-measurements74
Contents
4.2 M mAChR ..................................................................................................................89 4
4.2.1 Cloning of a functional M -construct...90 4
5 Discussion..........................93
5.1 Generation and optimization of a M mAChR FRET- sensor construct .....................93 2
5.2 Functionality of the M mAChR sensor ......................................................................94 2
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 M mAChR sensor construct ..........................................................99 4
6 Summary .........................................................................................100
7 Zusammenfassung..........102
8 Appendix104
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 signalling processes in the cell. With about 800 different receptors in the human
body, the G-protein coupled receptors (GPCR) represent the largest receptor family
(Lagerstrom and Schioth 2008) and thus represent an important drug target. GPCRs are
heptahelical transmembrane proteins consisting of seven transmembrane domains, three
extracellular and intracellular loops with a N- and C-terminal tail. The mammalian GPCRs
were divided into four different subclasses by means of their functional attributes and
homology by the International Union of Pharmacology (IUPHAR) (Foord et al. 2005). Class
1 contains the rhodopsin like GPCRs, such as the adrenergic receptor family or the muscarinic
acetylcholine receptors (mAChR). The secretory receptors were allocated to Class 2, the
metabotropic glutamate receptors represent class 3. The latest class, frizzled family receptors,
contains Frizzled, an important receptor family for cell differentiation, social behaviour,
embryonic development and many other processes (Malbon 2004), and Smoothened, which
plays a role in embryonic development (Corbit et al. 2005). In non-vertebrates, two further
families (fungal mating pheromone receptors and cAMP receptors) have been identified
(Attwood and Findlay 1994).
This thesis will concentrate on class A GPCRs, specifically the subtypes of the muscarinic
acetylcholine receptor family (M mAChR). GPCRs transmit their signal via a 1-5
heterotrimeric G-protein, which is activated by conformational changes of the specific GPCR.
To describe the binding sites of the endogenous receptor on the receptor surface, the term
“orthosteric site” is used (Ehlert 1985). All ligands binding exclusively to this site are termed
“orthosteric ligands”. Among the muscarinic receptor family, the orthosteric binding site
seems to be highly conserved (Hulme et al. 2003). Site-directed mutagenesis has identified
several conserved amino acids (see Figure 1.1.1 for example for human M mAChR: C98, 1
D105, Y106, P158, W164, R171, T172, C178, Q181, F182, S184, T189, T192, P200, W378,
Y381, Y404, Y408, P415) that seem to play a key role in binding of orthosteric l