Role of the intracellular domains in the regulation and the signaling of the human bradykinin B_1tn2 receptor [Elektronische Ressource] / vorgelegt von Göran Wennerberg

Göran Wennerberg

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 91
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Aus der Abteilung für Klinische Chemie und Klinische Biochemie
in der Chirurgischen Klinik-Innenstadt
der Ludwig-Maximilians-Universität München


Leiterin der Abteilung: Prof. Dr. rer. nat. Dr. med. habil. Marianne Jochum










Role of the intracellular domains in the regulation and
the signaling of the human bradykinin B receptor 2







Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der
Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München






Vorgelegt von
Göran Wennerberg
aus Stockholm

2010



Mit Genehmigung der Medizinischen Fakultät
der Ludwig-Maximilians-Universität München









Berichterstatter: PD Dr. rer. nat. Alexander Faussner


Mitberichterstatter: Prof. Dr. Nikolaus Plesnila
Prof. Dr. Franz-Xaver Beck


Mitbetreuung durch den
promovierten Mitarbeiter:


Dekan: Prof. Dr. med. Dr. h.c. M. Reiser,
FACR,FRCR


Tag der mündlichen Prüfung: 27.01.2010
CONTENTS………………………………………………………………………………………I

ABBREVIATIONS………………………………………………………………………………V
A ZUSAMMENFASSUNG ............................................................................................ 1
B INTRODUCTION ....................................................................................................... 4
B.1 The kallikrein-kinin system (KKS)
B.1.1 Historic background............................................................................................................................................4
B.1.2 Kinins...................................................................................................................................................................4
B.1.3 Kinin receptors ....................................................................................................................................................5
B.1.4 The signaling pathways of the kinin receptors..................................................................................................6
B.1.5 Regulation of kinin receptors .............................................................................................................................7
B.2 G proteins and G protein-coupled receptors (GPCRs)
B.2.1 G proteins ............................................................................................................................................................8
B.2.2 Functionality........................................................................................................................................................8
B.2.3 Superfamily of GPCRs .......................................................................................................................................9
B.2.4 The highly conserved DRY motif ....................................................................................................................10
B.2.5 Crystal structures of GPCRs.............................................................................................................................11
B.3 Receptor antagonists
B.4 Functional selectivity
C AIMS OF THE THESIS............................................................................................ 16
D MATERIAL AND METHODS................................................................................... 17
D.1 Material
D.1.1 Equipment .........................................................................................................................................................17
D.1.2 Chemicals and materials...................................................................................................................................18
D.1.3 Strains and cell lines .........................................................................................................................................21
D.1.4 Expression vectors ............................................................................................................................................22
D.1.5 Oligonucleotides used as primers for PCR amplification ..............................................................................23
D.1.6 Computer programs and Data analysis............................................................................................................23
D.1.7 Solutions............................................................................................................................................................24

CONTENTS I D.2 Methods
D.2.1 Molecular biological methods..........................................................................................................................25
D.2.1.1 Polymerase chain reaction (PCR)............................................................................................................25
D.2.1.2 Attachment of the restriction sites ...........................................................................................................25
D.2.1.3 Site-directed mutagenesis.........................................................................................................................26
D.2.1.4 Generation of the B eYFP chimera .........................................................................................................28 2
D.2.1.5 DNA cleavage with restriction endonucelases .......................................................................................30
D.2.1.6 Agarose gel electrophoresis .....................................................................................................................30
D.2.1.7 Extraction of DNA fragments from agarose gels ...................................................................................30
D.2.1.8 Ligation of DNA fragments .....................................................................................................................31
D.2.1.9 LB growth medium and plates for culture of E. coli strains ..................................................................31
D.2.1.10 Transformation of E.coli...........................................................................................................................31
D.2.1.11 Colony-PCR ..............................................................................................................................................32
D.2.1.12 Plasmid preparation from E.coli...............................................................................................................32
D.2.1.13 Confirmation of correctness of DNA sequence ......................................................................................32
D.2.1.14 Determination of DNA concentration......................................................................................................32
D.2.2 Cell culture methods .........................................................................................................................................34
D.2.2.1 Culture of mammalian cells .....................................................................................................................34
D.2.2.2 Cell freezing and thawing ........................................................................................................................34
D.2.2.3 Flp-In expression system .........................................................................................................................34
D.2.2.4 Transfection of HEK 293 cells ................................................................................................................36
3D.2.3 [ H]Bradykinin binding studies .......................................................................................................................37
D.2.3.1 Expression levels of constructs................................................................................................................37
D.2.3.2 Equilibrium binding experiments at 37°C and 4°C................................................................................37
3D.2.3.3 Competitive inhibition [ H]BK binding to cold drugs. ..........................................................................38
D.2.4 Determination of second messengers after G activation ...........................................................................38 q/11
D.2.4.1 Measurement of total inositol phosphate (IP) release ............................................................................38
D.2.4.2 Basal and stimulated IP accumulation in fibroblasts HF-15 after pro-longed stimulation ..................39
2+D.2.4.3 Time and Concentration-dependent release of intracellular [Ca ].......................................................39
D.2.5 Receptor sequestration and ligand internalization assays ..............................................................................40
D.2.5.1 Down-regulation assay .............................................................................................................................40
3 3D.2.5.2 Internalization of [ H]BK or [ H]NPC17331................................................................