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 B2 receptor 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 B2eYFP chimera .........................................................................................................28 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 3 D.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 3 D.2.3.3 Competitive inhibition [ H]BK binding to cold drugs. ..........................................................................38 D.2.4 Determination of second messengers after Gq/11 activation ...........................................................................38 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 3 D.2.5.2 Internalization of [ H]BK or [ H]NPC17331..........................................................................................40 D.2.6 Protein biochemical methods ...........................................................................................................................41 D.2.6.1 SDS polyacrylamide gel electrophoresis.................................................................................................41 D.2.6.2 Immunoblotting after solubilization with RIPA .....................................................................................41 D.2.6.3 Protein levels of B2 receptor during prolonged agonist/antagonist treatment ......................................41 D.2.6.4