Characterisation of Metalloprotease-mediated EGFR Signal Transactivation after GPCR Stimulation [Elektronische Ressource] / Matthias Schneider. Betreuer: Georg Krohne

Schneider

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Characterisation of Metalloprotease-mediated EGFR Signal
Transactivation after GPCR Stimulation



Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg




vorgelegt von

Matthias Schneider
aus
Ochsenfurt



Würzburg , 2011








Eingereicht am: 21.03.2011

Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. T. Dandekar
Gutachter: Prof. Dr. G. Krohne
Gutachter: Prof. Dr. A. Ullrich

Tag des Promotionskolloquiums: 29. Juni 2011

Doktorurkunde ausgehändigt am:.......................................................................................



Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation mit dem Titel

„Characterisation of Metalloprotease-mediated EGFR Signal
Transactivation after GPCR Stimulation“

selbständig am Max-Planck-Institut für Biochemie in Martinsried unter der Anleitung
und Betreuung durch Herrn Prof. Dr. Axel Ullrich (MPI für Biochemie, Martinsried)
und Herrn Prof. Dr. Georg Krohne (Julius-Maximilians-Universität, Würzburg)
angefertigt habe und dabei keine anderen als die von mir angegebenen Quellen und
Hilfsmittel verwendet habe.
Ich erkläre außerdem, dass die vorliegende Dissertation weder in gleicher, noch in
ähnlicher Form bereits in einem Prüfungsverfahren vorgelegen hat. Des weiteren
habe ich außer den mit dem Zulassungsantrag urkundlich vorgelegten Graden keine
weiteren akademischen Grade erworben oder zu erwerben versucht.



Martinsried, den 21.03.2011

____________________________
Matthias Schneider






Dedicated to my Parents
and Martina
Contents

1. INTRODUCTION .................................................................................................... 1
1.1 Protein Tyrosine Kinases .............................................................................................. 2
1.1.1 Receptor Tyrosine Kinases (RTKs) ........................................................................................ 2
1.1.1.1 EGFR family........................................................................................................................ 3
1.1.1.2 EGF-like ligands and receptor activation ............................................................................ 4
1.1.2 Cytoplasmic tyrosine kinases ................................................................................................ 6
1.1.3 RTK Downstream Signaling and Protein Interaction Domains ........................................... 8
1.2 G Protein Coupled Receptors ......................................................................................10
1.2.1 Heterotrimeric G-proteins ..................................................................................................... 12
1.2.2 Mitogenic GPCR Signalling ................................................................................................... 13
1.2.3 The Function of Arrestins ..................................................................................................... 13
1.3 Mitogen-Activated-Protein-Kinase (MAPK) pathways ................................................15
1.4 Receptor tyrosine kinase transactivation ...................................................................19
1.4.1 EGFR signal transactivation ................................................................................................. 19
1.4.1.1 Metalloproteases .............................................................................................................. 22
1.4.1.1.1 ADAMs ...................................................................................................................... 23
1.4.1.1.2 MMPs and TIMPs ...................................................................................................... 25
1.4.2 Reactive Oxygen Species (ROS) .......................................................................................... 26
1.5 Aim of this study ..........................................................................................................28
2 MATERIALS AND METHODS .............................................................................. 29
2.1 Materials ........................................................................................................................29
2.1.1 Laboratory chemicals and biochemicals............................................................................. 29
2.1.2 Enzymes .................................................................................................................................. 31
2.1.3 "Kits" and other materials ..................................................................................................... 31
2.1.4 Growth factors and ligands .................................................................................................. 32
2.1.5 Media and buffers .................................................................................................................. 32
2.1.5.1 Media for E. coli bacteria .................................................................................................. 32
2.1.5.2 Cell culture media ............................................................................................................. 33
2.1.6 Stock solutions and buffers .................................................................................................. 34
2.1.7 Bacteria strains (E. coli) ........................................................................................................ 37
2.1.8 Cell lines.................................................................................................................................. 37
2.1.9 Antibodies ............................................................................................................................... 38
2.1.10 Plasmids and oligonucleotides .......................................................................................... 39
2.1.10.1 Primary vectors ............................................................................................................... 39
2.1.10.2 Constructs ....................................................................................................................... 40
2.1.10.3 Important oligonucleotides .............................................................................................. 41
2.1.10.4 siRNA nucleotides .......................................................................................................... 41
2.2 Methods in molecular biology .....................................................................................43
2.2.1 Plasmid preparation for analytical purpose ........................................................................ 43
2.2.2 Plasmid preparation in preparative scale ............................................................................ 43
2.2.3 Enzymatic manipulation of DNA ........................................................................................... 43
2.2.3.1 Digestion of DNA samples with restriction endonucleases .............................................. 43
2.2.3.2 Dephosphorylation of 5'-termini with calf intestine alkaline phosphatase (CIAP) ............ 43 Contents
2.2.3.3 DNA insert ligation into vector DNA .................................................................................. 44
2.2.4 Agarose gel electrophoresis ................................................................................................. 44
2.2.4.1 Isolation of DNA fragments using low melting temperature agarose gels ........................ 44
2.2.5 Introduction of plasmid DNA into E.coli cells ..................................................................... 45
2.2.5.1 Preparation of competent E. coli bacteria ......................................................................... 45
2.2.5.2 Transformation of competent E. coli bacteria ................................................................... 45
2.2.6 Enzymatic amplification of DNA by polymerase chain reaction (PCR) ............................ 45
2.2.7 RT-PCR analysis .................................................................................................................... 46
2.2.8 DNA sequencing .................................................................................................................... 47
2.3 Methods in mammalian cell culture ............................................................................48
2.3.1 General cell culture techniques ............................................................................................ 48
2.3.2 Transfection of cultured cell lines ........................................................................................ 48
2.3.2.1 Transfection of cells with calcium phosphate ................................................................... 48
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2.3.2.2 Transfection of cells with Lipofectamine ......................................................................... 49