Investigation on the EGFR signal transactivation by G protein coupled receptors in cancer cells [Elektronische Ressource] / Beatrice Marg

Beatrice Marg

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München







Investigation on the
EGFR signal transactivation
by G protein coupled receptors
in cancer cells






Beatrice Marg

aus

Bielefeld










2005

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Axel Ullrich betreut und von Herrn Prof. Dr. Horst
Domdey vor der Ludwig-Maximilians-Universität München vertreten.


Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, den 15.11.2004












Dissertation eingereicht am 15.11.2004
1. Gutachter Prof. Dr. Axel Ullrich
2. Horst Domdey
Mündliche Prüfung am 03.05.2005










Für meine Eltern


















On ne voit bien qu’avec le cœur. L'essentiel est invisible pour les yeux.

Antoine de Saint Exupéry I
Contents

Contents .................................................................................................................................I
1 Introduction................................................................................................... 1
1.1 Protein tyrosine kinases ...................................................................................... 2
1.1.1 Receptor tyrosine kinases (RTKs) ................................................................. 3
1.1.2 EGFR family.................................................................................................. 4
1.1.3 EGF-like ligand induced activation of RTKs ................................................ 4
1.1.4 Cytoplasmic tyrosine kinases......................................................................... 6
1.1.5 RTK downstream signaling and protein interaction domains........................ 7
1.2 Mitogen-activated-protein-kinase (MAPK) pathways..................................... 8
1.3 Protein kinase B (PKB)/Akt ............................................................................. 10
1.4 G protein coupled receptors 10
1.4.1 Heterotrimeric G-proteins............................................................................ 11
1.4.2 Mitogenic GPCR signaling.......................................................................... 12
1.4.3 The LPA receptors Edg 2, 4 and 7............................................................... 13
1.5 EGFR signal transactivation 15
1.6 Metalloproteases ................................................................................................ 18
1.6.1 ADAMs........................................................................................................ 19
1.6.2 The matrix metalloproteinases (MMPs) ...................................................... 22
1.7 Molecular oncology ........................................................................................... 23
1.8 Aim of the study................................................................................................. 24
2 Materials and Methods ............................................................................... 26
2.1 Materials............................................................................................................. 26
2.1.1 Laboratory chemicals and biochemicals...................................................... 26
2.1.2 Enzymes....................................................................................................... 27
2.1.3 Radiochemicals............................................................................................ 27
2.1.4 "Kits" and other materials............................................................................ 28
2.1.5 Growth factors and ligands .......................................................................... 28
2.1.6 Media and buffers ........................................................................................ 29
2.1.7 Stock solutions and buffers 30 II
2.1.8 Bacteria strains (E. coli)............................................................................... 32
2.1.9 Cell lines ...................................................................................................... 33
2.1.10 Antibodies.................................................................................................... 33
2.1.11 Plasmids and oligonucleotides..................................................................... 35
2.2 Methods in molecular biology .......................................................................... 38
2.2.1 Plasmid preparation for analytical purpose.................................................. 38
2.2.2 Plasmid preparation in preparative scale ..................................................... 38
2.2.3 Enzymatic manipulation of DNA ................................................................ 39
2.2.4 Agarose gel electrophoresis ......................................................................... 40
2.2.5 Introduction of plasmid DNA into E.coli cells ............................................ 40
2.2.6 Enzymatic amplification of DNA by polymerase chain reaction (PCR)..... 41
2.2.7 RT-PCR analysis.......................................................................................... 42
2.2.8 DNA sequencing 42
2.2.9 cDNA array hybridization............................................................................ 42
2.3 Methods in mammalian cell culture ................................................................ 43
2.3.1 General cell culture techniques.................................................................... 43
2.3.2 Transfection of cultured cell lines 44
2.3.3 Retroviral gene transfer in cell lines ............................................................ 45
2.4 Protein analytical methods ............................................................................... 46
2.4.1 Lysis of eucaryotic cells 46
2.4.2 Determination of protein concentration in cell lysates ................................ 46
2.4.3 Immunprecipitation and Isolation of Glycoproteins.................................... 46
2.4.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .............................. 47
2.4.5 Transfer of proteins on nitrocellulose membranes....................................... 47
2.4.6 Immunoblot detection .................................................................................. 48
2.5 Generation of polyclonal antibodies ................................................................ 48
2.5.1 Large scale expression of GST-fusion proteins........................................... 48
2.5.2 Immunisation of rabbits ............................................................................... 49
2.6 Biochemical and cell biological assays............................................................. 50
2.6.1 Stimulation of cells ...................................................................................... 50
2.6.2 Erk 1/2 and Akt/PKB phosphorylation........................................................ 50
2.6.3 Erk/MAPK activity 50
2.6.4 Focus formation assay.................................................................................. 51
2.6.5 Proliferation assay........................................................................................ 51 III
2.6.6 In vitro wound closure ................................................................................. 51
2.6.7 Migration of cancer cells ............................................................................. 52
2.7 Statistical analysis.............................................................................................. 52
3 Results.......................................................................................................... 53
3.1 ADAM-specific antibodies ................................................................................ 53
3.1.1 ADAM10,MP antibody 53
3.1.2 ADAM12 antibody ...................................................................................... 54
3.2 Reconstituted LPA receptor expression in McA-RH7777 cells..................... 55
3.2.1 Stable expression of LPA receptors in McA-RH7777 cells ........................ 56
3.2.2 Expression profile of different ADAM proteins in McA-RH7777 cells ..... 56
3.2.3 EGFR transactivation can not be restored in cells stably expressing Edg 2, 4
or 7 ............................................................................................................... 57
3.2.4 Restored Erk phosphorylation upon Stimulation with LPA ........................ 59
3.2.5 Akt activation upon stimulation with LPA .................................................. 60
3.3 EGFR transactivation in A498 .............................................