Mitogenic signaling by G_1tnq_1tn/_1tn1_1tn1-coupled receptors [Elektronische Ressource] / by Susanne Roelle

Susanne Roelle

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Introduction





























For my parents Mathilde and Wolfgang
For my sister Barbara


1 Introduction

From the Institute of Pharmacology and Toxicology
Philipps-University Marburg
Director: Prof. Dr. med. Thomas Gudermann


Mitogenic signaling by G -coupled receptors q/11





Inaugural dissertation
to obtain a degree of a doctor
in natural sciences
(Dr. rer. nat.)

presented to the
Department of Chemistry
Philipps-University Marburg

by
Susanne Roelle
born in Brühl/Rheinland













2 Introduction





Accepted by the Department of Chemistry on April 7, 2004

st1 referee: Prof. Dr. Thomas Carell
nd2 referee: Prof. Dr. Thomas Gudermann
Day of oral examination: April 15, 2004
3 Introduction
TABLE OF CONTENTS
1. Introduction ................................................................................................ 12
1.1. Signaling pathways emanating from G protein-coupled receptors (GPCRs)12
1.2. Mitogenic signaling by G -coupled receptors............................................ 15 q/11
2+1.2.1. Ca -mediated activation of Pyk2................................................................. 19
1.2.2. GPCR-dependent Epidermal Growth Factor receptor transactivation ......... 20
1.3. Neuropeptide-mediated ERK activation....................................................... 24
1.3.1. The Gonadotropin-releasing hormone receptor utilizes PKC to activate the
ERK/MAPK cascade .................................................................................... 25
2+1.3.2. Galanin mediates growth of small cell lung cancer cells via rises in [Ca ] i
and ERK activation....................................................................................... 27
1.4. Apoptosis ..................................................................................................... 30
2. Objectives................................................................................................... 32
3. Materials and Methods .............................................................................. 33
3.1. Materials....................................................................................................... 33
3.1.1. Reagents...................................................................................................... 33
3.1.2. Cell Culture Supply ...................................................................................... 33
3.1.3. Antibodies .................................................................................................... 34
3.1.4. Agonists and Drugs 35
3.1.5. Oligonucleotides........................................................................................... 36
3.1.6. Mutagenesis primer 36
3.1.7. Hammerhead ribozymes.............................................................................. 36
3.2. Methods ....................................................................................................... 37
3.2.1. Cell culture and transient transfection.......................................................... 37
3.2.1.1. SCLC cell lines............................................................................................. 37
3.2.1.2. Gene transfer into SCLC cells by a viral approach using retroviruses......... 38
3.2.1.3. PC12W cells................................................................................................. 38
3.2.1.4. αT3-1 cells ................................................................................................... 39
3.2.1.5. LβT2 cells..................................................................................................... 39
3.2.1.6. PC-3 cells 39
4 Introduction
3.2.2. Biochemical Methods................................................................................... 40
3.2.2.1. Standard procedures.................................................................................... 40
3.2.2.2. Immunoprecipitation..................................................................................... 40
3.2.2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western
blotting procedures....................................................................................... 41
3.2.2.4. Src kinase activity assay.............................................................................. 44
3.2.2.5. ERK activity assay 45
3.2.2.6. Ras activation assay 46
3.2.2.6.1. Preparation of GST fusion proteins containing the Ras-binding domain of
Raf-1........................................................................................................ 46
3.2.2.6.2. Ras activation Assay ............................................................................... 47
3.2.2.7. Gelatin zymography ..................................................................................... 47
3.2.2.8. Colony formation assay................................................................................ 48
3.2.2.9. Liquid growth assay 48
3.2.2.10. Detection of apoptosis............................................................................. 48
3.2.3. Molecular biological methods....................................................................... 49
3.2.3.1. Standard procedures.................................................................................... 49
3.2.3.2. Preparation of total RNA from αT3-1 and SCLC cells.................................. 49
3.2.3.3. RT-PCR of Pyk2 in SCLC cells.................................................................... 50
3.2.3.4. guanine nucleotide-releasing factors for Ras ............................ 50
3.2.3.5. Molecular cloning strategies......................................................................... 50
3.2.3.5.1. Cloning of Pyk2 and generation of Pyk2 mutants.................................... 50
3.2.3.5.2. Cloning of Pyk2 into the retroviral pLXSN vector 51
3.2.4. Reproducibility of results.............................................................................. 51
4. Results ........................................................................................................ 52
4.1. The gonadotropin-releasing hormone receptor utilizes PKC to activate the
ERK/MAPK cascade .................................................................................... 52
4.1.1. GnRH-induced ERK activation depends on gelatinase activity in
gonadotropic cells. ....................................................................................... 52
4.1.2. HB-EGF shedding is involved in GnRH-mediated ERK activation............... 59
4.1.3. Gelatinase activity is involved in GnRH-mediated EGF receptor
transactivation in gonadotropic cells. ........................................................... 62
4.1.4. HB-EGF shedding mediates EGF receptor phosphorylation in gonadotropic
cells.............................................................................................................. 67
5 Introduction
4.1.5. GnRH-mediated Src activation in αT3-1 cells depends on gelatinase activity.
..................................................................................................................... 68
4.1.6. GnRH-elicited Ras activation is dependend on EGF receptor transactivation
and gelatinase activity in gonadotropic cells................................................ 71
4.1.7. EGF receptor tyrosine kinase and MMP activities are not required to mediate
GnRH-induced activation of JNK and p38MAPK. ........................................ 72
4.1.8. The induction of c-fos and c-jun depends on gelatinase activity and EGF
receptor transactivation................................................................................ 74
4.2. Neuropeptides mediate growth of small cell lung cancer cells via rises in
2+[Ca ] and ERK activation............................................................................ 76 i
4.2.1. Neuropeptide-activated Pyk2 associates with Src kinases and PI3K in a
2+[Ca ] –dependent way. ................................................................................ 76 i
2+4.2.2. Galanin or elevation of [Ca ] stimulate Src activity in SCLC cells. ............. 80 i
2+4.2.3. Neuropeptide-induced Ras activation is dependent on [Ca ] and Src kinase i
activity with RasGEFs not being involved. ................................................... 82
4.2.4. Src kinase activity is required for neuropeptide-induced ERK activation and
anchorage-independent growth of SCLC cells............................................. 83
4.2.5. Overexpression of Pyk2 induces cell death in SCLC cells........................... 86
5. Discussion.................................................................................................. 91
5.1. Matrix metalloproteinases 2 and 9 mediate EGF receptor transactivation by
gonadotropin-releasing hormone ................................................................. 91
5.2. Pyk2 represents a cellular point of convergence by regulating neuropeptide-
medi