Analysis of two newly identified protease activated receptor-2-interacting proteins, Jab1 and p24A, and their role in receptor signalling [Elektronische Ressource] / von Weibo Luo

Luo

Sujets

• Biologie

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 109
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Analysis of Two Newly Identified Protease-Activated
Receptor-2-Interacting Proteins, Jab1 and p24A,
and their Role in Receptor Signalling






Dissertation

zur Erlangung des akademischen Grades
(To the acquisition of the academic degree)



doctor rerum naturalium
(Dr. rer. nat.)



genehmigt durch
die Fakultät für Naturwissenschaften
der Otto-von-Guericke-Universität Magdeburg



von M. Sc. Weibo Luo
geb. am 21.06.1976 in Zhejiang, China


Gutachter: Prof. Dr. Georg Reiser
Prof. Dr. Uwe-Karsten Hanisch


eingereicht am: 16 November, 2006
verteidigt am: 19 April 2007 ACKNOWLEGEMENTS

I am very grateful to those who gave me all kinds of help and support during this work at
the Institute of Neurobiochemistry, Otto-von-Guericke Universität Magdegurg.
First of all, I would like to sincerely thank my supervisor, Prof. Dr. Georg Reiser, for
providing me an opportunity to join his lab to complete my Ph.D. work. His invaluable
knowledge, constructive discussion, professional guidance and constant support enabled me
to achieve my goals efficiently and easily, and to be of great benefit to my scientific career
throughout my life. I am happy to be a member of the Graduate School directed by Prof.
Reiser, and the wonderful lectures, seminars and practical courses he organized opened my
scientific ken and stimulated my idea. Of course, many thanks also to our Graduate School
secretary Frau Manuela Dullin-Viehweg, who assisted the nice organization of the scientific
and social activities.
I would like to deeply thank Dr. Rolf Stricker and Dr. Theodor Hanck for the introduction
of the baculoviral expression system and GST pull-down assays, valuable scientific
suggestions and technical supports.
I also appreciate Frau Evelyn Busse for her excellent technical assistance at the late
period of this work, as well as other colleagues in the lab, Dr. Fariba Sedehizade, Dr. Abidat
Schneider, Frau Petra Grüneberg, Frau Anke Imrich, Dr. Gregor Zündorf, Dr. Stefan Kahlert,
Frau Annette Jürgen, Frau Denise Ecke, Frau Ewa Ostrowska, Dr. Elena Sokolova, Dr.
Mikhail L. Strokin, Frau Anastasia Galvita, Frau Dorothe Terhardt, Herr Mohan E.
Tulapurkar, Frau Claudia Baumann, Dr. Tanuja Rohatgi, Herr Rongyu Li, and Frau Sabine
Hein for their cooperation and support, and Frau Ines Klaes, Herr Peter Ehrbarth for their kind
support, during the course of this work.
I would like to thank Prof. Dr. Bernhard A. Sabel, Institute of Medical Psychology, who
enabled me to come to Magdeburg for study.
Finally, I would express my special gratitude to my close colleague and wife Frau
Yingfei Wang for her constant discussions, advice, encouragement and love, and to my
parents for their support and understanding.
CONTENTS


1 Introduction ................................................................................................................…....1
1.1 Protease ……………………………………………………………………………..…...1
1.2 Protease-activated receptor ………………………………………………………..…….1
1.2.1 Subtypes of PAR …………………………………………………………………..2
1.2.1.1 PAR-1 ……………………………………………………………………2 1.2.1.2 PAR-2 ……………………………………………………………………3
1.2.1.3 PAR-3 ……………………… 1.2.1.4 PAR-4 ……………………………………………………………………4
1.2.2 Agonists and antagonists of PAR ………………………………………………….4
1.2.3 Mechanisms of PAR activation ……………………………………………………9
1.2.4 PAR-mediated intracellular signal transductions ……………………………….…9
1.2.5 Termination of PAR signaling …………………………………………………...13
1.2.6 PAR trafficking ……………………………………………………..……………14
1.2.7 Post-translational modification of PAR ..........……...............................................16
1.2.7.1 Glycosylation .............................................................................................16
1.2.7.2 Palmitoylation ............................................................................................17
1.3 Scope and aims of project …………………………...…………………………………18
1.3.1 Background ………………………………………………………………………18
1.3.2 Specific aims ……………………………………………………………………18
1.3.3 Strategy ………………………………………………………………………..…19
2 Materials and Methods …………………………………………………………………..20
2.1 Materials ………………………………………………………………………………..20
2.1.1 Chemicals and reagents …………………………………………………………..20
2.1.2 Antibodies ………………………………………………………………………..20
2.1.2.1 Primary antibodies ……………………………………………………….20
2.1.2.2 Secondary …………………………………………………….21
2.1.3 Cells, medium and related reagents ……………………………………………...21
2.1.3.1 Mammalian cells …………………………………………………………21
2.1.3.2 Insect cells ………………………………………………………………..21
2.1.3.3 Bacterial cells …………………………………………………………….22 2.1.3.4 Yeast cells ………………………………………………………………..22
2.1.4 Vectors …………………………………………………………………………...22
2.1.5 Small interfering RNAs ………………………………………………………..23
2.1.6 Enzymes ………………………………………………………………………….23
2.1.7 Markers ………………………………………………………………………...23
2.1.8 Buffers ……………………………………………………………………………23
2.1.9 Kits ……………………………………………………………………………….25
2.1.10 Instruments …………………………………………………………………….26
2.2 Methods ………………………………………………………………………………...27
2.2.1 RT-PCR ………………………………………………………………………….27
2.2.2 Plasmid constructs ……………………………………………………………….27
2.2.3 Yeast two-hybrid screening ……………………………………………………...28
2.2.3.1 Small-scale LiAc yeast transformation …………………………………..28
2.2.3.2 Yeast mating ……………………………………………………………..28
2.2.3.3 β-galactosidase assay …………………………………………………….28
2.2.3.4 Plasmids isolation from yeast ……………………………………………31
2.2.3.5 Rescue AD/library plasmids by transformation of E. Coli ………………31
2.2.4 Cell culture and transfection …….……………………………………………….31
2.2.5 PAR-2 activation and inhibitor treatment………………………………………32
2.2.6 GST pull-down assays …………………………………………………………..32
2.2.7 Immunoprecipitation ……………………………………………………………..33
2.2.8 Western blot ……………………………………………………………………...34
2.2.9 Immunofluorescence analysis ……………………………………………………34
2.2.10 Reporter gene assays ……………………………………………………………35
2.2.11 SiRNA …………………………………………………………………..…….35
2.2.12 Cytosolic calcium measurements ……………………………………………….36
2.3 Statistical analysis ……………………………………………………………………...36
3 Results …………………………………………………………………………………...37
Part I. Identification of interacting proteins of human PAR-2
by using yeast two-hybrid screening ………………………………………………37
Part II. PAR-2 overexpression in insect and mammalian cells ……………………….40
3.2.1 PAR-2 overexpression in Sf9 cells ………………………………………………40
3.2.2 PAR-2 in HEK293 cells ……………………………………….43
Part III. Jun activation domain-binding protein 1 is involved in PAR-2-induced activation of AP-1 ………………………………………………..48
3.3.1 Multiple intracellular domains of PAR-2 are responsible for
interaction with Jab1…………………………………………………………….. 48
3.3.2 Jab1 interacts with PAR-2 in vivo ………………………………………………49
3.3.3 Colocalization of Jab1 with PAR-2 in vivo ………………………………………52
3.3.4 Jab1 interacts with PAR-2 in normal primary human astrocytes ……………….54
3.3.5 PAR-2 activation reduces interaction with Jab1 ……………………………….57
3.3.6 The effect of PAR-2 activation on Jab1 distribution and expression …………..61
3.3.7 Jab1 mediates PAR-2-induced c-Jun activation ………………………………..63
3.3.8 Jab1 potentiates AP-1 ………………………………65
Part IV. p24A binds to the intracellular PAR-2, and might regulate
resensitization of PAR-2 ……………………………………………………………68
3.4.1 PAR-2 interacts with p24A in vitro ……………………………………..……….70
3.4.2 PAR-2 with p24A in vivo ………………………………………………73
3.4.3 Colocalization of PAR-2 with p24A in vivo ……………………………………..76
3.4.4 PAR-2 interacts with p23, another member of the p24 family …………………..78
3.4.5 p24 interacts with further receptors: PAR-1 and P2Y receptor..………...………79 1
3.4.6 PAR-2 activation disrupts the interaction with p24A and p23 …………………..81
4 Discussion ………………………………………………………………………………….84
4.1 General background and scenario for assessing the significance of
the present findings ……………………………………………………………….….84
4.2 Jab1 as a signal messenger mediates the signalling of extracellular proteases
trypsin, tryptase and others to the nuleus …………………………………………….85
4.3 p24A interacts with intracellular PAR-2: possible functions of
p24A-PAR-2 interaction…………………………………………………………..…88
4.4 Conclusion ……………………………………………………………………………..91
5 Abstract …………………………………………………………………………………93
5.1