Functional expression of P2Y_1tn2 receptor and trafficking of the receptor [Elektronische Ressource] / vorgelegt von Mohan Eknath Tulapurkar

Tulapurkar Mohan

Sujets

• Biologie

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 74
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Functional expression of P2Y receptor and trafficking of the receptor 2



doctor rerum naturalium
(Dr. rer.nat.)



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


vorgelegt von Master of Science
Mohan Eknath Tulapurkar
Geb. am July 21, 1977 in Bombay, Indien

Gutachter:
Prof. Dr. Georg. Reiser, Magdeburg
Prof. Dr. Matthias Kassack, Düsseldorf

Eingericht am:

November 24, 2006

Verteidigum am:
Mai 15, 2007
1Functional expression of P2Y receptor and trafficking of the receptor 2


Thesis

For the award of the academic degree

Doctor rerum naturalium
(Dr. rer. nat.)

Corresponding to Doctor of Philosophy
(Ph.D.)

Approved by
The Faculty of Natural Science
Otto-von-Guericke-Universität Magdeburg



From Master of Science
Mohan Eknath Tulapurkar
Born on July 21, 1977, in Bombay, India

Reviewers:
Prof. Dr. Georg Reiser, Magdeburg,
Prof. Dr. Matthias Kassack, Düsseldorf

Submitted on : November 24, 2006

Defense on: May 15, 2007

2









Dedicated:
To my Parents
For all the encouragement, love and support

3Acknowledgements

This work was done from August 2000 till February 2006 in the Institute for
Neurobiochemistry, headed by Prof. Dr. G. Reiser. I would like to thank all of the
people who have helped me during this period of my work which has made the duration
of my stay in the lab an educational, memorable and a pleasurable one.
I would like to thank Professor Reiser, director of the institute who gave me the
opportunity to join here and be also a part of the Graduiertenkolleg program of
“Biological basis of central nervous system diseases”. I would like to whole heartedly
appreciate his constant support and encouragement in the progress of my work. I would
also like to add a special mention in terms of the effort that he put in during the last
stages of my thesis, where he took personal interest in helping me in successfully
completing it.
I am greatly indebted to Dr. Rainer Schäfer and Dr. Theoder Hanck, who have been my
practical advisors during the entire period of my work. Dr. Rainer Schafer has played
the most pivotal role through the entire period of my work. He was there at all time,
through all my highs and lows of my work and his long standing research experience
and in-depth knowledge in the field of P2Y receptors was crucial for channeling the
direction of my work and its successful completion. He was the one who was always
present to help me out when I found my self in a tight spot and in an uncomfortable
situation. I am thankful to Dr. Hanck for helping me with every step of my work
involving the molecular biology aspect. In addition to the help from Dr. Hanck, I am
also grateful to Dr. Fariba Sedehizade who helped me in the molecular biology aspect
and helped in the establishment of stable cell lines that were a key part of my complete
Ph.D. work. She was the one who also helped me out in settling down comfortably in
Magdeburg, which also helped me in successfully completing my work.
Dr. Gregor Zündorf and Dr. Stefan Kahlert too played an important role in my
thesis work as they were instrumental in introducing and helping me at every step with
the techniques of confocal laser scanning microscopy and calcium imaging respectively.
They helped me in the critical analysis of the data obtained by these techniques, which
was crucial to the completion of my work.
I am also thankful to Dr. Rolf Stricker and Dr. Werner Laubinger for helping me
out with the technique of western blotting and radioactive assay. The work would not
4have been complete without the expert technical from Frau Dr. Abidat Schneider, Frau
Annette Jürgen, Frau Annette Schultz and Frau K. Christoph.
I am also grateful for the excellent technical help from Mr. Peter Ehrbarth, on
which I could rely throughout my experimental work. He also played a crucial part in
the final preparation of my thesis and in helping me in moving over to USA for my
post-doc. My stay in Magdeburg was free of all the beaucratic hassles for which I am
thankful to Frau Ines Klaes, Frau Manuela Dullin-Viehweg and Frau Elke Schillings.

Labmates and colleagues are true companions, without whom a researcher is
incomplete. My special appreciation is for Denise Ecke, who was always there to cover-
up for me and face all the questions related to me when I was not there in the lab. She
did help me out in the last stages of the thesis and in ensuring that I could smoothly
leave from Germany to USA for my post-doc.
I would like to thank my colleagues who are and were there during the course of
my Ph.D.: Dr. Achim Ubl, Anastasia Galvatia, Claudia Borrmann, Daniela Walther,
Dorothee Terhardt, Evelyn Busse, Ewa Ostrowska, Hong Wang, Dr. Elena Sokolova,
Dr. Marina Sergeeva, Dr. Mikhail Strokin, Olga Krestinina, Sabine Hein, Dr. Tamara
Azarashvili, Tanuja Rohatgi, Weibo Luo and Ying Fei Wang who made my stay in the
lab enjoyable and memorable.
Last but not least I have to acknowledge the support from my friends in
Magdeburg: Ayan Kumar Bandayopahadhya, B.V. Mishra, Fahad and Ambrin, Jignesh,
Jitendar Kumar, Megha Shyam Kavuri, Shaida Andrabi and Dr.Suhel Parvez who were
always there for me. I am also thankful to others who have helped me out in my Ph.D.
work in their own ways, whom I might have unknowingly and unintentionally left out.

5Table of Contents
1 Introduction.................................................................................................................... 8
1.1 Purinergic receptors .............................................................................................. 10
1.1.2 Expression and function of the P2Y receptor............................................... 12 2
1.1.3 Functional coupling of the P2Y receptor to other receptors......................... 14 2
1.1.4 Regulation of P2Y receptor-mediated signal transduction ............................ 16
1.2. Aims of this study:............................................................................................... 22
2 Materials and Methods................................................................................................. 23
2.1 Materials ............................................................................................................... 23
2.1.1 Lab instruments and materials ....................................................................... 23
2.1.2 Chemicals....................................................................................................... 24
2.1.3 Buffer and Solvent......................................................................................... 25
2.2 Methods ................................................................................................................ 25
2.2.1 Cell Culture.................................................................................................... 25
2.2.2 Subcloning of the P2Y receptor DNA.......................................................... 25 2
2.2.3 Transfection and selection of cells................................................................. 26
2+2.2.4 Cytosolic Ca measurements........................................................................ 26
2.2.5 Live cell imaging ........................................................................................... 27
2.2.6 Agonist-induced internalization..................................................................... 27
2.2.7 Cell staining for immunofluorescence........................................................... 27
2.2.8 Confocal imaging 27
2.2.9 Cell Proliferation ELISA by 5-bromo-2’-deoxyuridine (BrdU) incorporation
................................................................................................................................ 28
2.2.10 Data analysis................................................................................................ 29
3. Results:........................................................................................................................ 30
3.1 Functional expression of GFP- and MH-tagged P2Y receptors in HEK-293 cells2..... 30
3.2 Effect of agonist concentration on receptor localization ...................................... 32
3.3 Compartmentalization of the receptor after stimulation with agonist .................. 34
3.4 UTP induced actin cytoskeletal rearrangements................................................... 36
3.5 Inhibition of receptor internalization .................................................................... 41
3.6 Colocalization of the P2Y receptor and clathrin ................................................. 41 2
3.7 Reappearance of the P2Y receptor after receptor internalization........................ 45 2
3.8 Role of kinases and phosphotases in modulating the translocation of the rP2Y -2
GFP receptor............................................................................................................... 47
3.8.1 Role of Calcium