Post translational modulation and regulation of glutamate transporters [Elektronische Ressource] / vorgelegt von Poonam Balani

Igs

Sujets

• Biologie

Informations

• Publié par: westfalische_wilhelms-universitat_munster
• Publié le: 01 janvier 2005
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Poonam Balani












Post Translational Modulation and Regulation of
Glutamate Transporters
















-2005-


Biochemie








Post Translational Modulation and Regulation of
Glutamate Transporters

Inaugural-Dissertation
zur Erlangung des Doktorgrades
in der NRW Graduate School of Chemistry
im Fachbereich Chemie und Pharmazie
der Mathematisch-Naturwissenschaftlichen Fakultät
der Westfälischen Wilhelms-Universität Münster
vorgelegt von

Poonam Balani

aus

New Delhi, India
-2005-








Dekan: Prof. Dr. B. Wünsch
Erster Gutachter: Prof. Dr. H.-J. Galla
Zweiter Gutachter: PD Dr. Thomas Rauen
Tag der mündlichen Prüfung: 26.10.2005
Tag der Promotion: 26.10.2005



st st
The work described in this thesis was carried out from 1 October 2002 to 31 July
2005 at the institute for biochemistry, Westfälischen Wilhelms Universität Münster
under the guidance of Prof. Dr. H.-J. Galla and PD Dr.Thomas Rauen.



























I am thankful to Prof. Dr. H.-J.Galla and Dr. Thomas Rauen for the interesting topic
and their cooperation in the form of research ideas and discussions during the course
of my research work.




















































My Parents






Acknowledgements

I would like to thank Prof. Dr. H.-J.Galla and Dr. Thomas Rauen for the interesting
theme given for my Ph.D. thesis. I am grateful to them for their valuable time and
worthy suggestions and ideas which made this work possible.

I would also like to thank all the members of the BBB as well as the lipidis group for
always being there whenever I needed help.
My special thanks are due to Markus, Patrick, Vanessa, Christina and Pariya for the
great scientific discussions we had during the course of last three years.

I also would like to acknowledge the help and support offered by PD Dr. Joachim
Wegener the best when I needed it the most.
I also express my heartfelt thanks to Sabina, Sandra, Steffi, Freddy and Wolfgang for
the innumerable times they offered their help.

I would also like to express my gratitude towards the graduate school of chemistry for
the financial support to carry out my work at Muenster, as well as for the travel grants
provided for the conferences. I especially would like to thank Dr. Hubert Koller and
Stephan Elbers for the cooperation offered throughout my stay in Muenster.
I also take this opportunity to thank my graduate school colleagues, especially Nitin,
Sanjeev, Rajani, Raje, Anne and all others, for being extremely supportive all
throughout.

Lastly, and most importantly, I am indebted to my parents for being wonderful,
understanding and always being patient with me and all my nuisances.






































































Abstract

Abstract


Transporter-mediated uptake is critical for terminating the actions of glutamate, the major
excitatory neurotransmitter in the central nervous system (CNS), preventing the sustained
activation of receptors that would otherwise disrupt signaling at synapses and lead to
excitotoxic neurodegeneration. At synapses in the CNS, glutamate transporters can
influence the occupancy of glutamate receptors by reducing the peak concentration of
glutamate within the synaptic cleft, by accelerating the decay of the glutamate transient, and
by restricting the diffusion of glutamate to perisynaptic receptors or receptors at adjacent
synapses. The regulation of these transporters can occur at two levels: transcription and
synthesis of new protein and by changes in the number of pre-existent transporters at the
plasma membrane by trafficking mechanisms, but the regulatory mechanisms are poorly
understood.
This work characterized the protein expression profile and the functionality of glial
glutamate transporter GLAST and neuronal glutamate transporter subtypes EAAC1 and
EAAT4 in HEK-293 cells. The protein expression profiles were established both, by western
blotting and immunocytochemical localization and the functionality of the glutamate
transporters was established by high affinity glutamate uptake measurements.
A massive increase in the glutamate uptake capacity and cell surface expression of EAAC1
was observed in response to phorbol esters as well as phosphatase inhibitors and the role of
PKC was conclusively established in the regulated trafficking mechanism and observed
increase in the surface expression.
Moreover, it was also shown that the trafficking mechanism for the neuronal glutamate
transporter EAAC1 also depends on the intracellular calcium, calmodulin and
Calcium/calmodulin dependent CaM Kinase II for the trafficking.
All together, activation of PKC is not the only mechanism responsible for the regulated
trafficking of EAAC1, but also requires the participation of downstream pathway of
2+
Ca /Calmodulin dependent CaM kinase II for the re-distribution of intracellular pools of
EAAC1 to the plasma membrane. Finally, it was also demonstrated that redistribution of
glutamate transporter upon stimulation by phorbol esters and phosphatase inhibitors causes
the transporter to be phosphorylated at a tyrosine residue.
All together, an involvement of PKC, phosphatase inhibitors, intracellular calcium and
2+
Ca /calmodulin dependent CaM kinase II and tyrosine phosphorylation regulate the
trafficking of neuronal glutamate transporter EAAC1.

Table of Contents I


TABLE OF CONTENTS


1 ABBREVIATIONS 1
2 INTRODUCTION 3
2.1 GLUTAMATE IN THE CNS 3
2.2 GLUTAMATE TRANSPORTER TYPES-AN OVERVIEW 5
+ +2.2.1 ‘High-affinity’ Na /K -dependent glutamate transporters 5
2.2.2 ‘Low-affinity’ glutamate transporters 6
2.2.3 Sodium-independent uptake and glutamate–cystine exchangers 6
2.3 IMPORTANCE OF GLUTAMATE UPTAKE 6
2.3.1 Uptake mechanism and stoichiometry of the process 7
2.4 THE GLUTAMATE TRANSPORTER TOPOLOGY 8
2.5 REGULATION OF GLUTAMATE UPTAKE 10
2.5.1 Regulation of glutamate transporters by arachidonic acid 11
2.5.2 Redox modulation of glutamate transporters 12
2.5.3 Glutamate transporter regulation by growth factors 13
2.5.4 Trafficking mechanism of glutamate transporters: regulation
independent of protein synthesis 14
2.5.5 Effects of direct phosphorylation of transporter proteins 15
2.5.6 Regulation of glutamate transporters by protein kinases 16
2.5.6.1 Protein kinases C: general overview 16
2.5.6.2 Structural arrangement of PKC isozymes 17
2.5.7 Mechanism of fusion of transporter molecules from the intracellular
pools to the membrane: role of interacting partners 19
2.5.8 Regulated exocytosis and role of calcium signaling 20
2.5.9 Calcium regulated proteins: Calmodulin 21
2.5.10 CaM kinases 22

3 OBJECTIVES 24

4 RESULTS 25
4.1 PROTEIN EXPRESSION PROFILE OF GLUTAMATE TRANSPORTERS
IN VIVO AND IN VITRO 25
4.2 FUNCTIONAL CHARACTERISATION OF GLUTAMATE TRANSPORTERS
IN VITRO 28
4.3 KINETIC COMPARISON OF GLUTAMATE UPTAKE FOR HEK-EAAC1
AND C6 GLIOMA CELL LINES 31
Table of Contents II

4.4 POST TRANSLATIONAL GLYCOSYLATION OF NEURONAL GLUTAMATE
TRANSPORTER EAAC1. 32
4.5 RATES OF BIOSYNTHESIS AND DEGRADATION OF EAAC1 33
4.6 CELL SURFACE DISTRIBUTION OF EAAC1 35
4.7 IMPACT OF SUBSTRATE ON TRAFFICKING OF GLUTAMATE
TRANSPORTER EAAC1 37
4.8 POST TRANSLATIONAL REGULATION BY PHOSPHORYLATION:
INFLUENCE OF PHORBOL ESTERS ON THE GLUTAMATE UPTAKE
BY EAAC1 39
4.9 INFLUENCE OF PHORBOL ESTERS ON KINETICS OF EAAC1 41
4.10 INFLUENCE OF PHORBOL ESTERS ON LOCALISATION OF EAAC1 43
4.11 CELL SURFACE BIOTINYLATION: INFLUENCE OF PHORBOL ESTERS
AND PHOSPHATASE INHIBITORS ON CELL SU