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Modulation of protein functions by homo- and heterophilic protein interactions as studied with P2X receptors and glutamate transporters [Elektronische Ressource] / von Sandra Gendreau

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Modulation of protein functions by homo- and heterophilic protein interactions as studied with P2X receptors and glutamate transporters Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Sandra Gendreau aus Ivry/seine (Frankreich) Frankfurt 2004 Vom Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. Schwalbe Gutachter: Prof. Lambrecht, Prof. Schmalzing, Prof. Steinhilber, Prof. Fendler Datum der Disputation: 15. Oktober 2004 On fait la science avec des faits, comme on fait une maison avec des pierres, mais une accumulation de faits n'est pas plus une science qu'un tas de pierres n'est une maison. Henri Poincaré (1854-1912) Je dédie cette thèse à mon grand-père et à mes parents. Vos encouragements et votre affection ont été essentiels à l’accomplissement de ce travail. Merci. Diese Doktorarbeit widme ich meinem Großvater und meinen Eltern. Die Unterstützung und Zuneigung meiner Familie war unentbehrlich für die Bewältigung dieser Aufgabe. Danke. Table of contents Table of contents I/ Introduction 5 I-1. Synthesis of membrane proteins: The secretory pathway 5 1.

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Published 01 January 2004
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Modulation of protein functions by
homo- and heterophilic protein
interactions as studied with P2X
receptors and glutamate transporters



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich
Chemische und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main


von
Sandra Gendreau
aus Ivry/seine (Frankreich)


Frankfurt 2004

















Vom Fachbereich Chemische und Pharmazeutische
Wissenschaften der Johann Wolfgang Goethe-Universität als
Dissertation angenommen.











Dekan: Prof. Schwalbe

Gutachter: Prof. Lambrecht, Prof. Schmalzing, Prof. Steinhilber,
Prof. Fendler

Datum der Disputation: 15. Oktober 2004









On fait la science avec des faits,
comme on fait une maison avec des
pierres, mais une accumulation de
faits n'est pas plus une science
qu'un tas de pierres n'est une
maison.

Henri Poincaré (1854-1912)







Je dédie cette thèse à mon grand-père et à mes parents.
Vos encouragements et votre affection ont été essentiels à
l’accomplissement de ce travail. Merci.


Diese Doktorarbeit widme ich meinem Großvater und meinen
Eltern.
Die Unterstützung und Zuneigung meiner Familie war
unentbehrlich für die Bewältigung dieser Aufgabe. Danke. Table of contents
Table of contents

I/ Introduction 5

I-1. Synthesis of membrane proteins: The secretory pathway 5
1.1 Membrane protein synthesis, folding and assembly 5
1.2 N-glycosylation 6

I-2. P2X receptors 7
2.1 Structure of cloned P2X receptors 8
2.2 Signal transduction mechanisms 11
2.3 Distribution and biological effects 14
a- P2X receptor 14 2
b- P2Xptor 15 5
c- P2X receptor 15 7
2.4 Methods to characterize protein complexes and aim of the
experiments performed with the P2X receptors 17

I-3. Glutamate transporters 23
3.1 Roles of amino acid transporters 23
3.2 Sodium- and potassium-coupled glutamate transporters 25
a- The importance of glutamate uptake 25
b- The EAAT family 26
c- Molecular structure of glutamate transporters 28
d- Mechanism of glutamate uptake 30
e- Localization of glutamate transporters 31
f- Ion channel activities 33
3.3 Roles of glutamate transporters in disease 34
3.4 Methods to investigate the oligomeric structure of membrane
proteins and aim of the experiments performed with the EAAT2 and
ecgltP glutamate transporters in this study 35
a- Quaternary structure in detergent solution 35
b- Quaternary structure in the membrane 37


II/ Materials and methods 39

II-1. Materials 39

II-2. Cloning of cDNAs in expression vectors 39
2.1 PCR amplification 39
a- fication using a pure plasmid as template 39
b- PCR amplification from a cDNA library 40
2.2 Digestion with restriction enzymes, ligation, transformation, and
DNA purification 40
2.3 Mutagenesis 41
2.4 Deletion and ligation with primers 42
2.5 Sequencing of DNA 42
- 1 - 1Table of contents

II-3. Protein expression and purification 43
3.1 Expression and purification in E.coli 43
a- Small-scale expression 43
b- Larger cultures 44
3.2 Expression and purification in Xenopus Laevis oocytes 45
a- in vitro cRNA synthesis 45
b- Isolation and maintenance of Xenopus Laevis oocytes 46
c- Micro-injections of cRNA in 47
35d- Metabolic labeling with [ S]-methionine 47
e- Purification of (His) -tagged proteins 48 6
f- Chemical cross-linking 49
g- Deglycosylation 51

II-4. Rat brain Synaptosomes preparation and interaction assays using
GST-fusion proteins 51
4.1 Rat brain Synaptosomes preparation 51
4.2 Rat brain Synaptosomes purification through a Ficoll gradient 53
4.3 “Pull-down” experiments:Interaction assays using GST-fusion
proteins 54

II-5. Protein separation: PolyAcrylamide Gel Electrophoresis 54
5.1 SDS-PAGE 54
a- Linear SDS-PAGE 54
b- Gradient SDS-PAGE 56
c- Tricine-SDS-PAGE 56
d- Tris-Borate-EDTA SDS-PAGE 57
5.2 BN-PAGE 59
5.3 2D-electrophoresis 61

II-6. Other techniques 62
6.1 Silver staining 62
6.2 Western-Blotting 64
6.3 Overlay assays 65

III/ Results 67

III-1. Identification of proteins interacting with the C-terminal tail of P2X
receptors 67
356-472 364-4571.1 Design and expression of GST-rP2X , GST-rP2X and 2 5
433-596GST-rP2X fusion proteins 67 7
a- Expression of the three GST-rP2X fusion proteins 68
b- Pull-down experiments: Tubulin binds specifically to the P2X 2
subunit 70
c- Results confirmed by 2D-electrophoresis 73
1.2 All three GST-P2X fusion proteins bind MBP 76
1.3 The P2X subunit binds tubulin directly 77 2
- 2 - 2Table of contents
1.4 Tubulin binding is mediated by a prolin-rich segment of the P2X 2
C terminal tail 78
1.5 Co-injections of cRNAs coding for P2X and tubulin in Xenopus laevis 2
oocytes 81
1.6 Pull-down of ßIII tubulin expressed in Xenopus oocytes by the GST-
356-472P2X fusion protein 84 2
1.7 Perspectives 86

III-2. Investigations of the quaternary structure of two glutamate
transporters: hEAAT2 and ecGltP 89
2.1 hEAAT2 and ecgltP transporters migrate as trimers in blue native
PAGE gels 89
a- His-tagged hEAAT2 transporters exhibit unaltered functional
properties 89
b-
PAGE gels 90
2.2 Results obtained with a concatenated ecgltP dimer are consistent
with a trimeric ecgltP structure 94
2.3 Cross linking of hEAAT2 or ecgltP generates covalently bound dimers
and trimers 97
2.4 hEAAT2 is complex glycosylated 99
2.5 Does hEAAT2 form hetero-oligomers with hEAAT3 or ecgltP? 104
2.6 Study on the assembly motif of ecgltP using deletion mutants 106
2.7 BN-PAGE analysis of TetA(B), a tetracycline cation/proton
Antiporter 108

IV/ Discussion 112

IV-1. Identification of proteins interacting with P2X receptors 112
1.1 ßIII tubulin and MBP as binding partners of the rP2X subunit 112 2
a- The rP2X subunit interacts directly with the cytoskeleton 112 2
b- All three GST-P2X fusion proteins bind MBP 114
1.2 Localization of the tubulin binding motif on the rP2X subunit 115 2
1.3 Co-expression of rP2X and ßIII tubulin in heterologous expression 2
systems 120
a- Expression in Xenopus laevis oocytes 120
b- on of rP2X -GFP in COS-7 cells 121 2
IV-2. Bacterial and human glutamate transporters share a trimeric
quaternary structure 123
2.1 Evidences for multimeric glutamate transporters 123
2.2 What is the impact of the trimeric structure on the functionality of
the EAAT glutamate transporters? Putative relationship between
oligomerization and the chloride channel activity of EAA transporters
126
2.3 Study of the assembly motif of ecgltP 129
2.4 Perspectives 132

V/ Bibliography 134
- 3 - 3Table of contents

VI/ Appendix 148

VI-1. Abbreviations 148

VI-2. Constructs and primers 150
2.1 DNA constructs 150
2.2 Primer used for sequencing 151

VII/ Summary 152

VIII/ Deutsche Zusammenfassung 154

IX/ References 160

X/ Curriculum Vitae 161

XI/ Acknowledgements 162

- 4 - 4I/ Introduction
I/ Introduction

I-1. Synthesis of membrane proteins: The secretory pathway

1.1 Membrane protein synthesis, folding and assembly

All proteins are synthesized in the cytosol by the ribosomes, but some
selected proteins are captured by the endoplasmic reticulum (ER) as they
are being synthesized. These proteins are of two types: membrane
proteins, which are only partly translocated across the ER membrane and
become embedded in it, and water-soluble proteins, which are fully
translocated across the ER membrane and are released into the ER lumen.
All of these proteins, regardless of their subsequent fate, are directed to
the ER by the same kind of signal sequence and are translocated across it
by similar mechanisms (Alberts et al., 1994).
Once their synthesis is complete, newly introduced polypeptides in the
membrane and lumen of the ER must be folded, sorted, and transported.
Secretory and membrane proteins undergo five principal modifications
during their transit to the cell surface: (1) formation of disulfide bonds;
(2) proper folding of the polypeptide; (3) addition and modification of
carbohydrates; (4) specific proteolytic cleavages, and (5) formation of
multichain proteins (assembly). Each modification takes place in a specific
organelle through which these proteins pass. The first two and the fifth of
these reactions take place exclusively in the ER, and addition of some
carbohydrates and some proteolytic cleavages also occur in this organelle.
The modifications that occur in the ER are essential for the protein to
reach its proper cellular location. Only properly folded and assembled
proteins are transported from the ER to the Golgi complex and to the cell
surface. Unfolded, misfolded, or partly folded and assembled proteins are
selectively retained in the ER, or are retrieved to the ER from the cis-Golgi
reticulum. Misfolded proteins and unassembled subunits of multiple-
polypeptide complexes are either degraded within the ER or translocated
- 5 - 5I/ Introduction
back into the cytosol, where they are deglycosylated, ubiquitylated, and
degraded in proteasomes.
Many important secretory and membrane proteins form oligomers, they
are built of two or more polypeptides. Several prerequisite must be met in
order for subunits to oligomerize. The associating subunits must co-exist
spatially and temporally, recognize each other, and provide sufficient
stabilization energy to form a stable structure (Deutsch, 2002). The
formation of oligomeric proteins occurs in the ER membrane. Fig. 1
illustrates the series of events contributing to the biogenesis of oligomeric
membrane proteins and the temporal organization of these events.


Fig. 1: Channel biogenesis in the endoplasmic reticulum (Deutsch, 2002).


1.2 N-linked glycosylation

N-linked glycans in glycoproteins are attached to the amide nitrogens of
asparagine side chains. Glycosylated asparagine residue are almost
invariably found in the sequences Asn-X-Ser or Asn-X-Thr, where X can be
any amino acid except proline. The different categories of N-linked
- 6 - 6I/ Introduction
glycans, such as high mannose and complex types, contain a common
core-structure but differ in the terminal elaboration that extend from this
core.
It is convenient to divide the biochemical pathway for N-linked glycan
synthesis into three stages (Fig. 2A): (1) formation of a lipid-linked
precursor oligosaccharide; (2) en bloc transfer of the oligosaccharide to
the polypeptide; and (3) processing of the oligosaccharide. Processing
steps include removal of some of the original sugar residues (trimming)
followed by addition of new sugars at the non-reducing termini of the
glycan (Fig. 2B).

A B
Complex- Core- glycosylated
glycosylated protein
protein (Golgi)
(ER)










Fig. 2: (A) Overview of the pathway for glycoproteins biosynthesis and its
location within a cell. (B) Processing of an initial high mannose N-linked glycan
to generate complex glycans (Taylor and Drickamer, 2003).


I-2. P2X receptors

P2X receptors are ligand-gated membrane ion channels that when
activated by extracellular ATP mediate fast excitation in various cells,
including central and peripheral neurons. P2X receptors belong to the
superfamily of purine receptors, which is divided in two sub-families: the
adenosine receptors called P1 receptors and the P2 receptors, recognizing
- 7 - 7