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MuSK, a previously known muscle specific receptor tyrosine kinase is expressed in retinal astrocytes and interacting with Erbin [Elektronische Ressource] / vorgelegt von Muhammad Amir Khan

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MuSK, a previously known muscle specific receptor tyrosine kinase is expressed in retinal astrocytes and interacting with Erbin Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Muhammad Amir Khan aus Abbottabad, Pakistan 2006 Als Dissertation genehmigt von den Naturwissen- schaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 14-12-2006 Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder Erstberichterstatter: Prof. Dr. Andreas Burkovski Zweitberichterstatter: PD S. Hashemolhosseini To my mother Table of contents ________________________________________________________________________ Table of contents Zusammenfassung 1 Sumary 2 1. Introduction 3 1.1. Synapse 3 1.2. Neuromuscular junction 3 1.2.1. Development of neuromuscular junction 3 1.3. Clustering of AChRs 5 1.3.1. Agrin 6 1.3.2. Muscle specific kinase (MuSK) 9 1.3.3. Rapsyn 13 1.4. Signaling downstream of MuSK 15 1.4.1. Src kinases 15 1.4.2. Rho-family GTPases 15 1.5. MuSK interacting proteins 17 1.5.1. MAGI-1c 17 1.5.2.
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MuSK, a previously known muscle specific receptor
tyrosine kinase is expressed in retinal astrocytes and
interacting with Erbin





Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades





vorgelegt von
Muhammad Amir Khan
aus Abbottabad, Pakistan
2006






Als Dissertation genehmigt von den Naturwissen-
schaftlichen Fakultäten der Universität Erlangen-Nürnberg










Tag der mündlichen Prüfung: 14-12-2006

Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder
Erstberichterstatter: Prof. Dr. Andreas Burkovski
Zweitberichterstatter: PD S. Hashemolhosseini
























To my mother
Table of contents
________________________________________________________________________
Table of contents
Zusammenfassung 1
Sumary 2
1. Introduction 3
1.1. Synapse 3
1.2. Neuromuscular junction 3
1.2.1. Development of neuromuscular junction 3
1.3. Clustering of AChRs 5
1.3.1. Agrin 6
1.3.2. Muscle specific kinase (MuSK) 9
1.3.3. Rapsyn 13
1.4. Signaling downstream of MuSK 15
1.4.1. Src kinases 15
1.4.2. Rho-family GTPases 15
1.5. MuSK interacting proteins 17
1.5.1. MAGI-1c 17
1.5.2. Synaptic nuclear envelope-1 (Syne-1) 17
1.5.3. Dishevelled 17
1.5.4. CollagenQ (ColQ)/Acetylcholinesterase (AChE) 18
1.5.5. Src homology 2-domain-containing
tyrosine phosphatase 2 (Shp2) 18
1.5.6. 14-3-3γ 18
1.5.7. Abelson tyrosine kinases (Abl) 19
1.5.8. Geranylgeranyltransferase 1 (GGT-1) 19
1.5.9. Protein interacting with C kinase (PICK1) 20
1.5.10. Docking Protein -7 (Dok-7) 20
1.5.11. Protein kinase CK2 20
1.5.12. Src-class kinases (SFKs) 21
1.5.13. Putative ariadne-like E3 ubiquitin ligase (PAUL) 21
1.6. Main players of synapses at NMJ:
in search of new roles at CNS 22
I Table of contents
________________________________________________________________________
2. Aim of the Study 24
3. Material and methods 25
3.1. Materials 25
3.1.1. Reagents 25
3.1.2. Devices 26
3.1.3. Oligonucleotides 27
3.1.4. siRNAs 32
3.1.5. Enzymes 33
3.1.6. Kits and Columns 34
3.1.7. Antibodies 34
3.1.8. Frequently used solutions 35
3.1.9. Cell culture 35
3.2. Methods 36
3.2.1. Molecular Biology Methods 36
3.2.1.1. Isolation ofplasmid DNA 36
3.2.1.2. Determination of DNA/RNA concentration 36
3.2.1.3. Electrophoretic separation of DNA fragments in
agarose gel 37
3.2.1.4. PCR amplification of DNA 37
3.2.1.5. Cloning techniques 37
3.2.1.6. Plasmid constructs 38
3.2.1.7. Transformation of E. coli competent cells 41
3.2.1.8. Site directed mutagenesis 42
3.2.1.9. Total RNA isolation 44
3.2.1.10. ComplementaryDNA-synthesis (Reverse Transcription) 44
3.2.1.11. Lightcycler PCR 45
2. Yeast-two-hybrid techniques 46
3.2.2. Protein Biochemistry methods 50
3.2.2.1. Preparation of protein extract from cells and tissues 50
3.2.2.2. Immunoprecipitation 50
3.2.2.3. Protein expression and extraction from bacteria 52
II Table of contents
________________________________________________________________________
3.2.2.4. GST-pulldown 53
3.2.2.5. Electrophoresis ofproteins 53
3.2.2.6. Staining of protein gels 54
3.2.2.7. Western blot 54
3.2.3. Cell culture methods 55
3.2.3.1. Cultivation of HEK293, Cos7, C2C12,
and MuSK-deficient myoblasts 55
3.2.3.2. Transient transfection of cells 55
3.2.3.3. Luciferase reporter test 57
3.2.3.4. Agrin treatment 58
3.2.3.5. Immunocytochemistry 58
3.2.3.6. Quantification analysis of AChR clusters 58
3.2.3.7. Immunohistochemistry 59
4.Result 61
4.1. Yeast-two-hybrid screens 61
4.1.1. Generation of MuSK baits 61
4.1.2. Outcome of the yeast-two-hybrid screens 62
4.2. Further confirmation of MuSK - CK2 interaction 63
4.3. Detailed investigation of MuSK - Erbin interaction 64
4.3.1. Quantitative determination of Erbin transcript
level in different tissues 65
4.3.2. Localization of Erbin at the NMJ 66
4.3.3. Verification of the interaction between Erbin
and MuSK in HEK293 cells, myotubes
and muscle extracts 68
4.3.4. Mapping of epitopes of MuSK interacting with Erbin 69
4.3.5. Mapping of epitopes of Erbin interacting with MuSK 71
4.3.6. Biological role of Erbin at the NMJ 73
4.4 MuSK: in search of new roles at CNS 75
4.4.1. Expression profile of MuSK in different tissues of
adult rodents 75
III Table of contents
________________________________________________________________________
4.4.2. Immunohistochemical localization of MuSK in
astrocytes of the rat retina 77
4.4.3. Developmental expression pattern of MuSK in
the rat retina 82
5. Discussion 85
6. Abbreviations 90
7. References 93
Curriculum vitae 112
Publications 113
Acknowledgments 114

IV Zusammenfassung
________________________________________________________________________
Zusammenfassung

Die Muskel-spezifische Tyrosinkinase (MuSK) wird in Muskelfasern exprimiert, wo sie
die Bildung der neuromuskulären Endplatten vermittelt. In der vorliegenden Arbeit
konnte gezeigt werden, dass MuSK auch im zentralen Nervensystem exprimiert wird.
Hier konnte MuSK im Gehirn und im Auge von Nagetieren nachgewiesen werden. In der
Retina wird MuSK im Alter von 7 bis 14 Tagen nach der Geburt in Astrozyten
exprimiert, was der Zeitspanne entspricht, in der sich die Augen öffnen.
Interessanterweise, wurde auch Agrin, ein Aktivator von MuSK in Muskelzellen, im
zentralen Nervensystem nachgewiesen. Wir konnten zeigen, dass Agrin in Nachbarschaft
zu den MuSK-exprimierenden Astrozyten lokalisiert ist, die sich nahe der inneren
begrenzenden Membran der Nagetier-Retina befinden. Diese Ergebnisse lassen die
Vermutung zu, dass MuSK zusätzlich zu seiner bekannten Funktion bei der Ausbildung
der neuromuskulären Endplatte auch bei der Entwicklung des Nervensystems eine Rolle
spielt.
Zur Aufklärung der Funktionen von MuSK ist es erforderlich, Proteine zu identifizieren,
die als Interaktionspartner und/oder Effektoren von MuSK agieren. Zu diesem Zweck
haben wir Hefe-2-Hybrid-Analysen durchgeführt. Unter den in diesen Untersuchungen
entdeckten Proteinen waren die ß-Untereinheit der Proteinkinase 2 (CK2ß) und der
carboxyterminale Bereich des ErbB2-interagierenden Proteins (Erbin). Weiterführende
Interaktionsstudien zeigten, dass nicht nur die regulatorische ß-Untereinheit der
Proteinkinase 2, sondern auch deren katalytische α-Untereinheit mit MuSK interagieren
kann. Durch Epitop-Kartierungsstudien ließ sich die Region von Erbin, die für die
Interaktion mit MuSK verantwortlich ist, auf die Aminosäure 1175 bis 1229 im
Carboxyterminus des Proteins eingrenzen.
1 Summary
________________________________________________________________________
Summary

Muscle specific tyrosine kinase (MuSK) has been shown to be expressed in muscle fiber
in which it mediates the formation of neuromuscular junctions. In this study we show that
MuSK is expressed in the central nervous system (CNS), particularly in the brain and eye
of rodents. In the retina MuSK was expressed in astrocytes between postnatal days 7 and
14, i.e. at the time when the eyes open. Interestingly, agrin an activator of MuSK in
muscle cells was also detected in the CNS. We found that agrin was localized adjacent to
MuSK-expressing astrocytes which in turn were detected close to the inner limiting
membrane of the rodent retina. These findings raise an interesting possibility that, in
addition to the known function in the formation of the neuromuscular junctions, MuSK
may be involved in neural development.
To get new insight into the functions of MuSK, a yeast-two-hybrid approach was
undertaken to identify partners and/or effectors of MuSK. Two of the identified proteins
interacting with the intracellular domain of MuSK were the β subunit of the protein
kinase 2 (CK2β) and the carboxy-terminal part of ErbB2 interacting protein (Erbin).
Further studies have shown that not only the regulatory β subunit but also the catalytic α
subunit of CK2 interact with MuSK. Epitope-mapping studies define the area of Erbin
comprising amino acid residues between 1175 and 1229 in the carboxy terminus of the
protein is necessary for its interaction with MuSK.









2 Introduction
________________________________________________________________________
1. Introduction

1.1. Synapse
The word synapse first appeared in 1897, in the seventh edition of Michael Foster’s
Textbook of Physiology and describes the point of contact between two cells. Synapses
are specialized structural units for cellular communication in the nervous system. The
formation of synapses requires a series of steps. First, the parts of the two cells have to
migrate to the place where the synapse will form. Second, these subcellular structures of
the cells have to differentiate to specialized presynaptic terminals and postsynaptic
membranes (Bowe and Fallon, 1995). Much of the knowledge about synapses came from
the study of neuromuscular junctions.

1.2. Neuromuscular junction
The neuromuscular junction (NMJ) is a synapse composed by a specialized part of a
motoneuron and a muscle fiber (Hall and Sanes, 1993). The NMJ consists of the
presynaptic nerve terminal, the postsynaptic muscle fiber and the presynaptic Schwann
cells (PSCs, also known as “terminal” Schwann cells). Additionally between the nerve
terminal and the muscle membrane, a synaptic basal lamina develops, which is composed
of extracellular matrix and factors produced and secreted by both nerve and muscle
(Bloch and Pumplin, 1988; Sanes and Lichtman, 1999).
Neuromuscular junctions have been widely used for analyses of synaptic structure,
function and development because of several advantages over the synapses from the
central nervous system (Burden, 1998; Sanes and Lichtman, 1999; Wyatt and Balice-
Gordon, 2003), namely size, accessibility and simplicity.

1.2.1. Development of the neuromuscular junction
During development starting at about embryonic day (E) 11 in mice multinucleated
skeletal muscle fibers form by fusion of precursor myoblasts. Shortly after myotubes
begin to form (at E12-13 in mice), motoneurons begin to contact muscle cells (Fig.1).
Motoneurons can innervate from one to over hundred muscle fibers, but each muscle
fiber receives input from only one motoneuron. In the terminal branches of the motor
nerve, at dense patches called active zones synaptic vesicles filled with the
neurotransmitter acetylcholine (ACh) start to accumulate (Hall and Sanes, 1993).
3

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