Biochemical and functional characterization of RhoSAP [Elektronische Ressource] : a RhoGAP of the postsynaptic density / eingereicht von Janine Dahl

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2008
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Institut für Anatomie and Zellbiologie
Direktor: Professor Dr. med. Tobias M. Böckers





Biochemical and functional characterization of RhoSAP:
A RhoGAP of the postsynaptic density










Dissertation zur Erlangung des Doktorgrades
Dr. biol. hum.
der Medizinischen Fakultät der Universität Ulm




Eingereicht von:
Janine Dahl
aus Ludwigslust



2008










































Dekan: Prof. Dr. med. Klaus-Michael Debatin

Erster Gutachter: Prof. Dr. med. Tobias M. Böckers

Zweiter Gutachter: Prof. Dr. rer. nat. Dietmar Fischer

Tag der Promotion:
Abstract

Biochemical and functional characterization of RhoSAP:
A Rho GAP of the postsynaptic density

By
Janine Dahl
Ulm University

Glutamatergic synapses in the central nervous system are characterized by an electron
dense network of proteins underneath the postsynaptic membrane including cell adhesion
molecules, cytoskeletal proteins, scaffolding and adaptor proteins, membrane bound
receptors and channels, G-proteins and a wide range of different signalling modulators and
effectors. This so called postsynaptic density (PSD) resembles a highly complex signaling
machinery. We performed a yeast two-hybrid (YTH) screen with the PDZ domain of the
PSD scaffolding molecule ProSAP2/Shank3 as bait and identified a novel interacting
protein. This molecule was named after its Rho GAP domain: RhoSAP (Rho GTPase
Synapse Associated Protein), which was shown to be active for Cdc42 and Rac1 by a GAP
activity assay. Besides its Rho GAP domain, RhoSAP contains an N-terminal BAR domain
that might facilitate membrane curvature in endocytic processes. At the C-terminus
RhoSAP codes for several proline rich motifs that could possibly act as SH3 binding
regions. Therefore, a second YTH screen was carried out with RhoSAP’s proline rich C-
terminus as bait to discover putative interacting proteins. As an interacting partner
syndapin I, a molecule involved in vesicle endocytosis via direct interaction with dynamin
I was found. Furthermore, syndapin I contains a C-terminal SH3 domain that is most likely
the binding motif for RhoSAP’s proline rich region. This novel interaction as well as the
interaction with the ProSAP2 PDZ domain was verified by pull-down assays and
coimmunoprecipitations.
Coming from the finding that RhoSAP is associated with an endocytosis molecule, an
FM4-64 endocytosis assay was performed in cell line to delineate the molecule’s
significance in endocytic processes in general.
Taken together, RhoSAP is a novel ProSAP2/Shank3 interacting PSD protein, which
displays several protein/protein interaction domains. Due to the N-terminal BAR domain,
and the interaction with syndapin I, RhoSAP might act within endocytic processes of the
postsynaptic membrane.























to my family




































Contents

Abbreviations

1 Introduction 1
1.1 The postsynaptic compartment 2
1.2 The Rho GTPases 6
1.3 Rho GTPases-activating proteins (RhoGAPs) 9
1.4 Rho GTPases effector proteins and signaling pathways 12
1.5 Rho GTPases in actin dynamics and membrane trafficking 13
1.6 Nadrin and Rich1 15
Aim 19

2 Materials and Methods 20
2.1 Declaration of suppliers and basic recipes 20
2.1.1 Materials
2.1.2 Recipes 22
2.2 Molecular biological methods 25
2.2.1 DNA/RNA concentration measurement 25
2.2.2 DNA preparation and purification 25
2.2.3 Plasmid DNA isolation from E.coli cell cultures 26
2.2.4 RNA purification, generation of cDNA and PCR 27
2.2.5 Generation of electrocompetent E. coli cells and 28
transformation of plasmids
2.2.6 TOPO cloning and restriction analysis 29
2.2.7 DNA sequencing 30
i 2.2.8 Northern blotting 30
2.2.9 in situ Hybridization 32
2.2.10 Yeast two-hybrid screen 34
2.3 Protein biochemical methods 42
2.3.1 Protein concentration measurement 42
2.3.2 Preparation of GST-tagged fusion protein lysate 43
from E. coli cell culture
2.3.3 Preparation of tissue and organ lysates 44
2.3.4 Subcellular fractionation of PSDs by differential centrifugation 45
2.3.5 SDS-PAGE, SDS gel staining and Western blotting 47
2.3.6 IgG purification of polyclonal antisera by (NH )SO 50 4 4
2.3.7 Coimmunoprecipitation and pull-down to analyse possible 51
protein-protein interactions
2.3.8 GAP activity assay 52
2.4 Cell biological methods 54
2.4.1 Cell passages of cell lines 54
2.4.2 Transient transfection of HeLa and COS-7 cells 55
2.4.3 Generation of primary hippocampal neurons 56
2.4.4 Transfection of prim 57
2.4.5 Immunohistochemical staining for 57
confocal fluorescence microscopy
2.4.6 DAB staining for confocal light microscopy 58
2.4.7 Immunohistochemical staining for electronmicroscopy 59
(pre-embedding method)
2.4.8 Endocytosis assay and statistical analysis 59

ii3 Results 61
3.1 Gene and protein structural characterization of RhoSAP 61
3.1.1 Identification of RhoSAP as a PDZ domain interacting protein 61
3.1.2 Gene and protein structure of RhoSAP 62
3.1.3 Sequence homologies of RhoSAP 64
3.1.4 Putative splice variants of
3.1.5 Generation and characterization of anti-RhoSAP antisera 66
3.1.6 Profiling RhoSAP’s expression in brain and other tissue 67
3.1.7 Recombinant expression of RhoSAP in COS7 cells 71
3.1.8 Localization of RhoSAP in hippocampal neurons 73
3.1.9 Subcellular distribution of RhoSAP in neurons 77
3.2 Isolation and analysis of RhoSAP's protein interaction partners 80
3.2.1 Verification of RhoSAP's association with ProSAP2Shank3 80
3.2.2 Identification of novel binding partners 81
by a yeast two-hybrid screen
3.2.3 Confirmation of RhoSAP’s interaction with syndapin I 82
3.3 Functional analysis of RhoSAP 83
3.3.1 Investigation of GAP activity 83
3.3.2 of endocytic 85
3.3.3 Investigation of intracellular membrane trafficking 89
3.3.4 Investigation of RhoSAP's effect on the cytoskeleton 90
in cell culture

4 Discusion 93
4.1 Neuronal localization of RhoSAP and Rho GTPase signaling 93
4.2. RhoSAP: membrane trafficking and actin dynamics 96
4.3. Hypothesis for the function of RhoSAP in neurons 99
iii4.4 Conclusion 102

5 Summary 104

6 References 106

Acknowledgements 121
Curriculum Vitae 122



































ivAbbreviations

aa amino acid

ab antibody

AD activating domain

AMPAR 1-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
receptor

Arp2/3 actin related protein 2/3

BD binding domain

bp base pair

CAZ cytomatrix at the active zone

Cdc42 cell division cycle 42

CME clathrin-mediated endocytosis

COT the COT1 fraction of human genomic DNA consisting of
rapidly annealing repetitive elements

Cortactin cortical actin binding domain

d day

DAB 2,3-deaminobenzidine

DIV days in vitro

DNA deoxynucleic acid

GAP GTPase-activating protein

GDI guanine nucleotide dissociation inhibitor

GEF guanine nucleotide exchange factor

GFP green fluorescent protein

mGluR metabotropic glutamate receptor

GST glutathione-S-transferase

GTPase small GDP/GTP-binding protein

IgG G-class of immunoglobulins
vkDA kilo Dalton

LTD long-term depression

LTP long-term potentiation

NMDAR N-methy