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 20 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 (NH4)SO4 50 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 primary hippocampal neurons 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 ii 3 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 RhoSAP 64 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 Investigation of endocytic activity 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 Discussion 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 iii 4.4 Conclusion 102 5 Summary 104 6 References 106 Acknowledgements 121 Curriculum Vitae 122 iv Abbreviations 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 v kDA kilo Dalton LTD long-term depression LTP long-term potentiation NMDAR N-methyl D-aspartate receptor PCR polymerase chain reaction PDZ PSD95/ Disc-large/ ZO-1 domain PI protease inhibitor PIP2 phosphatidylinositol-3-4-phosphate ProSAP proline rich synapse-associated protein PSD postsynaptic density PSD95 postsynaptic density protein 95 Rac Ras-related C3 botulinum toxin substrate Ras Roux avian sarcoma Rho Ras homologous member RNA ribonucleic acid RhoSAP RhoGAP synapse associated protein Shank SH3 domain and ankyrin repeat containing proteins SNARE soluble N-ethylmaleimide-sensitive component attachment protein receptor SOD superoxide dismutase TdT terminal deoxynucleotidyl transferase UAS upstream activating sequence WASP Wiskott-Aldrich syndrome protein WAVE WASP family verprolin-homologous Y2H yeast two-hybrid screen vi 1 Introduction 1 1 Introduction Information is integrated, processed and stored in the brain. Therefore, a complex network of trillions of neurons containing a variety of molecules, often unique and crucial to brain and its function, allows neurons to communicate with one another. Neurons are the largest and most complex cells in the body. They are highly polarized cells, with one axon housing the molecular machinery necessary for action potential propagation and neurotransmitter release and several dendrites containing receptors and signaling components that respond to neurotransmitter emitted. Neurons mostly communicate through highly specialized cell-cell junctions: the chemical synapses. At these sites, a specific cytoskeletal matrix is assembled at the active zone (CAZ) of an axon terminal organizing the synaptic vesicle release of neurotransmitter (Dresbach et al., 2001). It directly opposes dendritic protrusions called dendritic spines, which are located along the entire length of dendrites in most neurons. The synaptic membranes are separated by the synaptic cleft, a specialized extracellular space typically about 20-30 nm in width (Gundelfinger & tom Dieck, 2000). The dendritic spines harbor the postsynaptic densities (PSD) that contain a multiprotein complex responsible for anchoring neurotransmitter receptors near sites of neurotransmitter release. Thus, synaptic transmission occurs directionally over the synaptic cleft from the pre- to the postsynaptic compartment. In excitatory synapses, glutamate is the predominant neurotransmitter, in contrast to inhibitory synapses which do not show spiny dendrites and release mainly glycine or GABA as neurotransmitter. Excitatory synapses are mainly associated with dendritic spine structures, whereas inhibitory synapses are localized on the somata or dendritic shafts of neurons (Kreienkamp & Dityatev, 2004). In most regions of the developing brain, the formation of dendritic spines coincides with synaptogenesis in the first weeks after birth. Spine formation, morphogenesis and remodeling induced through synaptic activity, learning and memory or hormonal fluctuations remain a phenomenon, not only restricted to young neurons. Dendritic spines and synapses remain plastic in the adult brain as well (Ethell & Pasquale, 2005). Consequently, synaptic plasticity depends upon maturation and maintenance of the synapse which in turn, is based on multifaceted protein-protein and protein-lipid interactions within dynamic macromolecular complexes at both, the presynaptic and the postsynaptic side (Gundelfinger et al., 2003; Ziv & Garner, 2004; Kim & Sheng 2004).