Efficient targeting of proteins to post-synaptic densities of excitatory synapses using a novel pSDTarget vector system.
Pre- and post-synaptic targeting of synaptic molecules is depending upon specific targeting signals that are encoded within defined regions of the respective protein. For the post-synaptic scaffolding proteins of excitatory synapses, ProSAP1/Shank2 and ProSAP2/Shank3 this targeting information is located within about 460aa of the C-terminus. We found the C-terminal targeting signal to be bipartite composed of a 135aa stretch and the SAM (sterile alpha motif) domain embedding a relatively large variable spacer region. Based on this we developed a new GFP vector system called pSDTarget to easily clone proteins of interest as GFP fusion proteins flanked by a bipartite targeting signal for post-synaptic densities (PSDs) of excitatory synapses. The targeting signal has been derived from the PSD scaffolding protein ProSAP1/Shank2. In hippocampal neuron culture we could effectively localize and attach i.e. Glutathion-S-transferase (GST) at PSDs of excitatory synapses already during early synaptogenesis. Moreover, Gephyrin, an important scaffold molecule of inhibitory post-synapses was succesfully targeted to excitatory synapses followed by the subsequent recruitment of GABAergic receptors leading to hybrid synaptic contacts. In light of the role of specific protein domains for plastic changes of the post-synaptic compartment or investigations focusing on synaptogenesis, signalling and/or transsynaptic crosstalk this vector system provides a powerful and innovative tool for the functional analysis of molecular mechanisms and structural changes in a small but well defined neuronal compartment.
Grabrucker
Andreas M
AM
Institute for Anatomy and Cell Biology, Ulm University, Albert Einstein Allee 11, D-89081 Ulm, Germany.
Vaida
Bianca
B
Bockmann
Jürgen
J
Boeckers
Tobias M
TM
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005822:Genetic Vectors; D006624:Hippocampus / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism*; D014162:Transfection
D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011963:Receptors, GABA-A; C485686:Shank2 protein, rat; C073441:gephyrin; C033652:postsynaptic density proteins