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Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: Selective targeting to the neuromuscular junction in vivo

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Gensler,  Sven
Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society;

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Sander,  Andreas
Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society;

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Korngreen,  Alon
Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society;

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Giese,  Günter
Department of Biomedical Optics, Max Planck Institute for Medical Research, Max Planck Society;

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Witzemann,  Veit
Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society;
Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Gensler, S., Sander, A., Korngreen, A., Traina, G., Giese, G., & Witzemann, V. (2001). Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: Selective targeting to the neuromuscular junction in vivo. European Journal of Biochemistry, 268(8), 2209-2217. doi:10.1046/j.1432-1327.2001.02093.x.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0028-EB8B-3
Abstract
Acetylcholine receptor (AChR) gamma and epsilon subunits were tagged by green fluorescent protein (GFP) to analyse assembly and targeting in live muscle fibers at the neuromuscular junction. N- or C-terminal fusion polypeptides showed no fluorescence upon transfection of HEK cells. When GFP was inserted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the gamma/GFP and epsilon/GFP subunits were fluorescent and formed together with the alpha, beta, and delta subunits GFP-tagged AChR complexes that were integrated into the plasma membrane. As the AChR were also clustered by rapsyn, the results indicate that the cytoplasmatic domains of the gamma and epsilon subunits may not be required for assembly and rapsyn-dependent clustering. The gamma/GFP and epsilon/GFP subunit-containing receptors were expressed in X. laevis oocytes and have affinities for acetylcholine similar to that of the wild-type receptors. Direct gene transfer into single muscle fibers reveals that gamma/GFP or epsilon/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both gamma/GFP or epsilon/GFP subunits compete with endogenous epsilon subunits to assemble GFP-tagged receptors, which are selectively targeted to the postsynaptic membrane.