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

Acetylcholine receptor (AChR) gamma and epsilon subunits were tagged by gre en fluorescent protein (GFP) to analyse assembly and targeting in live musc le fibers at the neuromuscular junction. N- or C-terminal fusion polypeptid es showed no fluorescence upon transfection of HEK cells. When GFP was inse rted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the gamma /GFP and epsilon /GFP subunits were fluorescent and form ed together with the alpha, beta, and delta subunits GFP-tagged AChR comple xes that were integrated into the plasma membrane. As the AChR were also cl ustered by rapsyn, the results indicate that the cytoplasmatic domains of t he gamma and epsilon subunits may not be required for assembly and rapsyn-d ependent clustering. The gamma /GFP and epsilon /GFP subunit-containing rec eptors were expressed in X. laevis oocytes and have affinities for acetylch oline 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 endop lasmatic reticulum. When reaching subsynaptic regions, both gamma /GFP or e psilon /GFP subunits compete with endogenous epsilon subunits to assemble G FP-tagged receptors, which are selectively targeted to the postsynaptic mem brane.