Acetylcholine receptor (AChR) γ and ε 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 γ/GFP and ε/GFP subunits were fluorescent and formed together with the α, β, and δ 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 γ and ε subunits may not be required for assembly and rapsyn‐dependent clustering. The γ/GFP and ε/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 γ/GFP or ε/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both γ/GFP or ε/GFP subunits compete with endogenous ε subunits to assemble GFP‐tagged receptors, which are selectively targeted to the postsynaptic membrane.

Gensler, S., Sander, A., Korngreen, A., Traina, G., Giese, G., & Witzemann, V. (2001). Assembly and clustering of acetylcholine receptors containing GFP‐tagged ε or γ subunits. European Journal of Biochemistry, 268(8), 2209–2217. Portico.