We tested the hypothesis that the folding, assembly and insertion of neuronal nicotinic receptors are critically dependent on the host cell line. We used recombinant adenoviruses encoding either the rat α7, α4 or β2 subunits in which expression of the subunit is controlled by a tetracycline- dependent promoter to screen five cell lines (GH4C1, SH-EP1, CV1, SN-56, and CHO-CAR). All five lines do not express detectable nicotinic receptor but do express receptor for human adenovirus, and all expressed mRNA for α7, α4 and β2 subunits when infected with viruses. Each cell line expressed varying levels of α4β2 receptors that bound [3H]cytisine, but only the GH4C1 and SH-EP1 cell lines expressed either surface or internal α7 receptors that bound [125I]α-bungarotoxin ([125I]α-BGT). All five cell lines expressed a 60 kDa protein immunoblotted by anti-α7 antibodies when infected with the α7 virus, presumably representing unassembled α7 subunits. In addition, GH4C1 cells expressed over 10-fold more surface α7 receptor than SH-EP1 cells, even though the total α7 receptor in the two cell lines was similar. Sedimentation experiments indicate that SH-EP1 cells only partially assemble α7 receptors compared with GH4C1 cells and control α7 from rat brain. These data suggest that not only is surface α7 receptor expression a multistep process, but that each step may involve cell-specific assembly factors. (C) 2000 Elsevier Science B.V.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cellular and Molecular Neuroscience