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Title

Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera

AuthorsLee, H. K.; Gwalani, L.; Mishra, Vivek N.; Anandjiwala, P.; Sala, Francisco; Sala, Salvador; Ballesta, Juan J.; O'Malley, Donald M.; Criado, Manuel ; Loring, Ralph H.
Issue Date2009
PublisherElsevier
CitationBrain Research 1259: 7- 16 (2009)
AbstractTo test the hypothesis that cell-dependent expression of α7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat α7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of α7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. 125I-α-bungarotoxin (αBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of α7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant α-BGT binding compared to transfection with GFP. In contrast, α7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without αBGT binding. Flow cytometry of cells transfected with α7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with α7-GFP did not correlate with amounts of cell surface or immunoprecipitable αBGT binding. Therefore, GFP folding at the C-terminal of the α7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of α7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled α7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for α7 receptor assembly.
URIhttp://hdl.handle.net/10261/59516
DOI10.1016/j.brainres.2009.01.046
Identifiersdoi: 10.1016/j.brainres.2009.01.046
issn: 0006-8993
e-issn: 1872-6240
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