Investigating the role of protein folding and assembly in cell-type dependent expression of alpha 7 nicotinic receptors using a green fluorescent protein chimera

• Published in: Brain research 2009-03, Vol.1259, p.7-16
• Main Authors: Lee, H K, Gwalani, L, Mishra, V, Anandjiwala, P, Sala, F, Sala, S, Ballesta, J J, O'Malley, D, Criado, M, Loring, R H
• Format: Article
• Language: English
• Subjects: Xenopus
• ISSN: 0006-8993
• DOI: 10.1016/j.brainres.2009.01.046

To test the hypothesis that cell-dependent expression of alpha 7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat alpha 7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of alpha 7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. super(1) super(2) super(5)I- alpha -bungarotoxin ( alpha BGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of alpha 7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant alpha -BGT binding compared to transfection with GFP. In contrast, alpha 7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without alpha BGT binding. Flow cytometry of cells transfected with alpha 7-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 alpha 7-GFP did not correlate with amounts of cell surface or immunoprecipitable alpha BGT binding. Therefore, GFP folding at the C-terminal of the alpha 7-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 alpha 7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled alpha 7 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 alpha 7 receptor assembly.