Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera.
To test the hypothesis that cell-dependent expression of alpha7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat alpha7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of alpha7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. (125)I-alpha-bungarotoxin (alphaBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of alpha7-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, alpha7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without alphaBGT binding. Flow cytometry of cells transfected with alpha7-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 alpha7-GFP did not correlate with amounts of cell surface or immunoprecipitable alphaBGT binding. Therefore, GFP folding at the C-terminal of the alpha7-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 alpha7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled alpha7 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 alpha7 receptor assembly.
Department of Pharmaceutical Science, Northeastern Univ, Boston, MA 02115, USA.
H K
HK
Lee
L
L
Gwalani
V
V
Mishra
P
P
Anandjiwala
F
F
Sala
S
S
Sala
J J
JJ
Ballesta
D
D
O'Malley
M
M
Criado
R H
RH
Loring
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials / Q000235:genetics; D009865:Oocytes; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; D049452:Green Fluorescent Proteins