Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems.
Different mammalian and insect somatic host cell systems were tested in their ability to express, fold, and assemble alpha7-type neuronal acetylcholine receptor (AChR) both at the transcriptional and translational level. For this purpose we employed clonal cell lines derived from the neural crest, such as PC12 cells from a rat adrenal pheochromocytoma, and GH3 cells isolated from a rat pituitary tumor, as well as non-neuronal cells such as NIH-3T3 fibroblasts from embryonic NIH Swiss mouse and Sf9 cells from ovary tissue of the Spodoptera frugiperda butterfly. Total RNA, isolated from either transfected or non-transfected PC12, GH3 or 3T3 cells, or recombinant AcNPV-infected and mock-infected Sf9 cells was analyzed by Northern blot. PC12 cells, which endogenously express alpha7 AChR, and all its heterologous alpha7-transfectant clones, exhibited variable but generally high amounts of a single transcript. GH3 and NIH-3T3 transfectant clones and recombinant AcNPV-infected Sf9 cells expressed variable levels of alpha7-mRNA, with a single transcript that co-migrated with the 28S rat rRNA. Only the neural crest-derived cell lines appeared to functionally express the alpha7 AChR, as measured by their [125I]alpha-bungarotoxin binding ability. The results suggest that heterologous expression of alpha7 is regulated not at the transcriptional, but at the postranslational level and that not all host cell systems appear to express the cellular factors needed for the correct postranslational modifications leading to mature and functional alpha7 AChR. Furthermore, the results suggest that tightly controlled expression mechanisms have evolved in parallel with this ancient cholinergic sequence.
Aztiria
E M
EM
Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur-CONICET, Bahía Blanca, Argentina.
Sogayar
M C
MC
Barrantes
F J
FJ
D016475:3T3 Cells; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015152:Blotting, Northern; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D007457:Iodine Radioisotopes; D051379:Mice; D016716:PC12 Cells; D010911:Pituitary Neoplasms; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins; D018411:Spodoptera / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D002038:Bungarotoxins; D007457:Iodine Radioisotopes; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins