Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant.
Torpedo californica acetylcholine receptor (AChR) alpha-, beta-, gamma-, and delta-subunit cDNAs were each stably introduced into muscle and/or fibroblast cell lines using recombinant retroviral vectors and viral infection, or using SV-40 vectors and DNA-mediated cotransfection. The expressed proteins were characterized in terms of their molecular mass, antigenicity, posttranslational processing, cell surface expression, stability in fibroblasts, stability in differentiated and undifferentiated muscle cells, and ability (of alpha) to bind alpha-bungarotoxin (BuTx). We demonstrated that the alpha, beta, gamma, and delta polypeptides acquired one, one, two, and three units of oligosaccharide, respectively. If all four subunits were expressed in the same cell, fully functional cell surface AChRs were produced which had a Kd for BuTx of 7.8 X 10(-11) M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual alpha polypeptides was only approximately 4 X 10(-7) M. The half-lives of the alpha, gamma, and delta subunits at 37 degrees C were all found to be quite short (approximately 43 min), while the half-life of the beta subunit was found to be even shorter (approximately 12 min). The unique half-life of the beta subunit suggests that it might perform a key regulatory role in the process of AChR subunit assembly. One stable fibroblast cell line was established by transfection that expressed beta, gamma, and delta subunits simultaneously. When this cell line was infected with a retroviral alpha recombinant, fully functional cell surface AChRs were produced. The successful expression of this pentameric protein complex combining transfection and infection techniques demonstrates one strategy for stably introducing the genes of a heterologous multisubunit protein complex into cells.
Claudio
T
T
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Paulson
H L
HL
Green
W N
WN
Ross
A F
AF
Hartman
D S
DS
Hayden
D
D
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology; D005822:Genetic Vectors; D007473:Ion Channels / Q000502:physiology; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009132:Muscles / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D011994:Recombinant Proteins; D012190:Retroviridae / Q000235:genetics; D013329:Structure-Activity Relationship; D014101:Torpedo; D014162:Transfection
D002038:Bungarotoxins; D007473:Ion Channels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins