A conserved disulfide loop facilitates conformational maturation in the subunits of the acetylcholine receptor.
To examine the structural determinants for the assembly of ligand-gated receptors, we constructed mutant alpha, beta, gamma and delta subunits of the Torpedo acetylcholine receptor (AChR), lacking one of the conserved cysteine residues which forms a 13-amino acid disulfide loop in the amino terminal domain of each subunit. Mutant subunits were co-expressed with complementary wild-type subunits in Xenopus oocytes. Using subunit-specific antisera and monoclonal antibodies that recognize the two distinct alpha-bungarotoxin (alpha-BuTX) sites on the AChR, we were able to distinguish immature subunit associations from conformationally mature AChR complexes. Removal of the disulfide loop on the alpha subunit completely destroyed the formation of the two toxin-binding sites, while removal of the structure on the beta subunit had little effect. While mutant gamma and delta subunits were capable of forming associations (immature assembly) with other subunits, the formation of alpha-BTX sites between alpha and mutant gamma or mutant delta subunits was diminished. Interestingly, assembly of alpha beta gamma subunits remained efficient in the presence of mutant delta subunits, whereas assembly of alpha beta delta subunits was inefficient in the presence of mutant gamma subunits. Thus, these results indicate that the formation of the disulfide loop facilitates the conformational maturation of alpha gamma and alpha delta complexes, which may be conditional for correct subunit coupling in assembling receptors. Furthermore, it seems likely that the correct coupling between the alpha and gamma subunits is the most important step in subunit assembly.
Walcott
E C
EC
Department of Psychobiology, University of California, Irvine 92697-4550, USA.
Sumikawa
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K
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry*; D000939:Epitopes / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011487:Protein Conformation; D012333:RNA, Messenger / Q000096:biosynthesis; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014101:Torpedo; D014162:Transfection; D014982:Xenopus laevis
D002038:Bungarotoxins; D004220:Disulfides; D000939:Epitopes; D012333:RNA, Messenger; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins