Binding of monoclonal antibodies against the carboxyl terminal segment of the nicotinic receptor delta subunit suggests an unusual transmembrane disposition of this sequence region.
Monoclonal antibodies (mAbs) specific for the carboxyl terminal region of the delta subunit of Torpedo nicotinic acetylcholine receptor (AChR), derived from mice immunized with AChR or a synthetic carboxyl terminal sequence of the delta subunit (C delta-mAbs), were used to determine the transmembrane disposition of their epitope(s) by immunoelectron microscopy, using AChR-rich postsynaptic membrane fragments from Torpedo electroplax. Some C delta-mAbs recognized only the cytoplasmic side of the membranes, some both sides to a similar extent, and others bound mostly, but not exclusively, to the cytoplasmic side. Binding of C delta-mAbs to the membranes was specifically blocked by synthetic peptides containing the carboxyl terminal region of the delta subunit. Control anti-AChR mAbs specific for the alpha or the delta subunits, whose epitopes have known transmembrane topology, uniquely recognized the expected side of the postsynaptic membrane. Residues involved in C delta-mAb binding were identified using single residue substituted peptide analogues of the sequence delta 481-501. All C delta-mAbs recognized epitopes within the same sequence segment, delta 485-493, at the carboxyl terminal of the AChR delta subunit. These results suggest that the delta subunit of the AChR might have alternative conformations, leading to exposure of the same sequence region on the extracellular or the cytoplasmic surface. Several Pro residues are present in this region. The alternative cis or trans conformation of one or more of them might result in different folding patterns of the carboxyl terminal sequence of the delta subunit, as described for a viral protein [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L., & Harrison, S. C. (1991) Nature 354, 278-284.
Lei
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Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
Okita
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DK
Conti-Fine
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BM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D010449:Peptide Mapping; D015531:Radioimmunoprecipitation Assay; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000276:immunology*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011978:Receptors, Nicotinic