Assembly of the nicotinic acetylcholine receptor. The first transmembrane domains of truncated alpha and delta subunits are required for heterodimer formation in vivo.
To investigate the mechanism of assembly of the mouse muscle acetylcholine receptor, we have expressed truncated N-terminal fragments of the alpha and delta subunits in COS cells and have examined their ability to fold, to associate into heterodimers, and to form a ligand-binding site. Truncated fragments of the alpha subunit that include all, part, or none of the first transmembrane domain (M1) folded to acquire alpha-bungarotoxin binding activity. Neither the full-length alpha subunit nor any of the fragments were expressed on the cell surface, although the shortest folded fragment lacking a transmembrane domain was secreted into the medium. When coexpressed with the delta subunit, the alpha subunit fragment possessing M1 formed a heterodimer containing a ligand-binding site, but shorter fragments, which lack transmembrane segments, did not associate with the delta subunit. N-terminal delta subunit fragments gave similar results. An N-terminal delta subunit fragment that contains M1 associated with the alpha subunit to form a heterodimer, while a fragment lacking M1 did not. These results show that a complete M1 domain is necessary for association of truncated N-terminal alpha and delta subunits into a heterodimer with high affinity ligand binding activity.
Wang
Z Z
ZZ
Department of Physiology, University of California, San Francisco, California 94143, USA.
Hardy
S F
SF
Hall
Z W
ZW
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary; D019281:Dimerization; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
D002038:Bungarotoxins; D018076:DNA, Complementary; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins