Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor.
Through specific intersubunit contacts, the four subunits of the nicotinic acetylcholine receptor assemble into an alpha 2 beta gamma delta pentamer. The specificity of subunit association leads to formation of proper ligand binding sites and to transport of assembled pentamers to the cell surface. To identify determinants of subunit association, we constructed chimeric subunits, transfected them into HEK 293 cells, and studied their association with wild-type subunits. We used beta gamma chimeras to determine sequences that associate with the alpha subunit to form a ligand binding site and found residues 21-131 of the gamma subunit sufficient to form the site. Residues 51-131 of the beta subunit do not form a binding site, but do promote surface expression of pentamers; of these residues, R117 is key for surface expression. We studied formation of tetramers by alpha and gamma subunits and dimers by alpha and delta subunits, and used gamma delta chimeras to identify sequences that result in either dimers or tetramers. The conserved residues I145 and T150 of the gamma subunit promote alpha gamma alpha gamma tetramer formation, whereas the corresponding residues in the delta subunit, K145 and K150, allow only alpha delta dimer formation.
Kreienkamp
H J
HJ
Department of Pharmacology, La Jolla 92093.
Maeda
R K
RK
Sine
S M
SM
Taylor
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins