Discovery of a protein sequence in the N-terminal region of the human neuronal alpha7 nicotinic acetylcholine receptor involved in homomeric interactions.
The neuronal nicotinic acetylcholine receptor subunit, alpha7, can form homopentameric receptor/ion channel complexes. Potential contributions of its N-terminal region to homomeric interactions were investigated, in comparison with the corresponding region of an analogous heteromeric subunit, alpha3. Recombinant chimeras were prepared upon engineering the N-terminal alpha7 (M1-V224) or alpha3 (M1-S232) sequence into the background of another homomeric mouse 5-hydroxytryptamine3 (5-HT)(3) receptor. The alpha7/5-HT(3) chimera, when expressed heterologously in a human epithelial cell line, SH-EP1, robustly expressed alpha-bungarotoxin binding sites as homooligomers while the alpha3/5-HT(3) did not produce epibatidine (non-selective ligand) binding sites, and did not interfere the alpha7/5-HT3 phenotype, upon co-expression. Yeast two hybrid assays with the N-terminal regions showed positive responses between alpha7:alpha7, but not between alpha7:alpha3 and alpha3:alpha3. Similar assays with the alpha7 N-terminal region and its five smaller fragments (G23-N46, D47-N90, V91-N133, S134-M182and Q183-V224) revealed that the G23-N46 sequence is involved in homomeric interactions. Replacement of the corresponding region of the alpha3/5-HT(3) chimera with the alpha7 G23-N46 sequence conferred a dominant negative role on the chimera, by abolishing the alpha7/5-HT(3) phenotype. These results support the view that the G23-N46 portion of the alpha7 N-terminal region may contribute to receptor homooligomerizations.
Chio
Christopher L
CL
Biology II/Neurobiology, Pharmacia & Upjohn, 301 Henrietta Street, Kalamazoo, MI 49007, USA.
Alberts
Glen L
GL
Im
Wha Bin
WB
Slightom
Jerry L
JL
Gill
Gurnam S
GS
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002678:Chimera; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D051379:Mice; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D015003:Yeasts
D010446:Peptide Fragments; D011978:Receptors, Nicotinic; C066297:acetylcholine receptor alpha-subunit (125-148)