Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica

被引:132
|
作者
Hansen, SB
Talley, TT
Radic, Z
Taylor, P [1 ]
机构
[1] Univ Calif San Diego, Dept Pharmacol, La Jolla, CA 92093 USA
[2] Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92093 USA
关键词
D O I
10.1074/jbc.M402452200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We generated an acetylcholine-binding protein from Aplysia californica by synthesis of a cDNA found in existing data bases and its expression in mammalian cell culture. Its subunit assembly and ligand recognition behavior were compared with the binding protein previously derived from Lymnaea stagnalis. The secreted proteins were purified by elution from columns of attached antibodies directed to the FLAG epitope encoded in the expression construct. Although the sequences of the two proteins from marine and fresh water mollusks exhibit the characteristic features of the extracellular domain of the nicotinic receptor, they only possess 33% amino acid identity. Both assemble as stable pentamers with five binding sites per pentamer, yet they show distinguishing features of stability and sensitivity to epitope tag placement. Both proteins exhibit changes in tryptophan fluorescence upon ligand binding; however, the magnitude of the changes differs greatly. Moreover, certain ligands show marked differences in dissociation constants for the two proteins and can be regarded as distinguishing or signature ligands. Hence, the two soluble proteins from mollusks, which can be studied by a variety of physical methods, become discrete surrogate proteins for the extracellular domains of distinct sub-types of nicotinic acetylcholine receptors.
引用
收藏
页码:24197 / 24202
页数:6
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