Itakura M, Suzuki H, Hirose S
Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan.
Biochem J. 1997 Mar 1;322 ( Pt 2)(Pt 2):585-90. doi: 10.1042/bj3220585.
Natriuretic peptide receptor-C (NPR-C) has a unique structure consisting of pre-existing covalent homodimers, but it is not known whether each subunit has ligand-binding activity or whether the dimeric structure is necessary for binding activity. To answer this question, a number of C-terminally truncated mutants were designed, subcloned into the mammalian expression vector pcDNA3 and expressed by transient transfection in COS-1 cells. Truncation at position 461, which eliminates the residue Cys469 that is involved in disulphide-linked dimerization, produced a soluble and monomeric form of NPR-C, as determined by gel filtration on Superose 12. Binding assays of the gel-filtration fractions clearly demonstrated that even monomeric NPR-C contains a high-affinity binding site for natriuretic peptides. Site-directed mutagenesis of the invariant residues (Asp407-Arg408 and Asp411-Phe412) in a region highly conserved among various species established that these invariant residues are essential for ligand-binding activity.
利钠肽受体-C（NPR-C）具有独特的结构，由预先存在的共价同型二聚体组成，但尚不清楚每个亚基是否具有配体结合活性，或者二聚体结构对于结合活性是否必要。为了回答这个问题，设计了一些C末端截短的突变体，亚克隆到哺乳动物表达载体pcDNA3中，并通过在COS-1细胞中瞬时转染进行表达。在第461位截断，消除了参与二硫键连接二聚化的半胱氨酸469残基，通过Superose 12凝胶过滤测定，产生了一种可溶性单体形式的NPR-C。凝胶过滤级分的结合测定清楚地表明，即使是单体NPR-C也含有一个利钠肽的高亲和力结合位点。对不同物种间高度保守区域中的不变残基（Asp407-Arg408和Asp411-Phe412）进行定点诱变，确定这些不变残基对于配体结合活性至关重要。
