Ligand binding sites on guinea pig C3aR: point and deletion mutations in the large extracellular loop and vicinity.
Ligand binding sites on guinea pig C3aR: point and deletion mutations in the large extracellular loop and vicinity.
复制标题
豚鼠 C3aR 上的配体结合位点：大细胞外环及其附近的点突变和缺失突变。
DOI：
10.1006/bbrc.1999.1372
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Hugli,TE
中科院分区：
文献类型：
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作者：
Fukuoka,Y;Ember,JA;Hugli,TE
人C3a受体(HuC3aR)属于G蛋白偶联受体家族，具有7个跨膜区。HuC3aR是这个家族中唯一的成员，在第4和第5个跨膜区之间有一个由175个氨基酸组成的胞外大环。基于对几个物种C3aR序列的比较，用定点突变的方法替换了豚鼠C3aR大EC环及其附近的一些带电和保守的氨基酸(Asp182、Asp309、Asp310和Arg331)。竞争结合分析表明，将豚鼠C3aR中的Arg331改变为Ala(或Gln)，而不将Asp182、Asp309或Asp310改变为Ala，则导致配体结合活性完全丧失。这些结果和主要的EC环缺失表明，在C3aR的跨膜部分存在必要的C3a结合位点，但在大的EC环中不存在。用不带电荷的残基取代Arg331足以消除配体-受体的相互作用。
Human C3a receptor (huC3aR) belongs to the G-protein coupled receptor family chacterized by having seven transmembrane domains. The huC3aR is a unique member of this family having a large extracellular (EC) loop of 175 amino acids between the 4th and 5th transmembrane domains. Based on a comparison of C3aR sequences from several species, a number of charged and conserved amino acids (Asp182, Asp309, Asp310, and Arg331) in and near the large EC loop of guinea pig C3aR were replaced using site-directed mutagenesis. Competitive binding assays showed that changing Arg331 in guinea pig C3aR to Ala (or Gln), but not changing Asp182, Asp309, or Asp310 to Ala, resulted in complete loss of ligand binding activity. These results and major EC loop deletions demonstrated that an essential C3a binding site is present in the transmembrane portion of C3aR, but not in the large EC loop. Replacement of Arg331 by a noncharged residue was sufficient to eliminate ligand–receptor interactions.