Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.
Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.
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人 G 蛋白偶联 C3a 受体跨膜区域中配体效应子结合位点的鉴定。
DOI：
10.1110/ps.8.11.2304
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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：
--
作者：
Sun,J;Ember,JA;Chao,TH;Fukuoka,Y;Ye,RD;Hugli,TE
人C3 a过敏毒素受体（C3 aR）是一种G蛋白偶联受体（GPCR），由7个跨膜α-螺旋通过亲水环连接而成。先前的嵌合C3 aR/C5 aR和C3 aR环缺失的研究表明，大的细胞外loop 2在非效应配体结合中起着重要作用，然而，C3 a的效应结合位点尚未确定。在这项研究中，选定的C3 aR的跨膜区的带电残基被替换为Ala使用定点突变，突变体受体在RBL-2 H3细胞系中稳定表达。配体结合研究表明，R161 A（螺旋IV），R340 A（螺旋V），和D417 A（螺旋VII）显示没有结合活性，虽然这些受体的完全表达建立了流式细胞术分析。C3 a在表达这三种突变受体的细胞中诱导非常弱的细胞内钙流。H81 A（螺旋II）和K96 A（螺旋III）显示配体结合活性降低。在H81 A和K96 A细胞中，C3 a诱导的钙流也持续降低。这些研究结果表明，带电荷的跨膜残基Arg 161，Arg 340，和Asp 417在C3 aR的配体效应器结合和/或信号耦合是必不可少的，残基His 81和Lys 96可能有助于不直接配体结合的总自由能。C3 aR中的这些跨膜残基识别用于解释C3 a诱导的受体活化的配体相互作用的特定分子接触。
The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane α-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.