Yu B, Simon M I
Division of Biology, 147-75, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 1998 Nov 13;273(46):30183-8. doi: 10.1074/jbc.273.46.30183.
We constructed a double mutant version of the alpha subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoalphaX). We investigated the interaction between GoalphaX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPgammaS for XDP in the GoalphaXbetagamma heterotrimer. Second, the GoalphaXbetagamma complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoalphaX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoalphaX, lacking a bound nucleotide and betagamma, formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoalphaX with receptor in COS-7 cells. The empty form of GoalphaX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoalphaX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that GoalphaX specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors.
我们构建了Goα亚基的双突变体版本，该版本由黄嘌呤核苷酸而非鸟嘌呤核苷酸调控（GoαX）。我们在体外研究了GoαX与G蛋白偶联受体之间的相互作用。首先，我们发现激活的M2型毒蕈碱胆碱能受体（MAChR）能够促进GoαXβγ异源三聚体中XTPγS与XDP的交换。其次，GoαXβγ复合物能够诱导N-甲酰甲硫氨酸肽受体（NFPR）形成高亲和力配体结合状态。这些实验表明GoαX能够与G蛋白偶联受体有效相互作用。第三，我们发现缺乏结合核苷酸和βγ的GoαX空形式与质膜相关的M2型毒蕈碱胆碱能受体形成了稳定的复合物。最后，我们研究了GoαX与COS-7细胞中受体的相互作用。GoαX空形式紧密结合受体且未被激活，因为细胞内无法获得XTP。我们测试了GoαX抑制转染的COS-7细胞中几种不同G蛋白偶联受体活性的能力，发现GoαX特异性抑制与Go偶联的受体。因此，修饰后的G蛋白可能作为显性负突变体来捕获并失活特定的受体亚群。
