G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.

译文

g蛋白偶联受体 (gpcr) 与异三聚体g蛋白相互作用,并启动多种信号通路。临床上重要的血栓烷A2 (TxA(2)) 受体 (TP) 和前列环素 (PGI(2)) 受体 (IP) 中gpcr-g蛋白相互作用的分子性质知之甚少。TP响应血栓烷的结合而激活其同源g蛋白 (G α q),而IP响应前列环素的结合而通过G α s发出信号。在这里,我们结合了由嵌合受体,分子建模和定点诱变组成的方法来精确研究g蛋白偶联的特异性。通过用IP的ICL区域替换TP细胞内环 (ICL) 来构建多个嵌合受体。我们的结果表明,ICL2和ICL3的序列和长度均影响g蛋白的特异性。重要的是,我们在前列腺素受体TP和IP上确定了一个精确的ICL区域,可以切换g蛋白特异性。通过引入临床相关的自然发生突变 (TP中的R60L和IP中的R212C),证实了嵌合技术和衍生分子模型的有效性。我们的发现为前列腺素受体-g蛋白相互作用提供了新的分子见解,这对于理解GPCRs在基础健康和心血管疾病中激活g蛋白的结构基础具有普遍意义。

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