Scott Daniel J, Rosengren K Johan, Bathgate Ross A D
Florey Neuroscience Institutes and the Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
Mol Endocrinol. 2012 Nov;26(11):1896-906. doi: 10.1210/me.2012-1188. Epub 2012 Sep 12.
Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leucine-rich repeat (LRR)-containing modules. Although relaxin can bind and activate both RXFP1 and RXFP2, INSL3 can only bind and activate RXFP2. To investigate whether this difference is related to the nature of the high-affinity ECD binding site or to differences in secondary binding sites involving the receptor transmembrane (TM) domain, we created a suite of constructs with RXFP1/2 chimeric ECD attached to single TM helices. We show that by changing as little as one LRR, representing four amino acid substitutions, we were able to engineer a high-affinity INSL3-binding site into the ECD of RXFP1. Molecular modeling of the INSL3-RXFP2 interaction based on extensive experimental data highlights the differences in the binding mechanisms of relaxin and INSL3 to the ECD of their cognate receptors. Interestingly, when the engineered RXFP1/2 ECD were introduced into full-length RXFP1 constructs, INSL3 exhibited only low affinity and efficacy on these receptors. These results highlight critical differences both in the ECD binding and in the coordination of the ECD-binding site with the TM domain, and provide new mechanistic insights into the binding and activation events of RXFP1 and RXFP2 by their native hormone ligands.
松弛素和胰岛素样肽3（INSL3）是肽类激素，在生殖、细胞外基质周转调节和心血管功能方面具有许多重要的生理作用。松弛素和INSL3分别通过密切相关的G蛋白偶联受体——松弛素家族肽受体1和2（RXFP1和RXFP2）介导其作用。这些受体具有大的细胞外结构域（ECD），在其10个富含亮氨酸重复序列（LRR）的模块中包含高亲和力配体结合位点。虽然松弛素可以结合并激活RXFP1和RXFP2，但INSL3只能结合并激活RXFP2。为了研究这种差异是与高亲和力ECD结合位点的性质有关，还是与涉及受体跨膜（TM）结构域的二级结合位点的差异有关，我们构建了一组将RXFP1/2嵌合ECD连接到单个TM螺旋上的构建体。我们表明，通过改变少至一个LRR（代表四个氨基酸替换），我们能够在RXFP1的ECD中设计出一个高亲和力的INSL3结合位点。基于大量实验数据的INSL3-RXFP2相互作用的分子模型突出了松弛素和INSL3与其同源受体ECD结合机制的差异。有趣的是，当将工程化的RXFP1/2 ECD引入全长RXFP1构建体时，INSL3对这些受体仅表现出低亲和力和效力。这些结果突出了ECD结合以及ECD结合位点与TM结构域协调方面的关键差异，并为RXFP1和RXFP2被其天然激素配体结合和激活的事件提供了新的机制见解。
