Ma Yitong, Wang Yijie, Tang Mengyuan, Weng Yuan, Chen Ying, Xu Yueming, An Shuxiao, Wu Yiran, Zhao Suwen, Xu Huanhuan, Li Dali, Liu Mingyao, Lu Weiqiang, Ru Heng, Song Gaojie
Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
Life Sciences Institute, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Zhejiang University, Hangzhou, Zhejiang, China.
Nat Commun. 2025 Jan 11;16(1):605. doi: 10.1038/s41467-025-55901-2.
G protein-coupled receptor 4 (GPR4) belongs to the subfamily of proton-sensing GPCRs (psGPCRs), which detect pH changes in extracellular environment and regulate diverse physiological responses. GPR4 was found to be overactivated in acidic tumor microenvironment as well as inflammation sites, with a triad of acidic residues within the transmembrane domain identified as crucial for proton sensing. However, the 3D structure remains unknown, and the roles of other conserved residues within psGPCRs are not well understood. Here we report cryo-electron microscopy (cryo-EM) structures of active zebrafish GPR4 at both pH 6.5 and 8.5, each highlighting a distribution of histidine and acidic residues at the extracellular region. Cell-based assays show that these ionizable residues moderately influence the proton-sensing capacity of zebrafish GPR4, compared to the more significant effects of the triad residues. Furthermore, we reveal a cluster of aromatic residues within the orthosteric pocket that may propagate the signaling to the intercellular region via repacking the aromatic patch at the central region. This study provides a framework for future signaling and functional investigation of psGPCRs.
G蛋白偶联受体4（GPR4）属于质子感应型G蛋白偶联受体（psGPCRs）亚家族，该亚家族可检测细胞外环境中的pH变化并调节多种生理反应。研究发现，GPR4在酸性肿瘤微环境以及炎症部位过度激活，其跨膜结构域中的三个酸性残基被确定为对质子感应至关重要。然而，其三维结构仍然未知，并且psGPCRs中其他保守残基的作用也尚未得到充分了解。在此，我们报告了活性斑马鱼GPR4在pH 6.5和8.5时的冷冻电镜（cryo-EM）结构，每种结构都突出了细胞外区域组氨酸和酸性残基的分布。基于细胞的实验表明，与三联体残基的更显著影响相比，这些可电离残基对斑马鱼GPR4的质子感应能力有适度影响。此外，我们揭示了正构口袋内的一组芳香族残基，它们可能通过重新排列中心区域的芳香族斑块将信号传递到细胞间区域。这项研究为未来psGPCRs的信号传导和功能研究提供了一个框架。
