Xu Jin-Gen, Huang Chunfeng, Yang Zhengfeng, Jin Mengmeng, Fu Panhan, Zhang Ni, Luo Jian, Li Dali, Liu Mingyao, Zhou Yan, Zhu Yongqun
From the Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, Zhejiang 310058 and.
the Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
J Biol Chem. 2015 Jan 23;290(4):2455-65. doi: 10.1074/jbc.M114.599134. Epub 2014 Dec 5.
Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs.
富含亮氨酸重复序列的G蛋白偶联受体（LGRs）是一类独特的G蛋白偶联受体，其特征在于具有一个大的细胞外结构域来识别配体并调节许多重要的发育过程。在LGRs的三组中，B组成员（LGR4 - 6）识别R - 斯波丁家族蛋白（Rspo1 - 4）以刺激Wnt信号通路。在本研究中，我们成功利用“杂交富含亮氨酸重复序列技术”，将LGR4与盲鳗VLR蛋白融合，获得了两种重组人LGR4蛋白，即LGR415和LGR49。我们确定了无配体LGR415和LGR49 - Rspo1复合物的晶体结构。LGR4呈现出扭曲的马蹄形结构。Rspo1采用扁平的β折叠结构，在复合物中通过静电和疏水相互作用结合于LGR4的凹面。所有Rspo1结合残基在LGR4 - 6中都是保守的，这表明LGR4 - 6通过相同的表面结合R - 斯波丁蛋白。我们的LGR4 - Rspo1复合物与先前确定的LGR4和LGR5结构的结构分析表明，LGR4的凹面是R - 斯波丁蛋白的唯一结合位点，这表明LGR4 - 6在配体识别中采用单一位点结合模型。LGR4 - 6的分子机制不同于A组受体LGR1 - 3的两步机制和C组受体LGR7 - 8的多界面结合模型，这表明LGRs利用不同的机制进行配体识别。我们的结构与先前的报道一起，提供了对LGRs配体识别的全面理解。
