Study Of Structural Basis Of Human RSPO1 Ligand Recognition By Its LGR4 Receptor

The Wnt signal transduction pathway plays a pivotal role in many physiological processes through embryonic development and self-renewing of adult tissues.Germline mutations in the Wnt signal cascade can give rise to several genetic disease and somatic mutations are also closely related to intestinal cancers and lots of other tissue diseases.R-spondins(RSPOs)are a group of secreted glycoproteins that synergize with Wnt proteins to potentiate Wnt/?-catenin signaling and have pleiotropic physiological roles in homeostasis and development.Recently the orphan receptors LGR4 and its homologues are demonstrated to be the receptors of R-spondins to mediate their roles in Wnt signaling pathway.Besides the canonical seven-transmembrane(7TM)domains,the group B leucine-rich repeat G-protein-coupled receptors(LGRs)including LGR4,LGR5 and LGR6 are also characterized by a large extracellular domain to recognize R-spondins and regulate developmental processes.Despite the critical roles of R-spondins and their LGR receptors in animals,the mechanism of R-spondins recognition by LGR receptors remains elusive.In our study,we successfully developed the "hybrid leucine-rich repeat technique" method which fused the extracellular domain of LGR4 to the C-terminal domain of hagfish VLR protein to obtain two hybrid recombinant proteins LGR415 and LGR49.We also solved the crystal structures of ligand-free LGR415 and the complex of RSPO1-LGR49 after many trials.Generally,the extracellular domain of LGR4 exhibits a twisted horseshoe-like structure.The two furin-like CRD domain of Rspol adopts a flat and ?-fold conformation and is bound in the concave surface of LGR4.There are many extensive electrostatic and hydrophobic interactions between them.We also notice that all the Rspol-interacting residues are conserved in LGRs,indicating that LGR receptors bind R-spondins through a uniform interface.Combining with the previously determined LGR5 structures we postulated that the concave surface of LGR4/5/6 is the sole binding site for ligands,which suggested a one-site binding model of LGR4/5/6 in R-spondin recognition.The recognition mechanism of LGR4/5/6 is not only different from the two-step recognition mechanism of group A receptors LGR1/2/3,but also the multiple-interface recognition mechanism of group C receptors LGR7/8,indicating LGRs adopt divergent manners to recognize ligands.In conclusion,our works,together with others' structures,promote a profound explanation of the ligands recognition by LGRs.