Structural Insights Into The Interaction Of R-spondin Family Proteins With Their Receptors

The canonical Wnt/β-catenin signaling pathway plays important roles in embryonicdevelopment and stem cell maintenance. The R-spondin family protein can enhance thecanonical Wnt/β-catenin signaling, abnormal expression of R-spondin is associated withsome genetic diseases and cancer. The two FU-CRD domains of R-spondins issufficient to promote Wnt signaling. The cell membrane receptors of R-spondins isLGR4, LGR5and LGR6of the LGR family, and E3ubiquitin ligase ZNRF3andRNF43. The mechanisms of their interaction is unclear. This research thesis determinedthe crystal structure of the FU-CRD domain of R-spondin1(RSPO1-2F) and theextracellular domain of LGR4(LGR4-ECD) with x-ray diffraction, and elucidated thecritical factors that affect their interactions with biochemical and cell-based assay.First, the FU-CRD domains of human R-spondins and the extracellular domains ofLGR4, LGR5and LGR6were expressed in insect cell with the bac-to-bac baculovirusexpression system. The recombinant protein was captured by nickel beads based onaffinity chromatography, purified with gel filtration column Superdex200, and thefractions from superdex200were analyzed with SDS-PAGE. The results showed thatRSPO1-2F, LGR4-ECD and LGR5-ECD can be expressed in insect cell, RSPO1-2F andLGR5-ECD can be purified as monomer, and LGR4-ECD is mixture of monomer anddimer which can be distinguished by SDS-PAGE loading buffer without DTT andSDS-PAGE.Then the RSPO1-2F/LGR4-ECD and RSPO1-2F/LGR5-ECD complex wereobtained by co-infecting recombinant baculovirus to insect cell and concentrated toscreen crystallization conditions. Crystal of RSPO1-2F/LGR5-ECD complex did notappeared. The crystallization conditions of the good-shaped crystals ofRSPO1-2F/LGR4-ECD complex were used for optimization by changing proteinconcentration and well-buffer ingredients. Single crystals were used to collect x-raydiffraction data, the structure was determined by molecular replacement. Then mutantsof RSPO1-2F based on the structural model were designed and purified, the criticalforce and amino acids that are important for the interaction of R-spondin1with LGR4 were determined based on biochemical and cell-based assay which was collaboratedwith Dr. Wei Wu’s lab.Moreover, the extracellular domain of ZNRF3and RNF43were expressed andpurified from insect cell, and ternary complex with RSPO1-2F/LGR4-ECD were alsoobtained, but no crystal appeared.This research thesis elucidated the interaction of R-spondin family proteins withtheir receptors LGR4, LGR5and LGR6, which not only could be helpful to understandthe activation of Wnt/β-catenin signaling, but also possess scientific and applicablevalues.