Fig. 3: Structure of the LGR4-RSPO2-ZNRF3 complex.

a Overall structure of the LGR4-RSPO2-ZNRF3_1:1:2 complex. The cryo-EM map (left) and ribbon models (middle and right) with LGR4 (blue), RSPO2 (green), and ZNRF3 (orange/red). The asterisk indicates a second protomer in the dimer. b Overall structure of the LGR4-RSPO2-ZNRF3_2:2:2 complex. The cryo-EM map (left) and the ribbon models (middle and right). c Close-up view of the interface between ZNRF3 and LGR4 (ZNRF3-LGR4*) in the extracellular region. The dashed lines indicate salt bridges and hydrogen bonds. d TOPflash reporter assays using full-length wild-type (WT) or mutant (ΔLRR15-LRRCT) LGR4. HEK293T cells were transfected with siRNA against LGR4 and LGR5, followed by plasmid transfection, and then treated with 5% Wnt3a-conditioned medium in the presence or absence of 3 ng/mL RSPO2. EV: empty vector. n = 3 biological replicates. Bars represent mean ± standard error of the mean (SEM), and dots show individual data points. Statistical significance was determined using two-way ANOVA with two-sided Tukey’s test. *P = 0.038, ***P < 0.001. ns not significant. e Pull-down assay of LGR4 and ZNRF3 in the presence or absence of RSPO2. Expi293F cells were transfected with ZNRF3 ECDTMD (Flag-tagged), LGR4 ECDTMD (Myc-tagged), and RSPO2 Fu1-2 (Strep-tagged). Cell lysates were incubated with anti-FLAG affinity beads, and bound proteins were detected by western blotting. Data are representative of three independent experiments. f The TMD region of the LGR4-RSPO2-ZNRF3_1:1:2 complex (front view). The TM helix of ZNRF3* is in contact with TM1 and TM7 of LGR4, and the distances between them are indicated. g The TMD region of the LGR4-RSPO2-ZNRF3_2:2:2 complex (bottom view). The two TM helices of ZNRF3 are sandwiched between TM1, TM6, and TM7 of LGR4, and the distances between them are indicated. h TOPflash reporter assays using full-length wild-type (WT) or mutant (CD4 TMD) LGR4. HEK293T cells were stimulated as in (d). n = 3 biological replicates. Bars represent mean ± SEM, and dots show individual data points. Statistical significance was determined using two-way ANOVA with two-sided Tukey’s test. ***P < 0.001. ns, not significant. Source data are provided as a Source data file.