Fig. 6. NTN4 protein modulates Wnt signaling through binding to Wnt ligands.
(A) Schematic diagram of the NTN4 protein’s full length (NTN4), Laminin_N domain (NTN4LN), Laminin_EGF domain (NTN4LE), and NTR domain (NTN4NTR) of the NTN4 protein with (SP) or without (ΔSP) signal peptide, used in coimmunoprecipitation, glutathione S-transferase (GST) pull-down assay, and conditional medium (CM) assays. (B) 293T cells were transiently cotransfected with the indicated plasmid. Coimmunoprecipitation was conducted using anti–HA-tag antibody–conjugated beads followed by immunoblotting with anti–HA-tag and anti–Myc-tag antibodies. The immunoglobulin G (IgG) antibody was used as a negative control. (C) GST pull-down assays were performed to determine the interaction of GST-tagged full-length or truncated NTN4 protein with indicated Myc-tagged Wnt ligands. The interaction was detected by immunoblotting with an anti–Myc-tag antibody. (D) Visualization of the NTN4-WNT3A docking model predicted by ClusPro. The protein structure of NTN4 and WNT3A was obtained from SWISS-MODEL. NTN4 (orange red) and WNT3A (blue) structures are shown as a ribbon diagram. (E) TOP/FOPFlash luciferase reporter assays were performed in MDA-MB-231 cell lines after treatment with different mixtures of CM. Data are represented as means ± SEM. ***P ≤ 0.001. n = 3. (F) Western blotting analyzing p-LRP6, LRP6, p-GSK3β, GSK3β, and β-catenin in total cell lysates and nuclear fractions prepared from the MDA-MB-231 cell line treated with different mixed CMs. (G) Graphical representation of NTN4 transcriptional regulation by the rs11836367 locus and its interaction with Wnt ligands. The protective T allele at the rs11836367 locus increases GATA3 binding to a distal enhancer of the NTN4 gene elevating NTN4 expression. NTN4 protein interacts with Wnt ligands and antagonizes Wnt/β-catenin signaling, decreasing breast cancer risk and tumor progression.