Varp Interacts With LRP6 And Negatively Regulates Wnt/b-catenin Signaling Pathway

Wnt signals play critical roles in embryogenesis and diseases. In the canonical Wnt signaling pathway, Wnt ligands bind to Frizzled and LRP6 receptor complex and activate a cellular process to stabilizeβ-catenin. The accumulatedβ-catenin is translocated into cell nucleus where it interacts with TCF/LEF-1 family transcriptional factors and activates Wnt targeted gene expression. How Wnt signal is transduced from receptors to downstream mediators remains largely unknown. LRP6 is a key receptor in the canonical Wnt signaling pathway, and the intracellular region of LRP6 has complete activity to transduceβ-catenin signals. In order to shed light on mechanism by which Wnt signal is transduced and regulated through LRP6, we searched for the proteins that associate with LRP6.Through a yeast two-hybrid screening, we cloned a novel LRP6 interacting protein, Varp (VPS9-Ankyrin Repeats Protein). Results from yeast two-hybrid and co-IP assays showed specific interaction between Varp and LRP6. The COOH-terminal region containing the second ankyrin domain of Varp and the COOH-terminal region containing the PPP(S/T)P motifs of LRP6 were responsable for their interaction.In the cultured cells, overexpression of Varp attenuated Wnt3a and LRP6 inducedβ-catenin accumulation and TCF/LEF-dependent transcriptional activation, whereas siRNA mediated depletion of the endogenous Varp enhanced Wnt signaling. We also demonstrated that Varp promote LRP6 degradation probably through lysosome pathway. Further study showed that the ectopically expressed LRP6 was rapidly internalized in a ligand independent manner, and this process might not be directly regulated by Varp. Wihle, the internalized was LRP6 co-localized with the ectopically expressed Varp on early endosomes, indicating that Varp might regulate the transport process of the internalized LRP6.As VPS9 domain was conserved in several well characterized Rab5 guanine nucleotide exchange factors, we speculated that Varp might be a novel Rab5GEF. Insterestingly, our data showed that Varp interacts preferentially with GDP-bound...