Mouse Cristin/R-spondin Family Proteins Are Novel Ligands for the Frizzled 8 and LRP6 Receptors and Activate -Catenin-dependent Gene Expression*

Wnt signaling plays critical biological roles during normal embryonic development andhomeostasis in adults. In the canonical pathway, binding of Wnt ligands to the Frizzled (Fzd) receptor and the low density lipoprotein-related receptor (LRP) 5 or LRP6 coreceptor initiates downstream signaling events leading to gene activation by -catenin and the T-cell factor (TCF)-lymphoid enhancer factor (LEF) family transcription factor complex. In this study, we provide several lines of evidence that the mouse Cristin/R-spondin family proteins function as Fzd8 and LRP6 receptor ligands and induce the canonical Wnt/ -catenin signaling pathway, leading to TCF-dependent gene activation. First, conditioned medium containing Cristin/R-spondin proteins effectively induced reporter activity in a TCF-binding site-dependent manner. Second, stimulation of cells with Cristin/R-spondin was accompanied by stabilization of endogenous -catenin proteins and induction of canonical Wnt target genes. Third, Cristin/R-spondin proteins physically interacted with the extracellular domains of the LRP6 and Fzd8 receptors in vivo and in vitro. Interestingly, unlike canonical Wnt ligands, Cristin/R-spondin failed to form a ternary complex with both LRP6 and Fzd8 receptors, suggesting that R-spondinmay activate the canonicalWnt signaling pathway by differentmechanisms. Furthermore, Cristin/R-spondin proteins possess an intriguing positive modulatory activity on Wnt ligands, possibly through a direct interaction. Our findings expand the repertoire of ligands that induce -catenin/TCF-dependent gene activation and implicate the presence of active -catenin-dependent gene activation in a Wnt-free biological context.