Center for Translational Cancer Research, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 1825 Pressler St., Suite 330E, Houston, TX, 77030, USA.
Sci Rep. 2023 Jul 4;13(1):10796. doi: 10.1038/s41598-023-37856-w.
LGR4 and LGR5 are two homologous receptors that potentiate Wnt/β-catenin signaling in response to R-spondin (RSPO) ligands. The RSPO and LGR4 complex binds to and inhibits activities of two related E3 ubiquitin ligases, RNF43 and ZNRF3, and thus protects Wnt receptors from the E3 ligase-mediated degradation. The RSPO and LGR5 complex, however, does not interact with the E3 ligases, and the structural basis of this difference remained unknown. Here we examined the affinities of monovalent and bivalent RSPO ligands in binding to LGR4, RNF43/ZNRF3, and LGR5 in whole cells and found unique features among the receptors and E3 ligases. Monovalent RSPO2 furin domain had much lower affinity in binding to LGR4 or RNF43/ZNRF3 than the bivalent form. In contrast, monovalent and bivalent forms had nearly identical affinity in binding to LGR5. Co-expression of ZNRF3 with LGR4 led to much higher binding affinity of the monovalent form whereas co-expression of ZNRF3 with LGR5 had no effect on the affinity. These results suggest that LGR4 and RNF43/ZNRF3 form a 2:2 dimer that accommodates bivalent binding of RSPO whereas LGR5 forms a homodimer that does not. Structural models are proposed to illustrate how RSPOs bind to LGR4, RNF43/ZNRF3, and LGR5 in whole cells.
LGR4 和 LGR5 是两种同源受体，它们能响应 R- 分泌蛋白（RSPO）配体增强 Wnt/β- 连环蛋白信号通路。RSPO 和 LGR4 复合物与两个相关的 E3 泛素连接酶 RNF43 和 ZNRF3 结合并抑制其活性，从而防止 Wnt 受体被 E3 连接酶介导的降解。然而，RSPO 和 LGR5 复合物不与 E3 连接酶相互作用，其结构基础尚不清楚。在此，我们检测了单价和二价 RSPO 配体在 whole cells 中与 LGR4、RNF43/ZNRF3 和 LGR5 结合的亲和力，发现了受体和 E3 连接酶之间的独特特征。单价 RSPO2 弗林蛋白酶结构域与 LGR4 或 RNF43/ZNRF3 的结合亲和力远低于二价形式。相比之下，单价和二价形式与 LGR5 的结合亲和力几乎相同。ZNRF3 与 LGR4 共表达导致单价形式的结合亲和力大大提高，而 ZNRF3 与 LGR5 共表达对亲和力没有影响。这些结果表明，LGR4 和 RNF43/ZNRF3 形成 2:2 二聚体，可容纳 RSPO 的二价结合，而 LGR5 形成同源二聚体，不允许二价结合。提出了结构模型来阐明 RSPO 如何在 whole cells 中与 LGR4、RNF43/ZNRF3 和 LGR5 结合。
