Center for Translational Cancer Research, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
Sci Signal. 2020 Dec 1;13(660):eaaz4051. doi: 10.1126/scisignal.aaz4051.
and encode two homologous receptors with critical, yet distinct, roles in organ development and adult stem cell survival. Both receptors are coexpressed in intestinal crypt stem cells, bind to R-spondins (RSPOs) with high affinity, and potentiate Wnt-β-catenin signaling, presumably by the same mechanism: forming RSPO-bridged complexes with the E3 ligases RNF43 and ZNRF3 to inhibit ubiquitylation of Wnt receptors. However, direct evidence for RSPO-bound, full-length LGR5 interacting with these E3 ligases in whole cells has not been reported, and only is essential for the self-renewal of intestinal stem cells. Here, we examined the mechanisms of action of LGR4 and LGR5 in parallel using coimmunoprecipitation, proximity ligation, competition binding, and time-resolved FRET assays in whole cells. Full-length LGR4 formed a tight complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 did not interact with either E3 ligase with or without RSPO. Domain-swapping experiments with LGR4 and LGR5 revealed that the seven-transmembrane domain of LGR4 conferred interaction with the E3 ligases. Native LGR4 and LGR5 existed as dimers on the cell surface, and LGR5 interacted with both FZD and LRP6 of the Wnt signalosome to enhance LRP6 phosphorylation and potentiate Wnt-β-catenin signaling. These findings provide a molecular basis for the weaker activity of LGR5 in the potentiation of Wnt signaling that may underlie the distinct roles of and in organ development, as well as the self-renewal and fitness of adult stem cells.
并编码两个具有重要但不同作用的同源受体，这些受体在器官发育和成年干细胞存活中发挥作用。这两种受体都在肠隐窝干细胞中表达，与 R 分泌蛋白（RSPO）具有高亲和力结合，并增强 Wnt-β-连环蛋白信号，可能通过相同的机制：与 E3 连接酶 RNF43 和 ZNRF3 形成 RSPO 桥接复合物，抑制 Wnt 受体的泛素化。然而，尚未有报道表明全长 LGR5 与这些 E3 连接酶直接结合 RSPO 并在整个细胞中相互作用，并且只有 对于肠干细胞的自我更新是必需的。在这里，我们使用共免疫沉淀、邻近连接、竞争结合和全细胞时间分辨 FRET 测定法平行研究了 LGR4 和 LGR5 的作用机制。全长 LGR4 甚至在没有 RSPO 的情况下与 ZNRF3 和 RNF43 形成紧密复合物，而 LGR5 则无论是否有 RSPO 都不与任何一种 E3 连接酶相互作用。LGR4 和 LGR5 的结构域交换实验表明，LGR4 的七跨膜结构域赋予了与 E3 连接酶相互作用的能力。天然 LGR4 和 LGR5 存在于细胞表面作为二聚体，并且 LGR5 与 Wnt 信号体的 FZD 和 LRP6 相互作用，以增强 LRP6 磷酸化并增强 Wnt-β-连环蛋白信号。这些发现为 LGR5 在增强 Wnt 信号中的较弱活性提供了分子基础，这可能是 和 在器官发育以及成年干细胞的自我更新和适应性方面的不同作用的基础。
