Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.
The Wnt signaling pathway plays a critical role in the developmental and physiological processes of metazoans. We previously reported that the Frizzled4 (FZD4) linker domain plays an important role in Norrin binding and signaling. However, the question remains whether the FZD linker contributes to Wnt signaling in general. Here, we show that the FZD linker is involved in Wnt binding and affects downstream Wnt signaling. A FZD4 chimera, in which the linker was swapped with that of the non-canonical receptor FZD6, impairs the binding with WNT3A and suppresses the recruitment of LRP6 and Disheveled, resulting in reduced canonical signaling. A similar effect was observed for non-canonical signaling. A FZD6 chimera containing the FZD1 linker showed reduced WNT5A binding and impaired signaling in ERK, JNK, and AKT mediated pathways. Altogether, our results suggest that the FZD linker plays an important role in specific Wnt binding and intracellular Wnt signaling.
Summary
Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances.
For complete details on the use and execution of this protocol, please refer to
Lee et al. (2021)
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