Project description:Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.
Project description:Transcriptional profiles of the embryonic yolk sac from embryos with ectopic Norrin expression were compared to their wild type littermate controls. The goal is to identify the transcriptional response to Norrin-Frizzled 4 signaling during embryonic angiogenesis.
Project description:This SuperSeries is composed of the following subset Series:; GSE16691: Transcriptional regulation by Norrin-Frizzled4 signaling in the embryonic yolk sac; GSE16703: Long-term effect on the transcriptome of a decrement in Norrin/Frizzled4/Lrp signaling in retinal endothelial cells; GSE16705: Transcriptional response to Frizzled4 signaling in cultured retinal endothelial cells; GSE16707: Long-term effect on the transcriptome of loss of Frizzled4 signaling in cerebellar endothelial cells Experiment Overall Design: Refer to individual Series
Project description:Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease.