The E3 Ubiquitin Ligase Trip12 attenuates Wnt9a/Fzd9b signaling during hematopoietic stem cell development.

Wnt signaling is essential for both the development and homeostasis of diverse cellular lineages, including hematopoietic stem cells. Organism-wide, Wnt signals are tightly regulated, as overactivation of the pathway can lead to tumorigenesis. Although numerous Wnt ligands and Frizzled (Fzd) receptors exist, how particular Wnt/Fzd pairings are established and how their signals are regulated is poorly understood. We have previously identified the requirements of the cognate pairing of Wnt9a and Fzd9b for early hematopoietic stem cell proliferation. However, the specific signals governing activation, but equally important, the molecular mechanisms required to turn the signal 'off,' are unknown. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) is specifically required to ubiquitinate the third intracellular loop of Fzd9b at K437, targeting it for lysosomal degradation. In contrast to other ubiquitin ligases described to regulate the cell surface availability of multiple Fzds broadly, our data indicate that Trip12 is selective for Fzd9b. We further demonstrate that this occurs through ubiquitination at K437 of Fzd9b in the third intracellular loop, ultimately leading to a decrease in Fzd9b receptor availability and in Wnt9a/Fzd9b signaling that impacts hematopoietic stem cell proliferation in zebrafish. Our results point to specific mechanisms driving the availability of different Fzd receptors. Determining how particular Fzd abundance is regulated at the membrane will be critical to developing specific therapies for human intervention.