A molecular triad governing adult stem cells activation: Crystallographic studies of LGR5, R-spondin 1 and E3 ligase ZNRF3

The small intestinal tissue is divided into two compartments: the ‘villus’ (with finger-like projection) and the ‘crypt’ (invagination between villi). The small intestine is responsible for absorption of nutrients in the food ingested and is constantly damaged due to exposure to biological and physical stress. As a result, the intestinal epithelium undergoes rapid apoptosis and needs to be renewed constantly. The intestinal crypt is the ‘cell production factory’, where stem cells actively divide to produce more cells. Newly produced daughter cells migrate upwards into the villus where they differentiate to perform specific functions. Some daughter cells remain in the crypt as stem cells. The major driving force of this stem-cell renewal activity is driven by a molecular mechanism, referred to as Wnt signaling. When Wnt (a signaling molecule) binds to receptor LRP6 and co-receptor Frizzled on the stem-cell surface, Wnt signaling pathway is activated. This leads to the transcription of genes required for stem-cell proliferation. It is important that the stem-cell proliferation is strictly regulated to maintain homeostasis. Over-production of new cells is undesirable, as excess tissue growth can cause tumour formation.

Wnt signal strength is regulated by a molecular trio on the stem-cell surface: LGR5, a 7-transmembrane receptor; single-pass transmembrane E3 ligase ZNRF3; and R-spondin, a secreted ligand. E3 ligase ZNRF3 functions as an antagonist for Wnt signaling complex by ubiquitinating Frizzled receptor and targeting it for degradation, hence diminishing Wnt signal strength. On the other hand, R-spondin is a stem-cell growth factor that binds to its receptor LGR5. Together LGR5-R-spondin complex targets E3 ligase ZNRF3 for removal from the stem-cell surface, hence increasing Wnt signal strength and stimulate stem-cell proliferation. This thesis describes the molecular interaction of LGR5-Rspondin and R-spondin-ZNRF3 by crystallographic studies, combined with functional analysis. In summary, this study provides us insights into regulation of Wnt signaling by LGR5, R-spondin, and E3 ligase ZNRF3.