Functional Wnt Binding Sites of LRP5/6 Proteins in Bone

Wnt signaling is an essential component of intracellular signaling cascades in bone and muscle cells. This mechanism plays a role in various developmental and degenerative diseases, and understanding functional pathway components can guide research for targeted treatments for these diseases. It is currently known that Wnt proteins bind to receptor-like LRP5/6 proteins on the cell  membrane and trigger an intracellular signaling cascade, resulting in increased expression of genes necessary for bone cell growth and survival.  Although the interactions and implications of Wnt binding are an area of great interest, the specific binding sites of Wnt molecules on LRP5/6 proteins are currently unclear. We hypothesize Wnt proteins have differential binding sites on Lrp5 and Lrp6 proteins.

To map out these binding sites, wild-type MLO-Y4 TopFlash cells, a luciferase reporter bone cell line, were cultured in media and treated with one of eight Wnt proteins in conditioned media. To detect the signaling of these proteins in the TopFlash cells, the Luciferase Bio-Glo Assay System was utilized to measure luciferase activity. Once Wnt activity was confirmed, the degree of activity was compared between cells treated with differing Wnt proteins. The cells treated with Wnt1, 2, 2b, 7a, 7b, and 10 had increased Luciferase activity. Luciferase activity increased 2-3 fold in the presence of these Wnt proteins relative to control cells. In future studies, we will investigate luciferase activity in the presence of modified LRP5/6 proteins to determine if altered Lrp5/6 Wnt binding sites alter binding of specific Wnt proteins.