Role of LRP6 in the Wnt/beta-catenin pathway and its regulation by heterotrimeric G proteins

Abstract

The Wnt/beta-catenin signaling pathway is a well-conserved signal transduction pathway that is highly regulated during metazoan development and is associated with various human diseases. In the current model of Wnt/beta-catenin signaling, Wnt ligands bind to its receptors, Frizzled and LRP6 (low-density lipoprotein receptor-related protein 6). These receptors transmits an intracellular signal that ultimately increases steady-state levels of cytoplasmic beta-catenin by inhibiting the phosphorylation of beta-catenin by the kinase GSK3. Elevated beta-catenin translocates to the nucleus and induces Wnt target gene expression. A major question in the field is how a Wnt/beta-catenin signal is transmitted from the receptors to mediate transmission of the Wnt signal from the plasma membrane. To understand how signaling through LRP6 inhibits beta-catenin degradation we expressed and purified the LRP6 intracellular domain and found that it stimulated beta-catenin stabilization while also stimulating the degradation of the negative regulator, Axin. Through additional egg extract and biochemical reconstitution experiments we found that LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin. The Frizzled receptor is predicted to have seven transmembrane domains, a feature that is characteristic of G protein-coupled receptors. To identify a role for heterotrimeric G proteins in Wnt/beta-catenin signaling, we screened major families of recombinant G protein alpha subunits in our egg extract system and found that Galphao, Galphaq, Galphai2, and Gbetagamma inhibit β-catenin degradation. We find that Gbeta1gamma2 is required for and promotes LRP6 phosphorylation and activation by directly recruiting GSK3 to the membrane and enhancing its kinase activity. We propose that heterotrimeric G protein activation results in formation of free Gbetagamma and Galpha, which act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription. Together, these studies provide insight on the molecular mechanism of the early intracellular events of Wnt/beta-catenin signaling.