Wnt Signaling Cascades: Pointing in the Right Direction

A reoccurring theme in signal transduction is for a single spanning trans-membrane receptor to form a homodimer upon signal ligand binding, this step is often crucial for the signal to be transduced into the cell. This applies to numerous hormone receptors, such as the PDGF receptor and the insulin receptor, although the insulin receptor pre-exists as a covalently bound homodimer regardless of an extracellular signal. Based upon a recent publication from Niehrs et al, it appears that the single trans-membrane spanning receptor LRP6 in the Wnt signaling pathway takes this theme up a notch and doesn’t just form a dimer but a large multi-protein oligomer, an in vitro protein aggregate that is an essential for signal transduction in the Wnt pathway. 1
The Wnt signaling proteins, a family of paracrine signaling growth hormones, are crucial to embryogenesis and limb formation in animals and perhaps unsurprisingly has also been implicated in several forms of cancer. Abnormal levels of the Wnt second messenger, Β-catenin, correlate to basal cell carcinoma. The abbreviation Wnt is an aggregation of Wg, as Drosophila Melanogaster flies that had genetic mutations in this pathway were “wingless” and INT, genes found to be involved in vertebrate integration in mice.
At the biomolecular level the Wnt pathway is activated when a Wnt signaling protein binds to two localized receptors; low-density lipoprotein receptor related protein (LRP) and frizzled (FRZ).2 Niehrs specifically analyzed the Wnt3a signaling ligand, which binds to LRP6. After this binding event, LRP6 undergoes phosphorylation by CK1-γ. Once activated, axin binds to the phosphorylated LRP6. For axin to bind to the activated receptor complex it is recruited from a β-catenin degradation complex, that includes axin, GSK-3 and APC; removing axin from this complex results in a loss of this quaternary protein complex’s catalytic power. As a result local Β-catenin concentration increases, permeates into the nucleus and interacts with various TCF/LEF transcription factors, ultimately upregulating gene expression.
Although it was known that Wnt signaling ligands bound to FRZ, it was unclear why it did so as the transduction mechanism appeared to be solely mediated via LRP6 phosphorylation. Why would a cell express two unique receptors, when only one was involved in signal transduction? Furthermore, an additional protein disheveled (Dvl) was implicated in this pathway as a scaffold protein that bound axin and FRZ, but as activated LRP6 also bound axin, the role of Dvl was clearly not well elucidated and seemed somewhat redundant. Additionally, how LRP6 activation occurred by CK1-γ was not clearly defined. With the presence of numerous rogue scaffolding proteins involved in this pathway it was clear that although the overall picture was in focus, the details had still yet to be refined.
Visualizing Wnt signaling by fluorescent labeled Wnt pathway proteins via real-time confocal microscopy, Niehrs found that this pathway involves the formation of ribosome sized aggregates near the cell membrane in Xenopus embryos, HeLa, P19 cells. Niehrs found that these aggregates included: the receptors, LRP6 and frizzled; the sequestered adaptor protein, axin; caveolin, a protein known to be involved in endocytosis and GSK3, an additional protein from the Β-catenin degradation complex and Dvl.
Upon further analysis of these aggregates, Niehrs found that the scaffolding protein Dvl was crucial for the phosphorylation of LRP6. In RNAi knockdown experiments of dvl in MEF cells and the equivalent homolog dsh in Drosophila cells, decreasing dvl levels strongly inhibited phosphorylation of LRP6 through Wnt ligand activation, even though CKI-γ levels were still at a natural level, showing that Dvl is necessary for CK1-γ action. Further experimentation needs to be conducted to determine how Dvl mediates CK1-γ activity towards LRP6; it may act by recruiting CK1-γ to LRP6 or by possibly inducing a conformational change in CK1-γ, inducing activation.
The exciting aspect of this research is the sheer magnitude of the signaling complex. The mass of the signalosome was determined by sucrose density sedimentation via centrifugation, without Wnt3a signaling LRP6 sedimented similarly to a 670 kD protein but upon Wnt3a signaling phosphorylated LRP6 aggregates cosedimented with ribosomal rich fractions. Signal transduction mediated by large-scale protein aggregation is rather unorthodox in the current signal transduction. As a result, our view of signal transduction may need to undergo an expansion.
Most phosphorylated receptors need to undergo endocytosis to remove the phosphate group and return to an inactivated state. Past research by Kikuchi shows that phosphorylated LRP6 undergoes endocytosis by a caveolin-mediated pathway.3 Would this entire complex then undergo endocytosis? It would be interesting to measure the size of these LRP6 endocytosed vesicles, which would allow for an inference of how much of this, as Niehrs states, “LRP6 signalosome” accompanied LRP6. It seems unlikely that the entire aggregate would undergo endocytosis due to the high energy costs associated with forming a lipid bilayer around such a large object. Furthermore, assuming that endocytosis only occurs when the cell wants to terminate the signal, it does not seem plausible that axin would be sequestered, as it would be inhibited from reforming the β-catenin degradation complex.
As this signaling system is crucial to limb formation, it is likely that some sort of gradient or oriented response would be mediated inside the cell. Could the formation of these aggregates, these highly concentrated and localized bundles of activated receptors, be the biomolecular mechanism that governs and orients proper limb formation on an organismal level? Although one can ask numerous questions related to the mechanisms and nature of interactions between these “signalosome” proteins the real issue would be to determine if and how this system creates a gradient of β-catenin into the nucleus and if that then correlates to specific uni-directional cell proliferation.
In highlighting a unique feature in the Wnt signaling pathway, Niehrs discovered a interesting signaling phenomena, which may happen to be the signaling mechanism that mediates oriented limb growth.

References:
1. Wnt Induces LRP6 Signalosomes and Promotes Dishevelled-Dependent LRP6 Phosphorylation. Niehrs et al., Science STKE. June 2007: 1619-1622
2. LDL Receptor-related proteins 5 and 6 in Wnt/B-catenin Signaling: Arrows point the way. Zeng et al. Development. April 2004. 131(8): 1663-77
3. Regulation of Wnt Signaling by Receptor-mediated Endocytosis. Kikuchi et al. J Biochem. April 2007. 141: 443-451