Editors' ChoiceCell Biology

Enhancing Wnt Signaling in Rafts

Science Signaling  03 Sep 2013:
Vol. 6, Issue 291, pp. ec210
DOI: 10.1126/scisignal.2004689

Binding of the ligand Wnt to its transmembrane receptor Frizzled and co-receptor low-density lipoprotein receptor–related 5 or 6 (Lrp5/6) results in phosphorylation of Lrp5/6 and subsequent internalization of the ligand-receptor complex, leading to intracellular signaling that culminates in nuclear accumulation of the transcription factor β-catenin. Lrp6 is found throughout the plasma membrane, but it is specifically phosphorylated in organized membrane raft domains (lipid rafts) in which the receptor complexes cluster before being endocytosed. Özhan et al. report that the Ly6 family member Lypd6 promotes Wnt signaling by stimulating phosphorylation of Lrp6 specifically in rafts. Ly6 proteins are either secreted or anchored to the plasma membrane through covalently linked glycosylphosphatidylinositol (GPI) moieties, and they participate in diverse processes, including immune cell function, cell adhesion, and neurotransmitter release. Wnt/β-catenin signaling promoted expression of lypd6 in zebrafish embryos, and lypd6 was required for Wnt8-mediated cell-fate specification in various tissues. Overexpression of lypd6 in fish embryos increased the severity of wnt8 and wnt3a overexpression phenotypes, and morpholino-mediated knockdown of lypd6 caused phenotypes similar to those caused by wnt8 knockdown and reduced the expression of endogenous Wnt target genes. Similar to some other members of the Ly6 family, Lypd6 contains a secretion signal and a GPI attachment site that mediates membrane anchorage. The authors generated fluorescently tagged versions of Lypd6 (GFP-Lypd6) that lacked the GPI attachment site (GFP-Lypd6-ΔGPI) or had the GPI attachment site replaced with the transmembrane domain of human CD44 (GFP-Lypd6-ΔGPI-CD44). GFP-Lypd6 and GFP-Lypd6-ΔGPI-CD44 localized to the plasma membrane in fish embryos and CHO cells and coimmunoprecipitated with Lrp6 in HEK 293T cells, but GFP-Lypd6-ΔGPI was secreted and did not coimmunoprecipitate with Lrp6. Therefore, the ability of Lypd6 to interact with Lrp6 did not depend on the mode of Lypd6 membrane localization. In plasma membrane vesicles generated from CHO cells, GFP-Lypd6 localized to lipid rafts, whereas Lypd6-ΔGPI-CD44 localized to disordered membrane regions, indicating that the GPI anchor was important for restricting Lypd6 to rafts. Whereas GFP-Lypd6 promoted expression of Wnt target genes and worsened wnt8 overexpression phenotypes in cultured cells and fish embryos, Lypd6-ΔGPI-CD44 had the opposite effect, implying that Lypd6 enhances Wnt signaling only when it is localized to lipid rafts. Finally, experiments with detergent-resistant membrane fractions from Wnt3a-stimulated HEK 293T cells indicated that GFP-Lypd6 promoted phosphorylation of Lrp6 in rafts but that GFP-Lypd6-ΔGPI-CD44 promoted Lrp6 phosphorylation only outside of rafts. These results are consistent with a model in which Lypd6 is both a transcriptional target and positive regulator of Wnt signaling that restricts Lrp6 phosphorylation to membrane rafts, from which it is internalized as part of the active receptor signaling complex.

G. Özhan, E. Sezgin, D. Wehner, A. S. Pfister, S. J. Kühl, B. Kagermeier-Schenk, M. Kühl, P. Schwille, G. Weidinger, Lypd6 enhances Wnt/β-catenin signaling by promoting Lrp6 phosphorylation in raft plasma membrane domains. Dev. Cell 26, 331–345 (2013). [Online Journal]