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Fig. 6
Lypd6 Knockdown or Mislocalization to Nonraft Membrane Domains Shifts Lrp6 Phosphorylation to These Domains and Inhibits Wnt Signaling
(A) pBAR activity in HEK293T cells transfected with Wnt8 (20 ng) plus spGFP-Lypd6 (100 ng) or spGFP-Lypd6ΔGPI-CD44 TMD (95 ng) or equimolar amounts of spGFP-GPI control. Error bars, SEM.
(B) Classes of phenotypes in wnt8-overexpressing embryos injected with wnt8 (20 pg) plus spGFP-lypd6 (150 pg) or spGFP-lypd6ΔGPI-TfR TMD (140 pg) or equimolar amounts of spGFP-GPI control RNA. spGFP-lypd6ΔGPI-TfR TMD significantly rescues Wnt8-induced phenotypes. Class 5, hyperdorsalization.
(C) spGFP-Lypd6ΔGPI-CD44 TMD reduces Wnt3a-induced phosphorylation of Lrp6 at S1490 in HEK293T cells assayed at 6 hr poststimulation with Wnt3a CM.
(D) lypd6 esiRNA reduces Wnt3a-induced Lrp6 phosphorylation in HEK293T cells.
(E) Fractionation of plasma membrane derived from HEK293T cells treated with Wnt3a into soluble and detergent-resistant fractions (DRMs). Endogenous TfR2 (a marker for soluble fractions), Caveolin-1 (Cav1, marking DRMs), P-Lrp6 (S1490), Lrp6, and overexpressed GFP in spGFP-Lypd6 (5 μg) or spGFP-Lypd6ΔGPI-TfR TMD (4.6 μg) or control spGFP-GPI (2.9 μg) transfected cells were detected by western blotting.
(F) Fractionation of plasma membrane derived from HEK293T cells transfected with 2 μg EGFP control or lypd6 esiRNAs and treated with Wnt3a CM into soluble and DRM phases.
(G) Imaging and quantification of Wnt3a- or Dkk1-induced Lrp6 speckles in HEK293T cells stably expressing Lrp6-EGFP. Scale bar, 10 μm.
See also Figure S5.
Reprinted from Developmental Cell, 26(4), Özhan, G., Sezgin, E., Wehner, D., Pfister, A.S., Kühl, S.J., Kagermeier-Schenk, B., Kühl, M., Schwille, P., and Weidinger, G., Lypd6 Enhances Wnt/beta-Catenin Signaling by Promoting Lrp6 Phosphorylation in Raft Plasma Membrane Domains, 331-345, Copyright (2013) with permission from Elsevier. Full text @ Dev. Cell