We investigated the potential role of PtdIns(4,5)P2
in LRP6 signalosome formation by examining whether endocytic structural proteins are part of the LRP6 signalosomes, given PtdIns(4,5)P2
has an important role in endocytosis (Di Paolo and De Camilli, 2006
; Poccia and Larijani, 2009
), a process also implicated in Wnt signal transduction (Blitzer and Nusse, 2006
; Yamamoto et al., 2006
; Cruciat et al., 2010
). Sucrose gradient centrifugation of HEK293 cell lysates was performed to isolate the LRP6 signalosomes as previously described (Bilic et al., 2007
; Pan et al., 2008
). Four heavy fractions that contained the signalosomes and four light fractions as a control were pooled (). LRP6 from these two pools was immunoprecipitated using an LRP6 antibody. As a positive control (Bilic et al., 2007
), Axin1 was detected only in the immunocomplexes from the heavy fraction pool, albeit more LRP6 protein was in the input and immunocomplex of the light fraction pool than those of the heavy pool (). Like Axin1, clathrin heavy chain (CHC), AP2 α and µ subunits were also exclusively detected in the immunocomplexes from the heavy fraction pool (). To the contrary, caveolin-1 was detected in the immunocomplexes from both heavy and light fraction pools, whereas early endosome antigen 1 (EEA1) was not detected in the heavy pool immunocomplex (). In agreement with coimmunoprecipitation of CHC or AP2 with LRP6 in the heavy fraction pool, we detected the presence of CHC and AP2µ in the heavy fractions of sucrose density gradient centrifugation (). Moreover, LRP6 and Axin1 were only detected in the immunocomplexes pulled down from the heavy fraction pool (). WNT3A induced PtdIns(4,5)P2
accumulation that was primarily localized in the heavy fractions and could be rapidly diminished by rapamycin-induced recruitment of the FKBP and INPP PtdIns 5′-phosphatase fusion protein to membrane-bound FRB (Fig. S1 A
; Pan et al., 2008
). Elimination of PtdIns(4,5)P2
also abrogated the presence of LRP6 (Pan et al., 2008
) as well as CHC and AP2 in the heavy fractions (). Furthermore, LPR6 coimmunoprecipitated with CHC, AP2α, or AP2µ in a ligand-dependent manner in whole-cell extracts (Fig. S1, B and C). These interactions strengthened as the function of Wnt stimulation and were correlated with intensity of LRP6 phosphorylation (Fig. S1 B). Importantly, the interaction between CHC and LRP6 could be disrupted by PtdIns(4,5)P2
hydrolysis (Fig. S1 C). These results together support a conclusion that LRP6 and endocytic structural proteins clathrin and AP2 form a complex that appears to share the characteristics of the LRP6 signalosome.
Figure 1. Interaction of LRP6 with CHC and AP2 in its signalosome. (A and B) CHC or AP2 interacts with the LRP6 aggregates. Cells were treated with 50 ng/ml WNT3A for 3 h, and fraction aliquots were analyzed by Western blot (A). Fractions 1–4 (H) and 8–11 (more ...)
Next, we assessed the importance of clathrin and AP2 in WNT3A-induced formation of the LRP6 signalosome. Knockdown of either CHC or AP2µ () reduced the amount of LPR6 detected in the heavy fractions of sucrose density gradient centrifugation (), suggesting that CHC and AP2 are important for the formation of the LRP6 signalosome. However, the presence of CHC in the heavy fractions was not affected by the knockdown of LRP5/6 (), even though LRP5/6 knockdown abrogated WNT3A-induced LRP6 phosphorylation () and β-catenin accumulation (). On the other hand, knockdown of Dvl1/2/3, Fz2/4/5, or PIP5K1, which were previously shown to reduce Wnt-stimulated PtdIns(4,5)P2
accumulation in HEK293 cells (Pan et al., 2008
), decreased the presence of CHC in the heavy fractions (), suggesting that the presence of CHC in the heavy fraction may depend on PtdIns(4,5)P2
. Both AP2 α and µ subunits bind to PtdIns(4,5)2
, which is required for the formation clathrin-coated pits during endocytosis (Haucke, 2005
; Ohno, 2006
). Consistent with this knowledge, knockdown of AP2µ reduced the presence of CHC in the heavy fractions (). Therefore, we concluded that clathrin and AP2 were required for the formation of LRP6 signalosomes. Consistent with the importance of LRP6 signalosome in LRP6 phosphorylation and β-catenin stabilization, siRNA-mediated knockdown of CHC or AP2µ significantly inhibited WNT3A-induced LRP6 S1490 phosphorylation (), β-catenin accumulation (), and Wnt reporter gene activity (Fig. S1 D).
Figure 2. Requirement of clathrin and AP2 for LRP6 signalosome formation and Wnt signaling. Cells were transfected with siRNAs, treated with 50 ng/ml WNT3A for 3 h, and subjected to Western analysis (A), sucrose density gradient centrifugation (B and C), or a β-catenin (more ...)
AP2, in addition to its role in PtdIns(4,5)P2
-dependent clathrin coat assembly, recognizes cargos through conserved sequences that include the YXXΦ (X, any amino acid; Φ, a bulky hydrophobic residue [Leu, Ile, Met, or Phe]) motif (Ohno et al., 1995
; Boll et al., 1996
). Examination of amino acid sequences of the intracellular domains of LRP5 and 6 revealed that LRP6 contains one and LRP5 contains three such motifs (Fig. S2, A and B
). In addition, these motifs are highly conserved across species (Fig. S2, A and B). Mutating Tyr-1522, the Y residue in the LRP6 motif, disrupted the interaction of LRP6 intracellular domain with AP2 (). The AP2µ mutations (D176A,W421A) that disrupt its interaction with the YXXΦ motif also disrupted its interaction with LRP6 intracellular domain (). Together with the observation that cells expressing the mutant LRP6 (Y1522A) had reduced Wnt signaling activities (), we concluded that the YXXΦ motif has an important role in AP2 interaction and Wnt signal transduction.
Figure 3. Role of Tyr-1522 in Wnt signaling. (A) The interaction of LRP6 C terminus with AP2µ depends on the YXXΦ motif. Cells were transfected with plasmids encoding LRP6 intracellular domain (CT), AP2µ, or their mutants as indicated. Immunoprecipitation (more ...)
In cells expressing exogenous LRP6, the signalosomes could be observed by confocal microscopy at the plasma membranes (Bilic et al., 2007
; Pan et al., 2008
). To determine how much of the endogenous LRP6 signalosomes are localized at cell surface, we used a reversible cell surface protein biotinylation approach as described in Fig. S2, C and D. We found that cell surface LRP6, rather than internalized LRP6, was detected in the heavy fractions of sucrose density gradient centrifugation (). This result suggests that LRP6 signalosomes are primarily localized at the cell surface. Next, we examined the effect of WNT3A on overall LRP6 internalization using a similar biotinylation approach, but without the step of sucrose density gradient centrifugation (Fig. S2 C), and observed no significant decreases in surface LRP6 contents or increases in internalized LRP6 contents in HEK293 cells after Wnt stimulation ( and Fig. S2 E). There was a low level of constitutive LRP6 internalization, but independently of Wnt stimulation ( and Fig. S2 E). In addition, the increases in the levels of phosphorylated LRP6 in whole-cell lysates or on cell surfaces could be detected even at 5 min of WNT3A treatment, but there were little increases in the phosphorylation of internalized LRP6 (). This result is consistent with the idea that LRP6 phosphorylation may primarily occur at the cell surface (some internalized phosphorylated LRP6 was detected at 3 h of WNT3A stimulation in Fig. S2 E, which may be due to the constitutive internalization). In contrast to LRP6, cell surface EGF receptors rapidly disappeared or were internalized upon EGF stimulation (Fig. S2 F). Of note, Wnt treatment did not affect EGF receptor endocytosis, nor did EGF treatment affect LRP6 endocytosis (Fig. S2 F). We also performed the biotinylation experiments in mouse embryo fibroblast (MEF) cells, and the results were the same as those in the HEK293 cells (Fig. S2 G). Thus, these data collectively indicate that LRP6 signalosomes are primarily localized at cell surfaces and that WNT3A does not induce significant LRP6 internalization in HEK293 and MEF cells.
Figure 4. WNT3A-induced LRP6 signalosomes are primarily localized at cell surfaces. (A) LRP6 signalosomes are localized at cell surfaces. HEK293 cells were stimulated with 50 ng/ml WNT3A and labeled with biotins, followed by sucrose density gradient sedimentation. (more ...)
Knowing that PtdIns(4,5)P2
hydrolysis is required for the late steps of clathrin-mediated endocytosis (Krauss and Haucke, 2007
; McMahon and Boucrot, 2011
), we hypothesized that the high concentrations of PtdIns(4.5)P2
formed upon Wnt stimulation might prevent LRP6 from being internalized. Consistent with this hypothesis, artificial induction in PtdIns(4,5)P2
hydrolysis using the rapamycin-inducible system in cells pretreated with WNT3A led to a reduction in the cell surface LRP6 (). Given that the short-term rapamycin treatment did not affect total or phosphorylated LRP6 contents in the whole-cell extracts (), we conclude that the reduction in the cell surface LRP6 by rapamycin is due to internalization.
Consistent with the biochemical evidence that WNT3A does not induce marked LRP6 internalization, WNT3A did not visibly reduce LRP6 staining at the surfaces of MEF cells examined by confocal fluorescence microscopy (). The specificity of LRP6 staining was confirmed by siRNA-mediated knockdown ( and Fig. S3 A
). In addition, Wnt treatment increased apparent cell surface CHC staining compared with mock or serum treatment (). We used one of the super-resolution fluorescence microscopic techniques, stochastic optical reconstruction microscopy (STORM; Rust et al., 2006
; Huang et al., 2008b
), to gain a higher resolution view of the staining at cells surfaces. Given the thickness of MEF cells and the relatively high nonspecific fluorescence background signal, we focused on the bottom surfaces that are more proximal to the objective. WNT3A induced appreciable clustering of LRP6 () and significant cluster size distribution shift toward larger size clusters upon Wnt treatment (Fig. S3 B). Knockdown of LRP6 significantly reduced the number of LRP6 clusters (; and Fig. S3, A and B).
Figure 5. Imaging of WNT3A-induced LRP6 and clathrin clusters at cell surface. (A) WNT3A treatment does not reduce membrane localization of LRP6. MEF cells were transfected with the control siRNA (siCtr) or LRP6 siRNA (siLRP6) for 2 d followed by serum starvation (more ...)
In serum-starved or -treated MEFs stained for clathrin, STORM revealed numerous clathrin patches with morphology and sizes (100–200 nm) consistent with those of endocytic clathrin-coated pits (). There were also a small number of larger patches of clathrin staining (>200 nm), which may be the previously described clathrin-coated plaques (Kirchhausen, 2009
). WNT3A treatment significantly increased the number of clathrin plaques, especially those with sizes greater than 400 nm (; and Fig. S3 C). Knockdown of AP2µ reduced the numbers of the large LRP6 clusters and clathrin plaques (Fig. S3, D–H), which is consistent with the important roles of AP2 in the formation of clathrin-coated structures and LRP6 signalosomes. Moreover, in agreement with the results of sucrose density gradient centrifugation (), knockdown of LRP6 did not affect the number of the large clathrin plaques (Fig. S3, F–H). This result once again supports the conclusion that clathrin clusters form upstream and independently of LRP6 cluster formation. In addition, these results support the conclusion that WNT3A may induce the aggregation of LRP6 and clathrin at the cell surface.
Next, we wanted to image the cell surfaces exposed to the culture medium rather than those attached to the coverslip, as the attachment to the coverslip glass surface may induce artifacts. We have previously imaged through BS-C-1 cells, a monkey kidney cell line with a thinner morphology, by 3D STORM (Huang et al., 2008a
). WNT3A could stimulate LRP6 phosphorylation, while not inducing marked LRP6 internalization, in these cells (Fig. S3 I). We performed 3D STORM imaging of BS-C-1 cells stained for LRP6 or clathrin. Similar to MEF cells, we observed increases in LRP6 and clathrin cluster sizes at both top and bottom surfaces of cells treated with WNT3A (; and Fig. S3 J). We also found that knockdown of Dvls blocked the formation of these clusters (Fig. S3 K). Using the two spectrally distinct photoswtichable dyes Alexa Fluor 647 and Atto 488 for STORM (Dempsey et al., 2011
), we also performed 3D imaging of BS-C1 cells co-stained for LRP6 and clathrin light chain (CLC) and observed some colocalization of LRP6 and clathrin clusters at cell surfaces (). The fractional colocalization of LRP6 with clathrin clusters increased substantially upon Wnt stimulation, from 6% in mock-treated cells to 42% upon stimulation for LRP6 clusters greater than 100 nm in size. The change was less pronounced for the small LRP6 clusters (from 10 to 30%).
In this study, we used biochemical and imaging approaches to characterize the LRP6 signalosome. Our results suggest that clathrin and AP2 may be the structural components of the LRP6 signalosomes and recruited to form a complex by Wnt-induced PtdIns(4,5)P2
, which in turn recruits LRP6 (Fig. S3 L). Despite the fact that the formation of this signalosome does not depend on LRP6, we continue to refer the complex in this article to as “LRP6 signalosome” both for the historic reason and for the reason that the downstream signaling events tested in this study depend on LRP6 aggregation. In HEK293 cells, MEF cells, and BS-C-1 cells, the LRP6 signalosomes seem to be primarily localized at cell surfaces, and WNT3A does not strongly induce LRP6 internalization in contrast to observations made in some tumor cell lines (F9 and Hela cells) or in cells overexpressing LRP6 (Yamamoto et al., 2006
; Jiang et al., 2012
; Sakane et al., 2012
). This apparent discrepancy in LRP6 internalization may be due to the difference in the stoichiometry of LRP6 signalosome components among these cells. LRP6 signalosomes in overexpressed cells were previously observed at the cell surface, but a balanced coexpression of Fz, Dvl, and Axin was required (Bilic et al., 2007
; Pan et al., 2008
). Thus, it is possible that LRP6 that cannot be incorporated into the signalosomes may be internalized. The second possible explanation is the differences in the kinetics of PtdIns(4,5)P2
metabolism among these cells, because the presence of LRP6 signalosomes on cell surfaces depends on high levels of PtdIns(4,5)P2
. Another possibility is differences in the levels of various endocytic components, which may skew LRP6 to caveolin-dependent endocytosis (Yamamoto et al., 2006
; Jiang et al., 2012
). Nevertheless, the failure of WNT3A to induce clear LRP6 internalization in the three cell lines we tested in this study suggests that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization. However, our results do not exclude the possibility that vesicle transport or even endocytic vesicle transport has a role in Wnt–β-catenin signaling.
In addition to LRP5/6–AP2 interaction, Dvl also interacts with AP2 (Yu et al., 2007
), which may provide another link of the signalosome to the clathrin-coated structure. It would be of interest to know whether this Dvl–AP2 interaction has a role in LRP6 signalosome formation and Wnt–β-catenin signaling, even though this link is important for noncanonical Wnt signaling. On the subject of noncanonical Wnt signaling, WNT5A, a prototypical noncanonical Wnt, can also stimulate PtdIns(4,5)P2
formation (Grumolato et al., 2010
) and Dvl aggregation (Nishita et al., 2010
). These findings together raised a possibility that PtdIns(4,5)P2
may also be involved in signalosome formation in noncanonical Wnt signaling. Thus, CHC/AP-2/PtdIns(4,5)P2
-dependent signalosomes, which may be more appropriately referred to as Wnt signalosomes, may be formed upon all types of Wnt stimulation, but the recruitment of the Wnt co-receptors to these complexes may vary depending on the nature of Wnt proteins (Nishita et al., 2010
). In this regard, it is also important to determine whether Fz and Dvl as well as other signalosome components, whose number has been growing (MacDonald et al., 2009
; Wu and Pan, 2010
; Tanneberger et al., 2011
), have a structural role in the signalosome formation and how these signalosome components are organized in the complex.