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. 2015 Aug 10:5:12916.
doi: 10.1038/srep12916.

PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2

Tadahiro Nagaoka  1 , Katsuhiko Tabuchi  2 , Masashi Kishi  1
Affiliations

PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2

Tadahiro Nagaoka et al. Sci Rep. .

Abstract

Postsynaptic density-95/Discs large/Zonula occludens-1 (PDZ) domain-mediated protein interactions play pivotal roles in various molecular biological events, including protein localisation, assembly, and signal transduction. Although the vertebrate regulator of planar cell polarity Van Gogh-like 2 (Vangl2) was recently described as a postsynaptic molecule with a PDZ-binding motif, the role of its PDZ interaction at the synapse is unknown. In this report, we demonstrate that the PDZ interaction was dispensable for the normal cluster formation of Vangl2 and not absolutely required for the synapse-associated localisation of Vangl2 in cultured hippocampal neurons. We further showed that the synaptic localisation of Vangl2 was categorised into two types: overlapping co-localisation with postsynaptic density (PSD)-95 or highly correlated but complementary pattern of association with PSD-95. Only the former was significantly sensitive to deletion of the PDZ-binding motif. In addition, the PDZ interaction enhanced the protein interactions between PSD-95 and Prickle2, which is another planar cell polarity factor that is localised at the postsynaptic density. Taken together with our recent report that the density of PSD-95 clusters was reduced in Vangl2-silenced neurons, these results suggest that Vangl2 determines the complex formation and clustering of postsynaptic molecules for synaptogenesis in mammalian brains.

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Figures

Figure 1
Figure 1. Interaction of Van Gogh-like 2 (Vangl2) with other postsynaptic density (PSD) proteins.
(a, b) HEK293T cells were transfected with the indicated expression constructs, and the cell lysates as well as immuno-precipitates (IP) were analysed by western blotting (WB) with the indicated antibodies (Abs). IP was performed with either α-FLAG (a) or α-green fluorescent protein (GFP) (b) Abs. 0.5% of the cell lysate was loaded as Input. IB: immunoblot. (c) Bar graphs showing the precipitated ratio of Prickle2 in the WB analyses presented in (b). Note the robust enhancement of the complex formation between Prickle2 and PSD-95 by Vangl2 wild type (WT) but not by the ΔETSV. The amount of Prickle2 in the cell lysate was highly sensitive to the introduction of Vangl2. The data are presented as mean ± standard deviation (SD). Significant differences (p < 0.05) versus control groups that were calculated with one-way ANOVA followed by Tukey’s multiple comparisons test are marked with *. **p < 0.005. a.u.: arbitrary unit.
Figure 2
Figure 2. Punctate and synaptic localisations of Vangl2 with or without PDZ-binding motif.
(a) Rat hippocampal neurons were transfected with the indicated expression plasmids (upper: GFP-Vangl2 WT, middle: GFP-Vangl2 ΔETSV, lower: mGFP) and cultured for 21 days. GFP fluorescence (green), α-PSD-95 immunofluorescence (IF; red) as well as their merged images are presented. Note the punctate localisation of GFP-Vangl2 ΔETSV and the mostly flat signals of mGFP. (b) Bar graphs showing the density of the GFP puncta that were formed by the indicated expression constructs along the dendrites. Note the similar levels of puncta formation between GFP-Vangl2 WT and ΔETSV. (c) Bar graphs showing the ratio of the PSD-95-associated population of formed GFP puncta for each expression construct. Note that the clusters of Vangl2 ΔETSV are more associated with PSD-95 than those of the simply membrane-tethered protein (mGFP). The highly magnified images of the delimited region are shown in the respective lower panels. Some examples of the co-localised puncta are indicated with arrowheads. The data are presented as mean ± SD. Significant (p < 0.05) and insignificant differences versus control groups that were calculated with one-way ANOVA followed by Tukey’s multiple comparisons test are marked with * and n.s., respectively. *p < 0.05, **p < 0.005, ***p < 0.0001. Scale bars: 20 μm and 5 μm for the upper and lower panels, respectively.
Figure 3
Figure 3. Identification of the three types of Vangl2 clusters in terms of association with PSD-95.
(a) The overall distribution of GFP-Vangl2 WT puncta on the dendrites of electroporated neurons in culture. An example of each type of punctum is indicate and marked with a capitalized letter (O: overlapped, C: complementary, U: unassociated). (b,c,d) The highly magnified images of the GFP signals of each GFP-Vangl2 WT punctum (green) merged with those of the IF signals of PSD-95 (red). Some examples of puncta with overlapped (b), highly associated but complementary (c) and essentially unassociated (d) localisation are presented. The puncta delimited in (a) are shown in the upper most row. (e,f,g) Plot-profile analysis of one of the merged images shown in (b,c,d). The images in the lowest row were analyzed. Note that the PSD-95 signals occupy the hollow of the GFP signals in this particular example (f). “O”s and the white lines shown in (b), (c) and (d) indicate the origins and tracks of the plot-profile analyses presented in (e), (f) and (g), respectively. Scale bars: 1 μm.
Figure 4
Figure 4. Statistical analysis of the synaptic localisations of Vangl2 with or without PDZ-binding motif.
(a) Overall distribution of GFP-Vangl2 ΔETSV and PSD-95 puncta on the dendrites of electroporated neurons in culture. An example of each type of punctum is indicated and marked with a capitalized letter (O: overlapped, C: complementary, U: unassociated). (b) The highly magnified images of the indicated regions in (a), showing the overlapped, complementary and unassociated localisation with PSD-95 IF. (c) Bar graphs showing the ratio of the GFP puncta with overlapped co-localisation with PSD-95. Note the significant reduction in the overlapped ratio by the deletion of PBM (ΔETSV). (d) Bar graphs showing the ratio of GFP puncta with a complementary pattern of association with PSD-95. Note that the deletion of the PBM did not affect the occurrence of the complementary association. (e) Bar graphs showing the ratio of GFP puncta without association with PSD-95. Scale bars: 20 μm (a) and 5 μm (b). The data are presented as mean ± SD. Significant (p < 0.05) and insignificant differences versus control groups that were calculated with one-way ANOVA followed by Tukey’s multiple comparisons test are marked with * and n.s., respectively. *p < 0.05, **p < 0.005, ***p < 0.0001.
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References

    1. Feng W. & Zhang M. Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density. Nat Rev Neurosci 10, 87–99, 10.1038/nrn2540 (2009). - DOI - PubMed
    1. Okabe S. Molecular anatomy of the postsynaptic density. Mol Cell Neurosci 34, 503–518, 10.1016/j.mcn.2007.01.006 (2007). - DOI - PubMed
    1. Sheng M. & Kim E. The postsynaptic organization of synapses. Cold Spring Harb Perspect Biol 3, 10.1101/cshperspect.a005678 (2011). - DOI - PMC - PubMed
    1. Songyang Z. et al. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275, 73–77 (1997). - PubMed
    1. Kornau H. C., Schenker L. T., Kennedy M. B. & Seeburg P. H. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 1737–1740 (1995). - PubMed

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