Vangl2 is a core component of PCP proteins, which are involved in cell-signaling that acts to divide and move cells along the tissue axis. Vangl2 comprises four transmembrane regions with both terminals exposed to the cytosol (Fig. 1A) (Tissir & Goffinet, 2013; Yoshioka et al., 2013). In a previous report, we showed that Vangl2 is involved in bidirectional regulation of neuronal dendritic branching; a Vangl2N deletion mutant showed a significant reduction in dendritic branching and spine density (Hagiwara et al., 2014). Therefore, in this report, we searched for molecules that interact with Vangl2N to better understand the molecular mechanism underlying neural dendritic development. From the Y2H screening, we obtained 61 candidate genes including pre- and post-synaptic proteins (Table 1) and confirmed the Vangl2N specific binding to adaptor protein Ap2m1(Fig. 1, 2, 3). Furthermore, shRNA KD of Ap2m1 and KD of Vangl2N resulted in less dendritic branching, although spine density increased after Ap2m1 KD (Fig. 4). From these results, we suggest that dendritic development associated with the clathrin-mediated endocytosis could be directed by the PCP pathway.
The C-terminal region of Vangl2 has multiple domains, including the looptail mutations D255E, S464N, and R259L, which cause neural tube defects, as well as interaction sites with numerous proteins, including Prickle (Pk) and PSD-95 (Yoshioka et al., 2013; Nagaoka et al., 2015; Bailly et al., 2018). Binding of Pk2 to the PCP protein binding region (Prickle binding domain; PKBD) of Vangl2 is required for dendritic spine formation (Nagaoka, Ohashi, et al., 2014). Considering synaptic proteins, Vangl2 forms a complex with PSD-95 through a conserved PDZ binding motif, which is required for localization of Vangl2 to the dendritic spines (Yoshioka et al., 2013). Thus, interacting molecules and functional roles of Vangl2 C-terminus have been investigated in terms of neuronal morphology. However, even though the N-terminus has an essential role in neuronal dendritic development, interaction at the N-terminal region has not been well demonstrated (Hagiwara et al., 2014).
In mice, cortical layer 2/3 neurons have immature dendrites at P3, and apical and basal dendrites become longer at P5 and more complex by P15 (Hoshiba et al., 2016). Because Vangl2 expression gradually decreases during this dendritic morphogenesis during the first 1–2 weeks (Yoshioka et al., 2013), we screened molecules interacting with the N-terminal region of Vangl2 using P9 mouse brain cDNA, and then identified Ap2m1 as a binding partner. In the pull-down assay using GST fused Vangl2 and Ap2m1-expressing HEK293T lysates, Ap2m1 strongly bound to Vangl2N and weakly bound to Vangl2C (Fig. 1C and 3B). Consistent with these data, in the pull-down assay using GST fused Ap2m1 and Vangl2-expressing HEK293T lysates, Ap2m1 bound to not only Vangl2ΔC but also Vangl2ΔN (Fig. 2B). These results suggested Ap2m1 preferentially bound to the N-terminus of Vangl2, although it bound to both the N- and C-terminal regions. A previous study showed that Ap1m1, an AP-1 subunit, binds to the C-terminal YYXXF motif of Vangl2, which is required for Vangl2 transport from the trans-Golgi network with the GTP-binding protein, Arfrp1 (Guo et al., 2013). In the present study, we showed that compared with GST-Vangl2-4 (corresponding to PKBD), Ap2m1 preferably interacted with the Vangl2 C-terminal, and hardly interacted with GST-Vangl2-3, which contains a YYXXF motif (279-283aa) (Fig. 3B). Therefore, the functional role of the interaction between AP-2 and Vangl2C might differ from that between AP-1 and Vangl2C.
Furthermore, in hippocampal neurons, binding of Vangl2 to N-cadherin through PKBD enhances its internalization in a Rab5-dependent manner (Nagaoka, Ohashi, et al., 2014), while in epithelial cells binding to E-cadherin is similar (Nagaoka, Inutsuka, et al., 2014). Because several endocytic adaptors including AP-2, Dab-2, and Numb associate with E-cadherin for the internalization of clathrin-mediated endocytosis (Bruser & Bogdan, 2017), AP-2 may play a role in Vangl2-related N-cadherin internalization by binding to Vangl2 and/or N-cadherin.
Because disruption of PCP genes, including Vangl2, Dishevelled1, and Dapper-antagonist of catenin-1 in mice, cause reduced dendritic arborization in neurons (Rosso et al., 2005; Okerlund et al., 2010, 2016; Hagiwara et al., 2014), the Wnt/PCP pathway is essential for the formation of the dendrite, dendritic spine, and excitatory synapse. In this study, we showed that Ap2m1 KD also decreased dendritic length, the total number of tips, and the branch number in cortical neurons and the Vangl2 KD (Fig. 4). Clathrin-mediated endocytosis (CME) regulated dendritic growth by mediating the internalization of receptors. Indeed, KD of the clathrin assembly protein CALM reduced dendrite length and the overall complexity of the neurites in cultured hippocampal neurons (Bushlin et al., 2008). KD of AP2b1, a subunit of the AP-2 complex, lowered the total number of dendritic tips in rat hippocampal neurons (Koscielny et al., 2018). Consistent with these results, the direct interaction between Vangl2 and Ap2m1 suggests that the Wnt/PCP pathway and CME functionally coordinate the development of dendritic morphology.