Abstract
In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.
Highlights
In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical– basal axis, a process mediated by several conserved signaling receptors
Our results suggest that VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the trans-Golgi network (TGN), which may contribute to their asymmetric localizations
We previously demonstrated that TGN export of Vangl2 and Fzd6 depends on distinct cargo sorting machineries
Summary
In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical– basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled (Fzd6), are located asymmetrically on opposite boundaries of the cell. Our results suggest that Vangl and Frizzled are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations. Corresponding to this, the alignment of noncentrosomal microtubules are reorganized so that the majority of noncentrosomal microtubules are aligned along the proximal– distal axis with an excess of the plus ends oriented distally prior to the onset of the PCP signaling events [13, 14, 17] This microtubule dynamics are controlled by Dachsous and Fat implicating that the Dachsous/Fat/Four-jointed pathway may provide long-range directional information to reorganize the microtubule cytoskeleton for polarized delivery of PCP proteins [17]
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