Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.
Most cells in multicellular organisms possess a property known as polarity that is reflected, in part, in the organization of the cell surface into distinct domains. One well-known axis in epithelial cells, such as those in the skin, divides the cell into an apical domain, which faces out, and a basal domain, which faces the underlying tissue. These cells rely on the distribution of structural components inside the cell, or within the cell membrane, to tell the difference between these two directions. Epithelial cells also possess a second type of polarity, planar cell polarity, that ensures that cells adjacent to each other in the plane parallel to the skin tissue are oriented correctly with respect to each other during development. This ensures, in turn, that hairs, scales, feathers and so on are all aligned.
All eukaryotic cells sort and process proteins within an organelle called the Golgi apparatus, and proteins that are required at a specific destination within the cell, such as the cell surface membrane, carry specific molecular sorting signals that act as address labels to convey the protein into and within the secretory pathway. As one of these proteins moves through the Golgi apparatus, its sorting signals are recognized by coat proteins, such as clathrin, that subsequently form a vesicle around it. The assembly of this vesicle is initiated by an enzyme from the Arf family, but the enzyme must first undergo a conformational change (by exchanging a molecule of GDP for one of GTP) before formation can begin. The resulting vesicle can then be sent on its way to the address indicated by its Golgi-to-cell-surface sorting signal. These sorting signals also help to establish planar cell polarity in cells by ensuring that proteins called signaling receptors are distributed asymmetrically within the cell membrane.
Guo et al. have now examined the mechanism behind the asymmetric sorting of two proteins that are involved in planar cell polarity: Vangl2 and Frizzled 6. In an effort to understand why these proteins are localized to opposite surfaces of epithelial cells, Guo et al. used genetic techniques to reduce the expression of Golgi-localized Arf proteins in epithelial cell cultures. They found that knockdown of a protein called Arfrp1 caused Vangl2 to accumulate in the last station of the Golgi complex instead of being transported to the cell surface membrane. Then, using a technique called affinity chromatography, they demonstrated that a coat protein called the clathrin adaptor complex (AP-1) had to be present for the formation of vesicles around Vangl2. Moreover, disrupting AP-1 and Arfrp1 did not prevent Frizzled 6 being transported to the cell surface membrane. This suggests that cells use several distinct adaptor proteins and coat complexes to ensure that proteins from the Golgi apparatus go to specific locations on the cell surface and, thus, help to establish planar cell polarity.