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Wingless-INT (WNT)

What Is Wingless-INT (WNT)?

Wnt proteins are characterized by the presence of a hydrophobic signal sequence followed by an invariant spacing pattern of 22 highly conserved cysteine residues. Most Wnt proteins range in size from about 350-400 amino acids and exhibit a molecular weight in the range of 40 kilodaltons. Two exceptions are Drosophila Wingless and Wnt3/5, which are larger at approximately 52 and 112 kilodaltons, respectively.
Many Wnt proteins are known to be N-glycosylated or possess potential glycosylation sites. However, the function of glycosylation on Wnt proteins is unclear. Mutating N-glycosylation sites individually or in combination on mouse Wnt1 did not affect its ability to signal, and there has been no study thus far that has conclusively demonstrated a function for glycosylation on other Wnt proteins. Since cell surface heparin sulfate proteoglycans (HSPGs) can influence the range of Wnt protein distribution in some tissues, one hypothesis is that Wnt proteins interact with HSPGs via post-translational glycosylation attachments.
Another characteristic of many Wnt family members is post-translational lipid modification. The specific lipid modifications observed on Wnt proteins include a palmitic acid (a saturated 16-carbon fatty acid chain) attachment on the most Nterminal conserved cysteine residue and a palmitoleic acid (monounsaturated 16- carbon fatty acid chain) attachment on a highly conserved serine residue. These sites correspond to cysteine 77 (Cys77) and serine 209 (Ser209), respectively, in mouse Wnt3a.
Lipid modification, or the covalent attachment of fatty acids, has been shown in different instances to regulate the biological activity of signaling proteins by affecting structural stability, membrane targeting, and protein-protein interactions. While it is more common for intracellular proteins to be lipid modified, secreted signaling proteins can also be regulated in this way. Hedgehog is one example of an important signaling molecule that is both secreted and lipid modified. Like Wnt, Hedgehog is used for cell-to-cell communication in a variety of processes during embryonic and larval development. Hedgehog is modified by the addition of cholesterol and palmitic acid. Loss of the cholesterol attachment affects the cell surface localization, range of activity, and asymmetrical trafficking of Hedgehog, while loss of the palmitic acid group reduces the signaling activity of the protein without affecting its processing, secretion, or distribution. Another more recent example of a secreted signaling protein that undergoes lipid modification is the Drosophila EGFR (epidermal growth factor receptor) ligand Spitz. Spitz is modified by the attachment of palmitic acid, which functions to enhance its association with the plasma membrane, thereby restricting its range of activity. 
These examples highlight the fact that altering the biochemical properties of a signaling protein by post-translational lipid modification can impact both its potency and range of signaling activity. This raises interesting questions about the role of lipid modification in Wnt signaling, with respect to both the mechanisms of signal transduction taking place at Wnt-receiving cells and the mechanisms of signal regulation taking place in Wnt-producing cells.

Processing and secretion of Wnt proteins by Wnt-producing cells
The processing and secretion of Wnt proteins is highly regulated, as evidenced by the discovery of several factors dedicated specifically to this task. One model system that has yielded many insights into this process is the fruit fly, Drosophila melanogaster. Genetic screens in Drosophila uncovered the first Wnt pathway member to play an autonomous role in Wnt-producing cells. This gene, porcupine, encodes a multipass transmembrane protein localized to the endoplasmic reticulum (ER). Embryos that lack both maternal and zygotic contributions of Porcupine phenocopy wnt1/wingless (wg) mutant embryos. In the absence of Porcupine, Wg protein is not secreted properly and becomes trapped in Wg-expressing cells. Thus far, Porcupine homologues have been found in C. elegans, Xenopus, mice, and humans. Studies of mouse Wnt3a and Drosophila Wnt5 demonstrate evidence of a conserved requirement for Porcupine in the secretion of various Wnt family members.
Because the absence of Porcupine disrupts the secretion of multiple Wnt proteins, it is hypothesized that lipid modification may be required for Wnt secretion. In addition, mutating the lipid modification site Ser209 to alanine on mouse Wnt3a results in reduced lipid modification as assessed by metabolic labeling with radioactive lipid substrates and a concomitant retention of Wnt3a in the ER. Since lipid modification of Drosophila Wnt1/Wingless is reported to target the protein to intracellular lipid rafts, an interesting hypothesis is that lipid modification might regulate secretion by directing the intracellular trafficking of Wnt proteins through specific membrane subdomains into the proper organelles along the secretory pathway. The trafficking of Wnt proteins along this particular route may be required for their efficient secretion. Regulating protein secretion is not necessarily the only function of lipid modification on Wnt proteins, however. It is possible that lipid modification on the serine residue corresponding to Wnt3a Ser209 is required for secretion, while the modification on the cysteine residue corresponding to Wnt3a C77 serves a different function.

Extracellular Wnt protein distribution
After growth factors such as Wnt proteins are secreted, they must often traverse the tightly packed mass of cells that comprises the developing tissue. Establishing the appropriate extracellular distribution of these signals in the tissue is critical for conveying accurate positional information to the cells receiving the signal. Understanding the mechanism of Wnt protein dispersal in an aqueous extracellular environment is especially interesting in light of the fact that many Wnt proteins are lipid modified.
One Wnt family member that has provided a useful system for addressing this topic is Drosophila Wntl/Wingless (Wg). As its name would suggest, one role of Wingless during development is to pattern the Drosophila wing. During larval development, Wingless functions as a morphogen in the wing imaginal disc primordia. Specifically, Wingless is produced and secreted by a narrow stripe of cells at the presumptive dorsal-ventral boundary of the wing, referred to as the source of the signal. A gradient of Wingless protein that decreases with increasing distance from the source is used to specify different cell fates in a concentration-dependent manner up to many cell diameters away. The existence of a known Wnt morphogen gradient in a model organism such as Drosophila, which is highly amenable to genetic manipulation, and in a tissue such as the larval wing disc, which lends itself well to immunostaining techniques, presents a powerful system for studying the mechanisms that regulate dispersal of Wnt ligands. In addition, Wingless is the only Wnt protein for which a highly effective monoclonal antibody is available for visualizing protein distribution in cells and in tissues.

Transport of Wnt Proteins Between Cells
Wnt proteins function as concentration-dependent molecules which act on longrange neighboring cells. Wnt proteins may also be transported by cytonemes; which are thin filopodial like processes that carry Wnt proteins from signaling cells. In case of C. elegans; mom-3 is required for production of Wnt proteins, however, overall there is not enough evidence throughout other species for specific exporters of Wnt molecules. As the Wnt proteins are secreted, a number of elements can modulate their activity. Evidences suggest that heparin-sulfated forms of proteoglycans (HSPG) plays a role in the transport or stabilization of Wnt. Secreted Wnts may also bind to the members of secreted Frizzled-related protein (SFRP) family and Wnt inhibitory factor (WIF) family. Both SFRPs and WIFs are thought to function as extracellular Wnt inhibitors.



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