User:Kinkreet/MCBII/Wnt

Wnt signalling is heavily involved in sexual reproduction and development - in the implantation of the blastocyst to the uterus, development in the proliferation of stem cells, induction of self-renewal of haematopoietic stem cells, survival and the specification of the neural crest; and also in embryogenesis and adult tissue homeostasis. Dysfunction of Wnt causes a lot of neural problems, such as an imcomplete Spina bifida (i.e. incomplete neural tube closure)

The activation of the Wnt pathway seems to be the major pathway in regulating cell motility by stimulating many cytoskeleton regulators, including the Rho family GTPase and Rho kinase. “Both Wnt1 and Wnt3a have been shown to activate RhoA, whereas the non-canonical Wnt5a promotes melanoma migration via RhoB” Inhibiting the action of Rho kinase blocks the effect of Wnt3a.

Wnt signals are pleiotropic, which means one gene can produce more than one phenotypic traits including mitogenic stimulation (encourages mitosis), cell fate specification, and differentiation.

Wnt signals are small (30-40kDa) proteins secreted by a source cell, which act by paracrine signalling. Many share amino acid sequence, suggesting they are proteins with important functions that persisted through evolution in most species. In humans, 19 Wnt proteins have been identified and they will each require many receptors (there are 10 different Fz, and two types of LRP - LRP5 and LRP6), antagonists, agonist etc and so the whole pathway includes many components, and any mutations in these will lead to a mutant (often cancerous) phenotypes. The roles of these components includes lipid modification, glycosylation and transport of the signal, though lipid modification and glycosylation already greatly affects the transport of the signal.

Canonical Pathway
Wnt proteins in the canonical pathway bind to Frizzled (Fz)/low density lipoprotein (LDL) receptor-related protein (LRP) complex on the plasma membrane of target cells. Fz is a 7-pass GCPR-like (in terms of structure) cell surface receptor, seven types have been found in humans so far. The binding of Wnt ligands to Fz/LRP activates the signal transduction pathways, which begins with the phosphorylation on serine and threonine residues of the cytoplasmic scaffolding proteins of the Dishevelled (Dsh) family (DVL1, DVL2 and DVL3). At least 16 different proteins bind to Dsh, and activates downstream elements; for the canonical pathway, this includes glycogen synthase kinase-3β (GSK-3), axin, Adenomatous Polyposis Coli (APC), and the transcriptional regulator, β-catenin.

In the canonical pathway, the levels of cytoplasmic β-catenin is usually kept low by continuous degradation by ubiquitylation in proteasomes (controlled by GSK-3/APC/Axin and CK-1). β-catenin forms a complexes with CK-1 and GSK-3/APC/Axin, CK-1 first prime the β-catenin by phosphorylating it, and then GSK-3 phosphorylates at a different site; this targets it for degradation in proteasomes. But when Wnt protein binds to the receptor, Fz transduce a signal to Dsh, and LRP transduce a signal to Axin, which may directly interact with each other. When LRP is bound, it is phosphorylated first by GSK-3 and then CK-1 (reverse order to the phosphorylation of β-catenin), to which axin can then bind. When axin binds, it brings the whole complex near the membrane before it breaks up the GSK-3/APC/Axin complex, releasing more GSK-3 and CK-1 to positively feedback phosphorylation of LRP, ensuring a quick activation of downstream effects. As the degradation complex is broken, the degradation mechanism of β-catenin is interrupted and thus β-catenin accumulates in the cytoplasm and nucleus. The β-catenin forms functional complexes with transcription factors of the lymphoid enhancer-binding factor 1/T cell-specific transcription factor (LEF/TCF) in the nucleus and displaces the transcriptional inhibitor Groucho and CtBP, altering the local chromatin structure so other more specific transcription factors can bind, enabling transcription.

β-catenin also exist at the cell surface as cadherin, and so a topic of research is to study whether a Wnt signal can induce the endocytosis of these cell surface cadherins, and add them to the cytoplasmic pool, inducing a positive feedback mechanism.

Non-canonical pathway
Wnt ligands can also bind to Ror2 or Ryk receptors to stimulate β-catenin independent pathways that is involved in cytoskeleton rearrangement and cell migration.

There are two non-canonical pathways - Planar cell polarity (PCP) and Ca2+. PCP signals for polarity (front/back, left/right, top/bottom) by regulating the arrangement of the cytoskeleton. JNK.

The Ca2+ regulated pathway have Ca2+ sensitive enzymes which when activated, alter gene transcription.

All three pathways uses Fz and Dsh, though we are uncertain how a specific ligand bind to a specific Fz.

Palmitoylation
Wnt proteins are defined by its sequence rather than its function. All Wnt proteins have a signal sequence followed by a conserved distribution of cysteines. Originally, it was difficult to isolate active Wnt proteins due to them being insoluble, which was later found to be caused by S-palmitoylation (the attachment of palmitic acid to cysteine residues of membrane proteins) on a conserved cysteine. This increases the hydrophobicity of Wnt proteins compared to those predicted by anaylsing its primary sequence alone. This allows for the Wnt proteins to better associate with the membrane. Because the bond between the palmitic acid and the protein is an ester bond, palmitoylation is reversible by using palmitoyl protein thioesterases. S-palmitoylation is a dynamic and post-translational process, and so the cell can use it to change the affinity of a protein for the membrane. When the conserved cysteine residue is removed by mutation, or if the palmitate is cleaved, Wnt3a loses its activity, suggesting that Wnt proteins work by association with the membrane.

However, it is found that un-palmitoylated Wnt proteins can still produce an attenuated signal when overexpressed in cells, suggesting that palmitate targets Wnt to the lipid membrane for it to function, and the lack targeting can be compensated by having a high concentration of Wnt.

The enzyme that carries out palmitoylation is thought to be encoded by the gene porcupine (por) in Drosophila, or mom-1 in Caenorhabditis elegans, as these only needs to be found in the signalling cell and not the target cell, plus the fact that they are phenotypically similar. Hofmann (2000) found similarities in the sequence of por and membrane-bound acyl transferases on the ER membrane, suggesting that por transfer palmitate onto the Wnt proteins.

Wingless is a gene in Drosophila akin to human Wnt3a (and is where the name of Wnt is derived from), and its hydrophobicity as well as membrane localisation is lost when we inhibit O-acyltransferase activity or when we remove the POR gene genetically. The MOM-3 gene product may have roles in the production and/or secretion of active Wnt.

Regulation of activated Wnt
After Wnt is secreted, the level of activated Wnt is modulated by inhibition by frizzled-related protein (SFRP) or Wnt inhibitory factor (WIF). SFRP has a similar sequence to the cysteine-rich domain (CRD, also the ligand-binding domain) of Fz, one of the receptors of the canonical pathway; while WIF resembles the extracellular portion of the Derailed/RYK class of non-canonical transmembrane Wnt receptors. It is largely agreed on that SFRP and WIF are both inhibitors of Wnt proteins, but it cannot be ruled out that they can also protect the Wnt proteins from degradation and facilitate its secretion and transport.

Other less-studied components
The Wnt inhibitors Dickkopf (Dkk) and Wise bind to the Wnt co-receptors Arrow and LRP. Dkk also interacts with Kremen to down-regulate LRP/Arrow from the cell surface. In Drosophila, Wnt can bind to the tyrosine kinase receptor Derailed [related to tyrosine kinases (RYK) in mammals]. This receptor has a domain similar to WIF. Heparin-sulfated forms of proteoglycans (HSPG) are also involved in Wnt stabilization, reception and/or transport. Boca/Mesd is specifically required for the transport of Arrow/LRP in the ER. A novel Frizzled ligand, Norrin, has also been identified. Similar to Wnt, Norrin bound to LRP and Frizzled can stimulate the canonical signaling pathway.

Experiments
The detection of Wnt proteins has been difficult due to the lack of antibody reagents, but the Wingless protein, analogue to Wnt3a, has been found to have a high concentration at the imaginal discs. Other experiments have suggested that Wnt proteins are concentration-dependent, long-range morphogenetic signals.

The mechanism for Wnt protein transport is not certain, it may be tethered to membranes and are slowly shuttered between cells, it may be secreted and made hydrophilic using carrier proteins. It may also be packaged into vesicles called argosomes, carrying the Wnt proteins as cargo. Cytonemes - long, thin tubes formed from the plasma membrane that connects different animal cells over long distances, can also be used to carry Wnt proteins and other growth factors.