Molecular Cell Biology II

Signalling During Vertebrate Development I - Wnts

Wnt and Shh are two highly studied developmental cell signals / ligands. They can act separately as well as together to regulate embryonic development and adult cell biology ² e.g. stem cells, cancer. Wnts 

   

Small secreted proteins (~40 kDa) that act as local mediators & morphogens Ancient, with shared amino acid sequence Made by all animals (Humans have 19 Wnts. Distinct functions but can overlap) Glycosylated & lipid modified (FA chain on N -terminus o binding to cell surfaces) Many molecules interact with Wnts

Involved in most cell signalling events during development and adult life Clinical importance ² e.g. inherited diseases in embryonic development Involved over many different timescales 3 major Wnt signalling pathways: 1. Planar Cell Polarity (PCP) 

 

Wnt / Jun Kinase (JNK) / cytoskeleton regulation / polarity Important for changing cell shape Important for cell migration ² involved in cancer metastasis

PCP pathway is important in Drosophila development. The pathway can be disrupted then the phenotype can be monitored for changes. e.g. In humans cilia will not develop properly in the ear, resulting in deafness. 2. Ca2+ 

Wnt / Ca2+ regulated enzymes / change in gene transcription

3. Canonical (Wnt / F-catenin) All 3 major Wnt signalling pathways start in the same way: 1. Wnt binds to a Frizzled cell surface receptor 

 

7-pass, GPCR (G-Protein Coupled Receptor) like Humans have 7 frizzleds Dishevelled is required for all 3 pathways

2. Frizzled can then bind Dishevelled (scaffold protein) Canonical Pathway (Wnt / F-catenin):

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Molecular Cell Biology II

Signalling During Vertebrate Development I - Wnts

Regulates proteolysis of F-catenin which functions in gene regulation as well as cell-cell adhesion Wnt binds to Frizzled protein and a co-receptor ² LDL-LRP 

(low-density lipoprotein receptor-related protein)

The degradation complex binds F-catenin and keeps it out of the nucleus, as well as promoting its degradation. The complex consists of« 

  

Casein Kinase 1 (CK1) ² A Ser / Thr kinase Glycogen Synthase Kinase 3 (GSK3) ² A Ser / Thr kinase Axin ² Scaffold protein* APC ² Scaffold protein*, involved in colon tumors

*Scaffold proteins hold complexes together

Canonical Signalling Pathway: In absence of Wnt signal 1. F-catenin binds degradation complex 2. CK1 phosphorylates F-catenin (priming) 3. GSK3 phosphorylates F-catenin 4. The double phosphorylated F-catenin is now targeted for degradation  Via ubiquitination and the proteasome 5. F-catenin cannot enter the nucleus 6. Wnt responsive genes are kept inactive by the Groucho co-repressor protein 

Groucho is bound to the gene regulatory protein LEF1/TCF

With Wnt Signal 1. Wnt binds to Frizzled and LRP, bringing the two receptors together 2. This recruits the degradation complex to the plasma membrane 3. GSK3 and CK1 phosphor ylate the cytosolic tail of LRP 4. Axin binds to the phosphorylated LRP and is inactivated / degraded 5. Loss of axin from degradation complex inactivates it 



F-catenin is no longer phosphorylated or ubiquitinated F-catenin will not be degraded, so it will accumulate and enter the nucleus

6. Within the nucleus, F-catenin binds to LEF1/TCF, displaces Groucho and acts as a co-activator to stimulate transcription on Wnt target genes. It can alter local chromatin structure and recruit other transcription factors.

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Molecular Cell Biology II

Signalling During Vertebrate Development I - Wnts

How does F-catenin enter the nucleus?  Does not enter via a nuclear localisation signal or importin 



It can bind to a nuclear pore Has an export signal and can ¶piggy back· out of the nucleus on axin

Wnt / F-catenin target genes: The downstream effect c-Myc is an example of a gene that is activated by F-catenin It encodes a protein that is a powerful stimulator of cell growth and proliferation

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Molecular Cell Biology II

Signalling During Vertebrate Development I - Wnts

Mutations of the Apc gene occur in 80% of colon caners These mutations will inhibit the proteins ability to bind F-catenin F-catenin will accumulate and enter the nucleus, stimulating transcription of c-Myc as well as other Wnt target genes, even in the absence of Wnt signalling. This results in uncontrolled cell growth and proliferation which will promote the development of cancer. Many Wnt targets will be effectors or repressors resulting in ± feedback loops Effects of Wnt Signalling: 

     

Proliferation (division) Self-renewal (e.g. stem cells, identical daughters need to be produced) Cell survival (overcoming programmed cell suicide) Cell fate / differentiation Cell polarity (e.g. neurons) Axis formation Synapse formation & functioning

When studying development with regards to Wnts, it is important to take a few facts into consideration. There are many possible points of regulation between ligand production and target response, which increases complexity. The response of a cell to a signal is context dependent, and down to a complex range of parameters. There are often differences between species, and in vitro studies do not always replicate what is going on in vivo. Wnts are modified (palmitolyated) in the ER by Porcupine 

 

Unsaturated fatty acids are added This is important for trafficking, secretion and signalling The lipid modification makes Wnts very insoluble, which isn·t common for signal peptides

Wnts are also glycosylated 

  

Necessary or secretion Might regulate folding during trafficking Might affect how Wnt spreads outside the cell Might affect the interaction with extracellular matrix

These two modifications will result in the Wnt having a larger mass than expected3 The Wnts can then be loaded onto a lipoprotein in specific endo / exocytic vesicles. Wnts can interact well with other proteins.

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Molecular Cell Biology II

Signalling During Vertebrate Development I - Wnts

A morphogen is a signal that forms gradients and has concentration dependent effects on responsive cells. The result of this signal is the choice of cell fate and differentiation. Positional value is interpreted by cells, which allows them to form a pattern. As Wnt is insoluble, it cannot create a gradient by diffusion. Possible methods« 



Lipid / lipoprotein vesicles / complexes Cytonemes (thin cell processes, look like fingers) 

Bound to heparin sulphate proteoglycans Dependent of ligand and tissue characteristics
The simplest way to control the amount of Wnt that reaches the target cell is to make more of less of the molecule. Another method is to produce extracellular molecules that bind to the Wnt and prevent it from binding to the target cell. Conversely, an extracellular molecule can bind to the Wnt, stabilise it and then present it to the target cell with greater affinity. Target Cell ² Wnt Receptors: Frizzleds (Fz) ² 7 transmembrane proteins Wnt binds to a cysteine-rich extracellular domain which may recruit it to other parts of the protein / coreceptors. A point of regulation ² can control receptor trafficking 

 

e.g. Prevent Fz transport in the ER e.g. Endocytose and remove Fz from cell surface LRP transport p surface requires chaperone, which will not function if mutated o Resulting in LRP null defects

LRPs- Single pass 

 

Large extracellular portion Small cytosolic portion Low affinity for Wnt

A point of regulation ² Dkk proteins bind LRP and link it to Kremens, triggering internalisation Some proteins can activate / block Wnts depending on context (receptors, receptor state, co-receptors, downstream enzymes, genes expressed, cell cycle etc.) There are many points of possible Wnt signalling regulation

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Molecular Cell Biology II

Signalling During Vertebrate Development I - Wnts

Others can facilitate its transport / secretion Wnts can bind many receptors (promiscuous) A receptor can bind many Wnts Important Clinical Example: Gut epithelial tissue is the most rapidly self-renewing tissue in the human body Wnt stimulates proliferation and terminal differentiation of gut cells ² can go wrong APC is involved in hereditary adenomatous polyposis, a form of colon cancer Mutant APC can no-longer regulate F-catenin, as discussed before Mutations in other components in the pathway can result in stable F-catenin p cancer Also involved in hair follicle tumors and baldness, Wnt and F-catenin are heavily involved in follicles for hair formation. Many pharma companies are looking for molecules that are Wnt modulators These are obviously toxic, so aim is to develop non-toxic inhibitors of Wnts for cancer treatment

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