Molecules & Cells - Martin Cann 4, Wnt and Hedgehog (Hh) receptors

Where did the evidence for the current Hh signalling pathway come from?

- Came from study of fly embryos with loss-of-function mutations in the hedgehog or smoothened genes. --> Both types of mutant embryos have very similar developmental phenotypes.-

How is Hh signalling regulated?

- Contol of the Hh pathway is important as unrestrained Hh signlling can cause cancerous overgrowth of cells. - In fruitflies (Drosophila) one of the genes induced by the Hh signal is patched. --> The subsequent increase in expression ofo Patched, antagonizes the Hh signal by reducing the pool of active smoothened protein (patched inhibits smoothened)

How were the compounds of the Wnt and Hedgehog signalling pathways elucidated?

- Elucidated mainly thruogh the analysis of mutations in fruit flies (Drosphophila) - This has also elucidated the signalling pathway in humans due to the fact that they are evolutionary conserved.

What occurs when Hh binds to Hh receptors on a cell?

- Following binding of Hh protein to the receptors Patched, both proteins move from the cell surface into internal vesicles (not shown in diagram), while smoothened moves from internal vesicles to the PM. --> The binding of Hh inhibits Patcheds ability to inhibit smoothened. - Some of the proteins in the cytosolic complex attached to microtubules, dissosciate from the microtubules. The proteins that phosphorylated Ci in the complex move to the C-terminal tail of smoothed. The resulting disruption of the cytosolic complex causes a reduction in both phosphorylation and cleavage of CI. --> The modified form of Ci, called Ci*, translocates to the nucleus, where it binds to a transciptional co-activator CREB, promoting the expression of target genes.

What is the purpose of Hh signalling?

- Hh signalling relieves repression of target genes --> Genes involved with tissue and organ formation, important in development.

Compare Hh signalling in Drosophila (fruitflies) and humans

- Humans express three hh and two ptc genes in contrast to one of each in Drosophila. - Humans also possess three Gli transcription factors that appear to perform the function of the single Ci of the fly. - It has been proposed that the PRIMARY CILLIUM (a single long immotile cilium found on most cells) may be a centre for Hh signal transduction in mammals/humans.

How is the Hh signalling protein produced?

- In contrast to Wnt, Hh is synthesized as a precursor that is subsequently cleaved into 2 parts 1/3 the way along. (autoproteolytic activyt) --> The thiol side chain of cysteine 258 performs a nucleophilic attack on the carbonyl carbon of Gly257 -Hedgehode is split in 2: an N-terminal fragmment that is subsequently secreted to signal to other cells after further modificantions, and a Cterminal fragment which is degraded. - MODIFICATIONS TO THE N-TERMINAL FRAGMENT: The C-terminal of the precursor catalyzes linkage of the c-terminus to cholesterol and a palmitoyl group is added to the N-terminus (N-terminus is the signalling part of the molecule). ---> This makes the protein highly hydrophobic and thus binds nonspecifically and reversibly (tethered) to cell PM, limiting its diffusion. This processing occurs intracellularly and Hh is subsequently tethered to the plasma membrane (like Wnt, went also has a hydrophobic pamitoyl group). These membrane interacting modifications probably restrict Hh mobility thus its range of action is limited to the order of 1-20 cells.

What occurs in the Wnt signalling pathway when Wnt protein is NOT bound to the Wnt transmembrane receptor? Draw a diagram

- In the abscence of Wnt signal, B-catenin is not attached to molecules in the membrane, but a CYTOSOLIC COMPLEX made up of multiple proteins. - In the resting state, 2 kinases in the complex sequentially phosphorylate B-catenin. The phosphorylated residues on B-catenine serve as a binding site for a ubiquitin-ligase protein, which uniquitinates B-catenine. B-CATENINE IS THEN RAPIDLY DEGRADED. --> Degradation of B-catenin prevents it from translocating to the nuclues, to activate transcription of particular repressed target genes (often ones that promote cell proliferation).

What occurs in a cell that has not yet recieved the Hh protein/signal, ie a cell that is in the resting state?

- In the absence of Hh, Ptc (the Hh receptor) represses Smo function and Smo is maintained in internal vesicles. - Patched is in the PM and Smoothened is in the membrane lining internal vesicles. The cytosolic complex is in takt in the cytosol bound to microtubules, consisting of multuple proteins including a transciption factor Ci. - Ci is phosphorylated by 3 components in the complex. Phosphorylated Ci is then proteolytically degraded by Slimb. The resulting Ci fragment, translocates to the nucleus and represses expression og Hh target genes.

Discuss the Wnt signalling system in relation to development of cancers

- Inappropriate acitvation of the Wnt pathway is characteristic of many human cancers. - In many tumors the level of B-catenin is abonormally high (which means that genes that promot cell proliferation are activated) - Mutations that inactive the genes that encoded the proteins/enzymes that form part of the cytosolic complex, are found in multiple human cancers (without these proteins encoded by these genes, B-catenin will always be free to translocate into the nuclues, even when Wnt isnt bound to the Wnt receptors and B-catenine will not be phosphorylated and therefore not tagged for degradation)

What occurs in the Wnt signalling pathway when Wnt protein is bound to the Wnt transmembrane receptor? Draw a diagram

- THE COMPLETE PATHWAY BY WHICH WNT SIGNALLING BLCOKS DEGRADATION OF B-CATENIN HAS NOT YET BEEN IDENTIFIED. We do know that: - Went binds to Frizzle membrane receptor protein. - In the presence of Wnt, Fz is able to interact with its co-receptor (LRP). - LRP in the Wnt-Fz-LRP complex is phosphorylated by free kinases (proably GSK3 or CK1) in the cytosol and this permits Axin binding. - Axin binds to phospho-LRP, which disrupts the cytosolic complex. - In turn this prevents phosphorylation of B-catenin, which prevents ubiquitination and subsequent degration. B-catenine is stabilised in the cytosol. This requuires dishelleved to bind to the frizzled protein - The freed B-catenin translocatesto the nuclues, where it assosciates with a transciption factor (TCF) and functions as a co-activator to induce gene activation/expression of particular taget genes (often including those that promote cell proliferation).

What does the Wnt pathway control?

- The activation of the Wnt pathway controls numerous critical developmental events, such as brain development, limb development and organ development (organogenesis) - Wnt also has a major role in bone formation, which was revealed by mutations in the Wnt pathway components, affect bone density. Wnt signalling is known to ocontrol formation of osteoplasts (bone-forming cells) - Wnt are also important in controlling stem cells

Why are the Wnt ligand and receptor called so?

- The word Wnt is an amalgamation of wingless, the corresponding fly gene, with int for the retrovirus integration site in mice.

Discuss the receptors in the Hh signalling pathway

- Two membrane proteins are required to recieve and transduce a Hh signal to the cell interior: 1.) Smoothened (Smo): has 7 membrane-spanning a helices (related to Wnt receptor Fz) 2.) Patched (Ptc): is predicted to contain 12 transmembrane a-helices

What receptor(s) do Wnt proteins/ligands bind to?

- Wnt proteins act rhough 2 cell-surface receptor proteins: 1.) Frizzled (Fz), which contains 7 transmembrane a helices and directly binds Wnt 2.) Co-receptor LRP, which assosciated with Frizzled when Wnt attache to Frizzled.

What type of signalling molecules are Wnt proteins (synpatic, endocrine, exocrine, or autonomic)? Explain.

- Wnt proteins are synaptic signalling proteins/ligands - Wnt proteins are secreted extracellularly, and then modified by addition of palmitate gorup (fatty acid) near their N-terminal. This hydrophobic group is thought to tether Wnt proteins to the PM of the Wnt-secreting cells. - This limits the their range of action to adjacent cells.

What are the two synaptic ligands and membrane bound receptors we discussed in this lecture?

1.) Wnt receptor binds the Wnt and family of signaling molecules. 2.) Hedgehog (Hh) receptor binds Hh family of signaling molecules. They are both multi-transmembrane receptors (like G-protein coupled receptors), but they do not couple to G-proteins. They are evolutionary conserved

In which processes do Wnt and Hedgehog ligands primarily function?

Developmental processes. - They often induce expression of genes rewuired for a cell to acquire a new identity or fate.

Compare and contrast the Wnt signalling pathway and Hh

SIMILARITIES: 1.) Both involve 2 membrane proteins, one with 7 transmembrane segments. 2.) Both involve disasembly of an intracellular complex containing a transcription factor DIFFERENCES: - Unlike Wnt, Hh protein undergoes distinctive post-translational processing - In Wnt both proteins are in the membrane all the time, in Hh one of them (patched) is in the membrane while smoothened is in the membrane of vesciles (when not activated) - In Wnt hen B-catenin enters the nucleus it binds to a trascription factor to upregulate expression of Wnt genes, in Hedghog when Ci enters the nucleus it reverts the repression of genes to activate their expression. - In Hh, unlike Wnt, Hh signalling is restricted to the primary cilium that protudes from the cell surface in MAMMALS.

Define Morphogens

Signals that induce different cell fates dependeing on their concentration at their target cells

Name two morphogens

Wnt and Hedgehog diFFERENT CONCENTRATIONS RESULTS IN TRANSCIPTION OF DIFFERENT GENES

Give a summary of the lecture

Wnt and Hh are powerful morphogens mediating signaling events in development. Both are modified by palmitoyl to limit their diffusion and reducing their signaling range. Both Wnt and Hh bind plasma membrane proteins, frizzled and patched respectively. Both Wnt and Hh cause the release of a protein from a complex, -catenin and Ci respectively.

What is the function of Wnt?

Wnt causes changes in gene transcription in target cells through the TCF transcription factor but TCF only activates transcription in the presence of B-catenin

What happens as Hh diffuses further and further away from the secreting cell (while still being tethered to it)?

- Although Hedgehog is a secreted protien, it moves only a short distance from a signalling cell, on the order of 1 to 20 cells, and is bound by receptors on recieving cells. --> Thus Hh has localised effects (like Wnt) - As Hh diffuses further away from secreting cells, its concentrations decrease and different Hh concentrations induce different fates in target cells (diffferent genes will be turned on) --> Signals that induce different cell fates dependeing on their concentration at their target cells are called MORPHOGENS

How is Wnt signalling controlled?

- Among the genes that the B-catenine activates (when Wnt is bound), are genes that control Wnt signalling. - Thus indicating a high degree of feedback regulation (when Wnt concentration is high, genes that regulate Wnt are activated, hence negative feedback).