Wnt Signalling Flashcards
What model organisms are used to study Wnt signalling?
Drosophila melanogaster
Mice
- Xenopus*
- C. elegans*
What is the overall flow of information during cell signalling?
- Ligand binds receptor on the cell surface
- Signal transduction occurs via second messengers
- Cellular responses, which may or may not include;
- Gene expression
What is the best studied Wnt signalling pathway?
The canonical Wnt signalling pathway
Outline the role of the Wnt protein family.
The Wnt family is a conserved group of signalling molecules that are important in cell fate decisions during development, and the maintenance of many stem cell populations in adults. The disruption of Wnt genes is implicated in many diseases and cancers.
What role does canonical Wnt signalling play in humans?
Canonical Wnt signalling plays an important role in development, tissue homeostasis, and cancer. At the cellular level, canonical Wnt signalling acts by regulating cell fate, cell growth, and cell proliferation.
What two gene discoveries lead to the naming of Wnt?
Wingless (Wg) was first identified as a mutation that caused one wing not developing in Drosophila melanogaster. About the same time this gene was cloned, another researcher cloned Int1. Int1 mutations in mice lead to mammary cancers, which was found to be caused by insertion of mouse mammary tumour virus (MMTV) causing overexpression of the gene.
The wingless mutation was found to be the same as Int1.
To avoid confusion, all those working on int1 and its relatives, including Wingless, consented to a new hybrid name ‘Wnt’ (for Wingless-related integration site) to denote genes belonging to the int1/Wingless family, with int1, now called Wnt1, as the founding member.
What is the Wnt1 homolog in Drosophila?
Wingless (Wg)
What events lead to the Wnt gene being identified in mammals?
It had been known since the 1930s that certain strains of laboratory mice are highly susceptible to breast cancer, and that the disease is usually transmitted from mothers to offspring mice through the milk. Later, the tumour-inducing activity was purified from the milk, and the milk-transmitted factor was shown to be a morphologically atypical retrovirus, called the Mouse Mammary Tumour Virus or MMTV.
Unlike the most intensively studied cancer-causing retroviruses, however, there was no readily identifiable oncogene in the viral genome. But, like other retroviruses, MMTV was known to insert a DNA copy of its RNA genome into the host cell genome during infection.
The insertion of viral DNA might cause a mutation of a gene in the vicinity of the integration site, and the change might confer a growth advantage to that cell. While it was possible that such mutations caused loss-of-function mutations by disrupting a host cell gene, an alternative and more attractive model was that a host cell gene was transcriptionally activated by the incoming provirus.
- Mutation screen: gathered over 30 MMTV-infected C3H (tumour strain) mice with mammary tumours, and found just one tumour (#18) with a single new provirus.
- Cloning: From the genome of this tumour, they cloned a ‘junctional’ fragment, containing a part of an MMTV provirus joined to adjacent chromosomal DNA, then derived smaller fragments containing only host cell DNA.
- Chromosome walking: With those fragments as probes, they ‘walked’ along the chromosome in either direction from the original proviral insertion site to obtain more probes for a broader region of the mouse genome.
- Mapping: a map was assembled of the relevant region of the mouse genome, with the various proviral insertions in different tumours scattered throughout a region spanning ∼30 KB.
- One of the probes detected a tumour-specific mRNA—that is, a species not found in normal mammary gland tissue—on northern blots. A host gene, a putative proto-oncogene, had been transcriptionally activated by MMTV proviruses in multiple, independent tumours. The gene was called int1 (to denote the first common integration site).
- Later, the structure and sequence of the int1 gene and its cDNA was determined.
How was the Int1 homologue in Drosophila melanogaster discovered?
During the 1970s, genetic screens in Drosophila had unveiled a set of genes that were essential for the development of the embryo. ‘Segment polarity genes’ describes one class of mutants that share a similar patterning phenotype during embryogenesis. One of the genes in this group was called Wingless; others, Armadillo and Arrow. The Wingless gene had actually been identified earlier as a weak mutant allele leading to loss of wing tissue, hence the name Wingless.
The Wingless gene was cloned by P-element transposon tagging, a method akin to the proviral tagging methods used for int1. The gene that was cloned had restriction maps matching the Drosophila int1 clone. The genes were identical; the int1 homologue in Drosophila was Wingless, one of the first examples of a gene involved in development and also activated in cancer
How many Wnt genes are there in vertebrates?
With the complete sequences of the genomes of many multicellular animals in hand, we now realize that vertebrates contain a family of 19 Wnt-related genes; pairs of these genes can often be placed in subfamilies that are highly similar to each other, perhaps reflecting gene duplications relatively recently in evolution. Each of these genes seems likely to have a specific role in development or other processes; they are generally expressed in different cells and at different times in maturation.
It is known that mutations in Wnt members cause defects such as Wingless in Drosophila and diseases such as mammary cancer in mice. What diseases arise from mutant Wnt signalling proteins in humans?
- Adenomatous polyposis coli (APC), a trait associated with multiple polyps in the colon, often leads to colon cancer at a relatively early age. The corresponding mutations—often non-sense or frameshift mutations that produce truncated proteins—were found in an enormous gene called APC.
- Bone density abnormalities with dysfunctional LRP receptors.
- Retinal degeneration with Frizzled mutations.
- Some metabolic disorders, including diabetes mellitus, have been associated with alterations in Wnt pathway genes.
What is seen in the Wnt mutants:
- Wg downregulation
- Ectopic Wg expression
- Porc mutant
- Downregulated Wg: Mutation in Wg leads to glazed eyes: Wg important for eye development. Loss of Wg function leads to loss of naked cuticles: Wg involved in this process.
- Ectopic Wg: Wg expression leads to excess naked cuticle production: this resembles APC, which is due to loss of a negative regulator.
- Porc mutant: Wingless is a morphogen (form generating substance) which forms a concentration gradient, causing transcription of different genes depending on the Wg concentration. Wg is secreted, and staining shows it is extracellular. It is therefore important to find mutations that affect the secretion of Wg/Wnt. Porc mutants do not secrete Wg, and this leads to an inactive Wg pathway.
What are the essential basic components of the Wnt signalling pathway?
Outline the canonical Wnt signalling pathway.
In the absence of Wnt signalling (left panel), β-catenin is in a complex with axin, APC and GSK3-β, and gets phosphorylated and targeted for degradation. β-Catenin also exists in a cadherinbound form and regulates cell–cell adhesion.
In the presence of Wnt signalling (right panel), β-catenin is uncoupled from the degradation complex and translocates to the nucleus, where its binds Lef/Tcf transcription factors, thus activating target genes.
What are the minimal molecules involved in Wnt signalling?
- Wnts
- Extracllular Antagonists: Frzbs, Dikkopf
- Receptors: Frizzled, Arrow
- Cytoplasmic: Dsh, APC, Axin, ZW3, Arm
- Nuclear: Groucho, TCF, Arm, Pan, Lgl
What model does Wnt signalling exist in?
Two state model: the pathway is either on or off, in some cases this is modulated to a certain degree.
Wnt absence: Frizzled and Arrow are not bound by Wnt, and the destruction complex (Axin, APC, GSK3) leads to the phosphorylation and breakdown of beta-catenin.
Wnt presence: Disheveled is activated, recruiting Axin out of the destruction complex, which falls apart and can no longer phosphorylate beta-catenin, which then enters the nucleus and transcribes Wnt genes.
Outline Wnts.
Wnts are 350-400 aa secreted gylcoproteins that contain conserved cysteine residues. There are at least 19 human Wnts. They are palmitylated in the ER by Porcupine, resulting in their being very hydrophobic.
Outline Frizzleds and FRP/FrzBs.
Frizzed is a family of G protein-coupled receptor proteins that has 7 transmembrane domains (TD) and Conserved Cysteine Rich Domain (CRD). It sits in the cell membrane and binds Wnts. Frizzled is a tissue polarity gene in Drosophila melanogaster.
The mammal homolog to Frizzled is a frizzled-related protein known as FRP/FrzB. There are at least 11 vertebrate Frzs. FrzB is a Wnt-binding protein important in embryonic development. FrzB is localized in the plasma membrane. Unlike Frizzled, FrzB lacks the 7 transmembrane domains normally found in G-protein-coupled receptors. It is still considered a homolog of Frizzled because it contains a Cysteine Rich Domain (CRD), and because of its intracellular C-terminus which is crucial for signaling. Secreted FRP/FrzB proteins modulate signalling, and extracellular loops may also bind Wnt and activate signalling.
Outline Arrow/LRP.
Arrow is a fly mutant that codes for a co-receptor for Wnt. Arrow contains 4 EGF repeats and a repeated YWTD motif that binds Wnt and Frizzled.
Cloning of Arrow mutants found homology to LRP5/6 in humans/vertebrates which codes for LRP (Low density lipoprotein receptor Related Protein), which binds Wnt and Frizzled.
Arrow/LRP binds the extracellular antagonist Dikkopf, which inhibits LRP6 forming a complex with Wnt/Frizzled. Normally it binds to Wnt and forms tertiary complexes with Frizzled and Wnt.
What inhibits Arrow/LRP binding to Frizzled/Wnt?
Dickkopf and Kremen
Outline Dishevelled.
Dishevelled has 18 possible protein interactions (currently identified). It is a cytoplasmi scaffolding protein associated with the plasma membrane that interacts with Frizzled, brings proteins together, is phosphorylated by several kinases. Dishevelled contains three conserved domains: DIX, PDZ and DEP. In the cytoplasm Dishevelled binds to FRAT/GBP which binds to GSK3
Outline Axin.
Axin is a negative regulator of Wnt signalling and is a product of the mouse fused locus. Axin contains a Regulator of G-Protein Signalling (RGS) domain and a DIX domain.
Outline APC.
APC is a tumour supressor gene with cellular roles including regulation of microtubules. It encodes a large protein, APC, which has different domains and interacts with several different proteins, including E-cadherin. APC antagonises Wnt signalling.
Outline GSK3.
Glycogen Synthase Kinase 3 (GSK3) is a serine/threonine kinase that phosphorylates Armadillo, APC, Axin and Arrow/LRP. By doing so it antagonises Wnt signalling. GSK3 has multiple non-Wnt related functions including its involvement in insulin signalling, NFAT phosphorylation and also in Hedgehog signalling.
Outline Armadillo/β-Catenin
β-Catenin is important for cell-cell interactions (epithelial cell contacts at junctional complexes), and in the absence of Wnt signalling β-catenin is kept at low levels. During Wnt signalling there is lots of beta-catenin at which time it can enter the nucleus and binds DNA with the help of other factors.
β-Catenin is the human homolog of Armadillo in Drosophila melanogaster.
Outline TCF and Groucho.
T-Cell Factor (TCF) can bind DNA, but can’t activate transcription. In the absence of signalling TCF and Groucho repress transcription of target genes. In the presence of Wnt signalling beta-catenin displaces Groucho from TCF and turns on transcription at the same promotors.
Name some of the interactions between Wnt signalling proteins.
- Wnt: Fz and Arr
- Frizzled: Dsh
- Arrow/LRP: Axin
- Dishevelled: Axin and GBP
- Axin: APC: b-catenin
- GSK3: GBP, Axin and b-catenin
- Armadillo/Beta-Catenin: TCF
Outline the destruction complex.
When Wnt signalling is inactive, APC and Axin act as scaffolding proteins to bring GSK3 and other kinases into proximity with beta-catenin.
Phosphorylation of beta-catenin by GSK3 tags it for destruction by the proteosome.
