Mohammed Kaleem PhD Candidate

 Wnt/  -Catenin  Frizzle receptor

Important in multiple developmental events Mutated: leads to disease Canonical pathway: Wnt signaling through Frizzled (Fz) to β-catenin Main intracellular proteins involved: Lipoprotein receptor-related protein 5/6 (LRP5/6) Dishevelled (Dsh) β -catenin interacts with T-cell transcription factor/lymphoid enhancer factor (TCF/LEF) Wnt signaling inhibits degradation of β -catenin CK1α: casein kinase 1α; Glycogen synthase kinase 3 (GSK-3) Axin - Adenomatous Polyposis Coli (APC) β ‑ TrCP E3: β-transducin repeat containing protein

 Wnt signaling: research on oncogenic retroviruses.  In 1982, Roel Nusse and Harold Varmus infected mice with mouse mammary tumor virus in order to mutate mouse genes to see which mutated genes could cause breast tumors.  Identified a new mouse proto-oncogene int1 (integration 1).

Dsh APC GSK3 Axin Β-c TCF Β-c TCF Axin Β-c WNT protein Frizzled LRP5/6 DNA

Transcriptional repression of Wnt target genes Activate expression of the target genes leads to proliferation and transformation

 Wnt ligands bind to the ROR-Frizzled receptor complex to recruit and activate Dvl.  Dvl binds to the GTPase Rho by de-inhibition of DAAM1 (Dvl associated activator of morphogenesis 1).  The GTPase Rac1 and Rho together trigger ROCK (Rho kinase) and JNK.  Leads to rearrangements of the cytoskeleton and/or transcriptional responses via ATF2 (activating transcription factor 2).  Vangl is activated by phosphorylation in a Wnt5a-dependent manner.  Wnt/Ca2+ signaling by G-protein activated phospholipase C activity leading to intracellular calcium fluxes and downstream calcium dependent cytoskeletal and/or transcriptional responses Non-canonical Wnt signaling

 Large family of secreted molecules ◦ 350 to 400 amino acids ◦ Signal sequence ◦ Invariant pattern of conserved cysteines  Involved in intercellular signaling during development ◦ Early mesodermal patterning of embryo ◦ Morphogenesis of brain and kidneys  Drosophila homologue – Wingless ◦ Loses hydrophobicity and activity when por eliminated ◦ Por is necessary for lipidation/membrane targeting

 Secreted from cells  Experiment in Drosophila ◦ Antibody to Wingless  Significant spread in imaginal discs  Concentration-dependent long-range morphogenetic signals acting on distant neighbours  Flies have vessicles in imaginal discs ◦ Argosomes ◦ Might carry Wingless as cargo

Name derived from first 2 members discovered: Drosophila Wingless Mouse int-1 Overexpression of Fz: No Wnt signal Co-overexpression of Wingless: Signaling Fz activation is ligand dependent Fz forms receptor complex with another single-pass transmembrane protein Mutations in the human Fz-4 receptor have been associated with familial exudative vitreoretinopathy

 Complex formation with cadherins and  -catenin at the plasma membrane is essential for the role of β-catenin in cell adhesion.  In vertebrate development, loss of a single Wnt gene can produce dramatic phenotypes that range from embryonic lethality and CNS abnormalities to kidney and limb defects  Wnt pathway has distinct transcriptional outputs.  In many cases, the cell determines the nature of the response, and up- or down-regulation of Wnt target genes is cell-type specific.

 Frizzled ◦ Binds to Dsh  Ubiquitously expressed ◦ C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif  Required for Fz signaling  LRP ◦ Binds to Axin ◦ Cytoplasmic tail has several Pro-Pro-Pro-(SerTrp)Pro motifs  Phosphorylated upon Wnt binding  Axin and Dsh: DIX domains ◦ Can heterodimerize ◦ LRP and Fz may promote interaction between Dsh and Axin

 Three ways of β-catenin accumulation ◦ Disruption of degradation complex 1. Recruitment of Axin to LRP or Fz/Dsh ◦ Amount of Axin in cell much lower than other complex proteins  Limiting factor 2. Protein phosphatases ◦ PP2A  Binds to Axin, dephosphorylates GSK-3 3. GBP/Frat ◦ GSK-3 binding protein ◦ Removes GSK-3 from degradation complex

 In adults, mis-regulation of the Wnt pathway also leads to a variety of abnormalities and degenerative diseases Mutations : such as breast and prostate cancer, glioblastoma, type II diabetes

 Current evidence indicates that the Wnt cascade is the single most dominant force in controlling cell fate along the crypt-villus axis.  In Tcf4 -/- neonatal mice, the villus epithelial compartment appears unaffected but the crypt progenitor compartment is entirely absent, inicates that physiological Wnt signalling is required for maintenance of the crypt progenitor phenotype.

 Study knock-outs ◦ Gene expression pattern correlates with mutant phenotype  Demonstrates Wnt requirement in developmental process  Wnt3 ◦ Expressed in primitive streak in mouse embryo ◦ Wnt3 mutants – gastrulation defects  Frizzled4 ◦ Cerebellar, auditory and esophageal defects  TCF1 ◦ Defects in limb bud development ◦ Mammary and gut tumours

 β-Catenin: cell–cell adhesion and gene transcription  Wnt signaling pathway: axis patterning, cell differentiation, cell proliferation and cell migration  Fz has an extracellular cysteine-rich domain and seven transmembrane helices similar to a G-protein-coupled receptor.  When activated, Fz leads to activation of Dsh in the cytosol.  Fz proteins play vital roles: ◦ cell polarity, embryonic development, formation of neural synapses, cell proliferation

Fz consist of cysteine-rich domain and tyrosine kinases. The Fz has 7TMR and have in their extracellular region a cysteine-rich domain that has been associated as the Wnt binding domain. Fz and tyrosine kinases: key roles in development such as muscle-specific receptor tyrosine kinase, the neuronal- specific kinase and ROR1 and ROR2. Has α-helices and domain contains ten cysteines that form five disulphide bridges.

 FAP patients, inheriting one defective APC allele, develop large numbers of colon polyps  Mutational inactivation of APC leads to overproduction of β- catenin, which indicates excessive cell division in Wnt/ β- catenin pathway  When APC is not mutated in colorectal cancer, and axin 2 is mutant, β-catenin remove its N-terminal Ser/Thr destruction motif.  Tcf4 target gene programme in colorectal cancer. The Wnt/ β-catenin pathway in colon cancer

GeneDisease Wnt3Tetra-amelia LRP5Bone density defects Fzd4Familial Exudative Vitreoretinopathy (FEVR) Axin2Tooth agenesis Predisposition to Colorectal Cancer APCFamilial adenomatous polyposis (FAP) Colon Cancer Extracellular Wnt ProteinTarget Cell Membrane ProteinIntracellular Protein

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