1. Mohammed Kaleem PhD Candidate

2.  Wnt/  -Catenin  Frizzle receptor

3. 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

4.  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).

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

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

7.  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

8.  Large family of secreted molecules ◦ 350 to 400 amino acids ◦ Signal sequence ◦ Invariant pattern of 23-24 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

9.  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

10. 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

12.  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.

13.  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

14.  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

16.  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

17.  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.

18.  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

19.  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

20. 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.

21.  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

23. 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

24.  Dann CE, Hsieh JC, Rattner A, Sharma D, Nathans J, Leahy DJ. 2001. "Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains". Nature. 412: 86–90.  Jeong WJ, Ro EJ, Choi KY. 2018. Interaction between Wnt/β-catenin and RAS- ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway. NPJ Precis Oncol. 2:5.  Logan CY, and Nusse R. 2004. The Wnt Signaling Pathway in Development and Disease. Annu. Rev. Cell. Dev. Biol. 20: 781-810  MacDonald BT, He X. 2012. Frizzled and LRP5/6 receptors for Wnt/β-catenin signaling. Cold Spring Harb Perspect Biol. 1;4 (12).  Schulte G. International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors. Pharmacol Rev. 2010 Dec;62(4):632-67.  Wodarz, A. and Nusse, R. 1998. Mechanisms of Wnt Signaling in Development. Annu. Rev. Cell Dev. Biol. 14:59–88  Zhan T, Rindtorff N, Boutros M. 2017. Wnt signaling in cancer. Oncogene. 36:1461-1473.