New insights into Ror1, Ror2, and Ptk7 mediated Wnt signaling
Perttilä, Robert (2021)
2021
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-05-26
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202105124912
https://urn.fi/URN:NBN:fi:tuni-202105124912
Tiivistelmä
Background and aims: Wnt signaling has central role during embryonic development and adult tissue homeostasis. Dysregulation of this important signaling can lead to different developmental disorders and other diseases such as cancers. Wnt signaling is divided into canonical and non-canonical branches, involving multiple partially overlapping receptors, ligands, and pathways. Wnt signaling is not fully understood due to its complexity and more studies are needed. This Master thesis work aims to characterize ligands and downstream signaling of the non-canonical Wnt receptors Ror1, Ror2, and Ptk7.
Methods: The expression and secretion profiles of 15 Wnt ligands were studied by transfecting Wnt-encoding plasmids into HEK293T cells and analysing Wnt levels in media and cell lysates by western blotting. To determine the Wnt ligands for Ror1, Ror2, and Ptk7 receptors, coimmuno-precipitation assay was performed in HEK293T cells. 15 Wnt ligands were individually over-expressed with either Ror1, Ror2, or Ptk7 and analysed which Wnt ligands become co-precipitated with the receptor. To understand which domains of the receptor are required for binding Wnts, deletion constructs of Ror1 that lack either Cysteine-rich domain or Kringle domain were ex-pressed in HEK293T cells together with Wnt5a or Wnt16 ligands and subjected to coimmunoprecipitation assay. In other part of the study, downstream signaling of Ror1, Ror2, and Ptk7 in response to Wnt binding was investigated. Canonical Wnt signaling was studied by luciferase assay with expressing Wnts alone or together with Ror1, Ror2, or Ptk7 in HEK293T cells. Non-canonical Wnt signaling was studied by stimulating BaF3 cells stably expressing Ror1, Ror2, or Ptk7 with Wnt ligands and analysing ERK/AKT phosphorylation levels by western blotting.
Results: Wnts were secreted at very variable levels by the transfected HEK293T cells. Wnt lig-ands exhibited highly overlapping binding profile to Ror1, Ror2, and Ptk7 – some Wnts interacted with all three receptors, while others had no interactions at all. However, there was some degree of specificity in which Wnts Ror1, Ror2, and Ptk7 bind, even though the binding profiles were rather similar between the receptors. It was shown that both Cysteine-rich and Kringle domains of Ror1 have indispensable role in the Wnt binding. Wnt3a was the only Wnt ligand that could activate the canonical Wnt signaling, and this activation was repressed by 62-82% when co-expressed with Ror1, Ror2, or Ptk7. On the other hand, it was shown that certain Wnt ligands act through these receptors to increase ERK/AKT phosphorylation levels, indicating the activation of non-canonical Wnt signaling.
Conclusions: This work answers the need for large-scale screens focusing into the components of the less known non-canonical Wnt signaling. The results are preliminary and should be repeated in other cell lines and experimental settings. Nevertheless, the findings show how complex and interconnected the Wnt signaling pathways are and provide important fundamental information about the Ror1, Ror2, and Ptk7-mediated Wnt signaling.
Methods: The expression and secretion profiles of 15 Wnt ligands were studied by transfecting Wnt-encoding plasmids into HEK293T cells and analysing Wnt levels in media and cell lysates by western blotting. To determine the Wnt ligands for Ror1, Ror2, and Ptk7 receptors, coimmuno-precipitation assay was performed in HEK293T cells. 15 Wnt ligands were individually over-expressed with either Ror1, Ror2, or Ptk7 and analysed which Wnt ligands become co-precipitated with the receptor. To understand which domains of the receptor are required for binding Wnts, deletion constructs of Ror1 that lack either Cysteine-rich domain or Kringle domain were ex-pressed in HEK293T cells together with Wnt5a or Wnt16 ligands and subjected to coimmunoprecipitation assay. In other part of the study, downstream signaling of Ror1, Ror2, and Ptk7 in response to Wnt binding was investigated. Canonical Wnt signaling was studied by luciferase assay with expressing Wnts alone or together with Ror1, Ror2, or Ptk7 in HEK293T cells. Non-canonical Wnt signaling was studied by stimulating BaF3 cells stably expressing Ror1, Ror2, or Ptk7 with Wnt ligands and analysing ERK/AKT phosphorylation levels by western blotting.
Results: Wnts were secreted at very variable levels by the transfected HEK293T cells. Wnt lig-ands exhibited highly overlapping binding profile to Ror1, Ror2, and Ptk7 – some Wnts interacted with all three receptors, while others had no interactions at all. However, there was some degree of specificity in which Wnts Ror1, Ror2, and Ptk7 bind, even though the binding profiles were rather similar between the receptors. It was shown that both Cysteine-rich and Kringle domains of Ror1 have indispensable role in the Wnt binding. Wnt3a was the only Wnt ligand that could activate the canonical Wnt signaling, and this activation was repressed by 62-82% when co-expressed with Ror1, Ror2, or Ptk7. On the other hand, it was shown that certain Wnt ligands act through these receptors to increase ERK/AKT phosphorylation levels, indicating the activation of non-canonical Wnt signaling.
Conclusions: This work answers the need for large-scale screens focusing into the components of the less known non-canonical Wnt signaling. The results are preliminary and should be repeated in other cell lines and experimental settings. Nevertheless, the findings show how complex and interconnected the Wnt signaling pathways are and provide important fundamental information about the Ror1, Ror2, and Ptk7-mediated Wnt signaling.