WNT-mediated signaling relay in stem cells and oncogenesis - from basic biology to applications

Biophysical analysis of FZD function

Early stage researcher 3 (ESR3) project

Supervision: Gunnar Schulte

Lab webpage:  http://ki.se/research/gunnarschulte

Aim:
Generate a cell-free membrane-based system to investigate allosteric interactions between WNT, FZD and intracellular signaling components during signaling specification.

Methodology:
ESR3 will generate and employ cell-free receptor reconstitution systems to study WNT binding using QCM technology (international secondment: Sunahara, private sector secondment: Attana). WNT-FZD interaction profiles will be assayed for commercially available WNTs, WNT-mimicking peptides and WNT fragments (collaboration Isogenica). ESR3 will relate the data to conformational rearrangement of FZD upon stimulation. Measurement of FZD activity and conformational changes by state-of-the-art imaging techniques will be integrated to provide information on agonist-specific FZD conformational changes (collaboration Hoffmann). ESR3 will reconstitute receptors with purified proteins (collaboration UPE) to address the role of associated signalling components (DVL and heterotrimeric G proteins).

Collaborators:
Hoffmann, Sunahara, Attana, Isogenica, UPE

Project goals:
ESR3 will characterise FZD complex composition and identify distinct structural features of WNT-FZD receptor complexes, determining signal specification. The developed QCM- and FRET-based FZD activity assays may provide new possibilities in drug development.

Risk assessment and contingency plans:
High risk-high gain project; support on receptor reconstitution technology will come from Sunahara. Putative technical obstacles such as unspecific WNT-lipid binding will be bypassed by using WNT-mimicking peptides and WNT fragments. Alternative routes may include established WNT-FZD binding assays in crude membrane preparations.

Key publications:

  1. Halleskog C, Dijksterhuis JP, Kilander MB, Becerril-Ortega J, Villaescusa JC, Lindgren E, Arenas E, Schulte G. (2012) Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation. J Neuroinflammation, 9:111.
  2. Schulte G. (2010) International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors. Pharmacol Rev, 62(4):632-67.
  3. Bryja V, Gradl D, Schambony A, Arenas E, Schulte G. (2007) Beta-arrestin is a necessary component of Wnt/beta-catenin signaling in vitro and in vivo. Proc Natl Acad Sci.,104(16):6690-5.