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

Detection of ligand dependent FZD conformational changes

Early stage researcher 7 (ESR7) project

Supervision: Carsten Hoffmann

Lab webpage: http://www.pharmakologie.uni-wuerzburg.de/startseite/arbeitsgruppen/ag_hoffmann/

Aim:
Understanding mechanisms of Frizzled (FZD) activation using conformational sensitive receptor sensors.

Methodology:
ESR7 will investigate the kinetics of ligand binding especially for FZD5 in living cells using FRET-microscopy and real time measurements at the millisecond time-scale (collaboration Maurice). These data will be combined with data from ligand binding techniques (private sector secondment: Attana, collaboration UPE). ESR7 will characterise the signalling behaviour of ligands using different FRET-probes selective for G protein dependent or independent signalling pathways (international secondment: Schulte). ESR7 will correlate the observed conformational changes with onset of signalling to gain insight into the activation process by different WNTs and how this information is translated into signalling specification.

Collaborators:
Schulte, Maurice, Attana, UPE

Project goals:
ESR7 will detect conformational changes using FZD sensor and gain of mechanistic insights into agonist modulation of receptor conformation

Risk assessment and contingency plans:
Sensitivity in FRET to differentially detect the conformational receptor changes relies on the appropriate selection of FlAsH site localization in loop regions. Functional receptor constructs for GPCRs will lead the design.

Key publications:

1. Hlavackova V, Zabel U, Frankova D, Bätz J, Hoffmann C, Prezeau L, Pin JP, Blahos J and Lohse MJ (2012) Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1. Sci Signal 5(237):ra59.

2. Hoffmann C, Gaietta G, Bünemann M, Adams SR, Oberdorff-Maass S, Behr B, Vilardaga JP, Tsien RY, Ellisman MH and Lohse MJ (2005) A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods 2(3):171-176.

3. Hoffmann C, Gaietta G, Zürn A, Adams SR, Terrillon S, Ellisman MH, Tsien RY and Lohse MJ (2010) Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat Protoc 5(10):1666-1677.