Journal
CELLULAR SIGNALLING
Volume 26, Issue 9, Pages 1943-1949Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cellsig.2014.05.012
Keywords
Fluorescence recovery after photobleaching; Heterotrimeric G proteins; GPCRs; Class Frizzled; WNT signaling
Categories
Funding
- Karolinska Institutet
- Swedish Research Council [K2008-68P-20810-01-4, K2008-333 68X-20805-01-4, K2012-67X-20805-05-3]
- Swedish Cancer Society [CAN 2008/539, 2011/690]
- Knut & Alice Wallenberg Foundation [KAW2008.0149]
- Board of Doctoral Education at Karolinska Institutet
- Engkvist's Foundations
- Foundation Lars Hiertas Minne
- Program KI-MU [CZ.1.07/2.3.00/20.0180]
- European Social Fund
- state budget of the Czech Republic
- Swedish Royal Academy of Sciences/Foundation Hierta-Retzius Fond
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The WNT receptors of the Frizzled family comprise ten mammalian isoforms, bind WNT proteins and mediate downstream signaling to regulate stem cell fate, neuronal differentiation, cell survival and more. WNT-induced signaling pathways are either beta-catenin-dependent or -independent, thereby dividing the 19 mammalian WNT proteins into two groups. So far hardly any quantitative, pharmacological information is available about WNT-FZD interaction profiles, affinities or mechanisms of signaling specification through distinct WNT/FZD pairings. This lack of knowledge originates from difficulties with WNT purification and a lack of suitable assays, such as ligand binding assays and FZD activity readouts. In order to minimize this gap, we employ fluorescence recovery after photobleaching (FRAP) to investigate WNT effects on the lateral mobility of FZD(6)-GFP in living cells. Pharmacological uncoupling of heterotrimeric G proteins by pertussis toxin and N-ethylmaleimide argues that changes in FZD(6) mobility are related to putative precoupling of heterotrimeric G(i/o), proteins to FZD(6). We show that recombinant WNT-1, -2, 3A, -4, -5A, -7A, -9B and -10B affect FZD(6) surface mobility and thus act on this receptor. WNT-5B and WNT-11, on the other hand, have no effect on FZD(6) mobility and we conclude that they do not act through FZD(6). We introduce here a novel way to assess WNT-FZD interaction by live cell imaging allowing further mapping of WNT-FZD interactions and challenging previous experimental limitations. Increased understanding of WNT-FZD selectivity provides important insight into the biological function of this crucial signaling system with importance in developmental biology, stem cell regulation oncogenesis, and human disease. (C) 2014 Elsevier Inc. All rights reserved.
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