Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

Biochem Biophys Res Commun. 2008 Apr 4;368(2):285-91. doi: 10.1016/j.bbrc.2008.01.088. Epub 2008 Jan 28.

Abstract

Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, differentiation, and cell fate. In the beta-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of beta-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the beta-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here, we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain of Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Frizzled Receptors / chemistry*
  • Frizzled Receptors / metabolism*
  • Green Fluorescent Proteins*
  • Humans
  • Protein Interaction Mapping / methods*
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / metabolism*
  • Signal Transduction / physiology*
  • Wnt Proteins / chemistry*
  • Wnt Proteins / metabolism*

Substances

  • FZD5 protein, human
  • Frizzled Receptors
  • Receptors, G-Protein-Coupled
  • WNT7A protein, human
  • Wnt Proteins
  • Green Fluorescent Proteins