Integrated analysis of receptor activation and downstream signaling with EXTassays

Nat Methods. 2010 Jan;7(1):74-80. doi: 10.1038/nmeth.1407. Epub 2009 Dec 13.

Abstract

The ability to measure multiple cellular signaling events is essential to better understand the underlying complex biological processes that occur in living cells. Microarray-based technologies are now commonly used to study changes in transcription. This information, however, is not sufficient to understand the regulatory mechanisms that lead to gene expression changes. Here we present an approach to monitor signaling events upstream of gene expression. We coupled different reporter gene assays to unique expressed oligonucleotide tags (EXTs) that serve as identifiers and quantitative reporters. Multiple EXT reporters can be isolated as a pool and analyzed by hybridization to microarrays. To test the feasibility of our approach, we integrated complementation assays based on a protease from tobacco etch virus (TEV protease) and transcription factor activity profiling. Thereby, we simultaneously monitored Neuregulin-dependent mouse ErbB receptor tyrosine kinase dimerization, effector recruitment and downstream signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Cell Survival
  • Electronic Data Processing
  • Enzyme Activation
  • Expressed Sequence Tags
  • Gene Expression Profiling
  • Gene Library
  • Genes, Reporter / genetics*
  • Genetic Complementation Test
  • Luciferases / analysis
  • Luciferases / genetics
  • Luciferases / metabolism
  • Mice
  • Neuregulins / genetics
  • Neuregulins / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Oligonucleotides / genetics
  • PC12 Cells
  • Phosphorylation
  • Protein Multimerization
  • Rats
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / metabolism*
  • Signal Transduction*
  • Transcription Factors / metabolism

Substances

  • Neuregulins
  • Oligonucleotides
  • Transcription Factors
  • Luciferases
  • Receptor, ErbB-2