The Genetic Design of Signaling Cascades to Record Receptor Activation

Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):64-9. doi: 10.1073/pnas.0710487105. Epub 2007 Dec 28.

Abstract

We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biochemistry / methods*
  • Calcium / metabolism
  • Cell Line
  • Cell Physiological Phenomena
  • DNA-Binding Proteins / metabolism
  • Gene Expression Regulation
  • Genetic Techniques*
  • Humans
  • Ligands
  • Models, Biological
  • Models, Genetic*
  • Plasmids / metabolism
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Signal Transduction
  • Transcriptional Activation

Substances

  • DNA-Binding Proteins
  • Ligands
  • Receptor Protein-Tyrosine Kinases
  • Calcium