Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 1, Pages 64-69Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0710487105
Keywords
cellular assays; G protein-coupled receptor; protein interaction
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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.
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