title: The genetic design of signaling cascades to record receptor activation.
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.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA.
authors.forename: Gilad
authors.initials: G
authors.lastname: Barnea
authors.forename: Walter
authors.initials: W
authors.lastname: Strapps
authors.forename: Gilles
authors.initials: G
authors.lastname: Herrada
authors.forename: Yemiliya
authors.initials: Y
authors.lastname: Berman
authors.forename: Jane
authors.initials: J
authors.lastname: Ong
authors.forename: Brian
authors.initials: B
authors.lastname: Kloss
authors.forename: Richard
authors.initials: R
authors.lastname: Axel
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Lee
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002468:Cell Physiological Phenomena; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D008024:Ligands; D020794:Receptor Protein-Tyrosine Kinases; D002118:Calcium