The genetic design of signaling cascades to record receptor activation.
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.
Barnea
Gilad
G
Howard Hughes Medical Institute, Department of Biochemistry and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA.
Strapps
Walter
W
Herrada
Gilles
G
Berman
Yemiliya
Y
Ong
Jane
J
Kloss
Brian
B
Axel
Richard
R
Lee
Kevin J
KJ
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
D004268:DNA-Binding Proteins; D008024:Ligands; D020794:Receptor Protein-Tyrosine Kinases; D002118:Calcium