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      The genetic design of signaling cascades to record receptor activation.

      Proceedings of the National Academy of Sciences of the United States of America

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

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          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.

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          Author and article information

          Journal
          18165312
          2224232
          10.1073/pnas.0710487105

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