The genetic design of signaling cascades to record receptor activation

doi:10.1073/pnas.0710487105

The genetic design of signaling cascades to record receptor activation

*Howard Hughes Medical Institute, Department of Biochemistry and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, NY 10032; and
^‡Sentigen Biosciences, 3960 Broadway, New York, NY 10032

Contributed by Richard Axel, November 14, 2007 (received for review October 9, 2007)

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.

Footnotes

^††To whom correspondence should be addressed. E-mail: ra27{at}columbia.edu
Author contributions: G.B., W.S., G.H., Y.B., J.O., B.K., R.A., and K.J.L. designed research; G.B., W.S., G.H., Y.B., J.O., B.K., and K.J.L. performed research; G.B. contributed new reagents/analytic tools; W.S., G.H., Y.B., J.O., B.K., and K.J.L. analyzed data; and G.B., R.A., and K.J.L. wrote the paper.
↵ ^†Present address: Department of Neuroscience, Brown University, Providence, RI 02912.
↵ ^§Present address: Sirna Therapeutics, Merck & Co., Inc., San Francisco, CA 94158.
↵ ^¶Present address: ARMGO Pharma, Inc., New York, NY 10032.
↵ ^‖Present address: Orthobond Corporation, Monmouth Junction, NJ 08852.
↵**Present address: New York Structural Biology Center, New York, NY 10027.
↵ ^‡‡Present address: The Ellison Medical Foundation, Bethesda, MD 20814.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0710487105/DC1.
Freely available online through the PNAS open access option.