Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand
- Departments of *Biochemistry,
- †Neurosciences, and
- §Psychiatry, Case Western Reserve University School of Medicine, Cleveland, OH 44106;
- ‡Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543-8000; and
- ¶Department of Pharmacology, University of North Carolina Medical School, Chapel Hill, NC 27705
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Communicated by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, January 15, 2007 (received for review October 19, 2006)
Abstract
We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a Gi-coupled designer receptor in hippocampal neurons (hM4D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo.
Footnotes
- ‖To whom correspondence should be addressed. E-mail: bryan_roth{at}med.unc.edu
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Author contributions: B.N.A., X.L., S.H., and B.L.R. designed research; B.N.A. and X.L. performed research; M.H.P. contributed new reagents/analytic tools; B.N.A., X.L., and S.H. analyzed data; and B.N.A. and B.L.R. wrote the paper.
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The authors declare no conflict of interest.
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See Commentary on page 4777.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0700293104/DC1.
- Abbreviations:
- GPCR,
- G protein-coupled receptor;
- DREADD,
- designer receptors exclusively activated by a designer drug;
- CNO,
- clozapine-N-oxide;
- ACh,
- acetylcholine;
- CCh,
- carbachol;
- mAChR,
- muscarinic ACh receptor;
- hM1–5,
- human mAChR subtypes 1–5;
- hM1–5D,
- human mAChR DREADD subtypes 1–5;
- rM3Δi3,
- rat M3 receptor containing a third intracellular loop deletion;
- hPASMC,
- human pulmonary artery smooth muscle cell;
- PI,
- phosphatidylinositol;
- GIRK,
- G protein inward-rectifying potassium channel.
- © 2007 by The National Academy of Sciences of the USA
