Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand

• Published in: Proceedings of the National Academy of Sciences - PNAS 2007-03, Vol.104 (12), p.5163-5168
• Main Authors: Armbruster, Blaine N, Li, Xiang, Pausch, Mark H, Herlitze, Stefan, Roth, Bryan L
• Format: Article
• Language: English
• Subjects: Animals; Bioavailability; Biochemistry; Biological Sciences; Cell Line, Transformed; Cell lines; Cellular biology; Chemical compounds; Cholinergic receptors; Clozapine - analogs & derivatives; Clozapine - pharmacology; Designer Drugs; Epitopes; Evolution, Molecular; G Protein-Coupled Inwardly-Rectifying Potassium Channels - metabolism; Gene expression; Genetic mutation; Hippocampus - drug effects; Humans; Hydrolysis - drug effects; Libraries; Ligands; Models, Molecular; Mutant Proteins - metabolism; Myocytes, Smooth Muscle - drug effects; Neurons; Neurons - drug effects; Pharmacology; Phosphatidylinositols - metabolism; Protein Engineering; Proteins; Pulmonary Artery - cytology; Pulmonary Artery - drug effects; Rats; Receptor, Muscarinic M3 - metabolism; Receptor, Muscarinic M4 - metabolism; Receptors; Receptors, G-Protein-Coupled - metabolism; Saccharomyces cerevisiae; Signal transduction; Smooth muscle myocytes; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0700293104

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.