title: Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.
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 G(i)-coupled designer receptor in hippocampal neurons (hM(4)D) 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.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Blaine N
authors.initials: BN
authors.lastname: Armbruster
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Mark H
authors.initials: MH
authors.lastname: Pausch
authors.forename: Stefan
authors.initials: S
authors.lastname: Herlitze
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Roth
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D003024:Clozapine / Q000031:analogs & derivatives / Q000494:pharmacology; D015198:Designer Drugs; D000939:Epitopes; D019143:Evolution, Molecular; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D006624:Hippocampus / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D010716:Phosphatidylinositols / Q000378:metabolism; D015202:Protein Engineering; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D043588:Receptor, Muscarinic M4 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012441:Saccharomyces cerevisiae
chemical_list: D015198:Designer Drugs; D000939:Epitopes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D008024:Ligands; D050505:Mutant Proteins; D010716:Phosphatidylinositols; D043587:Receptor, Muscarinic M3; D043588:Receptor, Muscarinic M4; D043562:Receptors, G-Protein-Coupled; D003024:Clozapine; C079149:clozapine N-oxide