![]() ![]() In this context, we sought to develop designer receptors exclusively activated by a designer drug (DREADD), or simply “designer” receptors, which represent receptors that are activated solely by a synthetic ligand(s) possessing minimal or no biologic activity. RASSLs, as in the case of Ro1, have been demonstrated to be valuable tools ( 4, 6) however, because the synthetic ligand frequently has high affinity and/or potency at the native receptor ( 5, 7, 8), this potentially limits their usefulness in vivo, at least in tissues with a wild-type receptor present. Such mutant receptors, like Ro1, have been classified as receptors activated solely by synthetic ligands (RASSLs), because they are activated by synthetic ligands but not by their endogenous ligands ( 5). At the forefront of such modified GPCRs is Ro1, a G i/o-coupled κ opioid receptor activated by a synthetic but not a native ligand, which has been conditionally expressed in transgenic mice to study cardiac function after its selective activation ( 4). One approach to this problem has been to rationally modify receptors to favor synthetic over natural substrate/ligand recognition, and subsequently, these mutant proteins have been used as bio-tools to study protein function in complex biological environments ( 2, 3). Selective activation of individual GPCR subtypes in a defined tissue, in either a knockout or wild-type animal, is currently problematic but, if possible, would serve to complement present findings by providing novel insights into disease states resulting from overstimulation of certain signaling pathways. Knowledge of the roles of the individual family members is being bolstered by the ongoing creation of knockout mice for many GPCRs. Genetic studies are frequently limited to loss-of-function phenotypes, whereas nonselectiveness of a drug often interferes with interpretation of pharmacological studies. However, the potential of this family is restricted by our ability to assess their function, which currently involves transgenic, knockout, and/or in vivo studies with selective drugs. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo.īecause of the assorted cellular responses directed by them, their number, and the ease of which they are pharmacologically screened, the superfamily of G protein-coupled receptors (GPCRs) is one of the most therapeutically important targets in the proteome ( 1). ![]() ![]() We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. We also expressed a G i-coupled designer receptor in hippocampal neurons (hM 4D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. 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. 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 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). ![]()
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