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.

Fig. 1.

Pharmacological profiles of an rM₃Δi3 receptor mutant selected during directed molecular evolution for CNO responsiveness. (A) Experimental design for directed evolution of mammalian GPCRs in yeast to create DREADDs. (1) Libraries of randomly mutated rM₃Δi3 receptors were produced by mutagenic PCR; (2) yeast-expressing mutant receptors activated by synthetic ligands (e.g., CNO) were selected for by growth on nutrient deficient medium; (3) mutants were verified by secondary liquid growth assays in 96-well plates; (4) plasmid DNA was isolated from yeast; (5) clones were retransformed into yeast to pharmacologically profile mutants by liquid growth assays, and those with desirable properties were sequenced and remutagenized for subsequent rounds of selection to yield receptors with higher potency. (B) Optical density at 650 nm of liquid cultures of yeast transformed with either wild type, clone “G2” (first library, 10 μM clozapine screen), clone “9” (second library, 1 μM CNO screen), or clone “118” (third library, 5-nM CNO screen) rM₃Δi3 receptors incubated with ACh (■), clozapine (□), or CNO (○). Data shown are mean ± SEM values of a representative experiment performed with two independent yeast transformants grown for each clone.

Fig. 2.

Focused screening of hM₃ receptor mutants to optimize CNO stimulation of PI hydrolysis in HEK T cells. (A) Receptor-activated PI hydrolysis in HEK T cells transfected with a third-generation clone “118” (□) identified by the yeast screen and wild-type rM₃Δi3 receptor (■) treated with ACh (Upper) or CNO (Lower). (B) Similarly, wild-type (■) human M₃ receptors with the indicated single Y149H (□) and Y149C (●) or multiple mutations [Y149C, A239G (○); hM₃ DREADD], found in yeast screen clones, were transiently expressed in HEK T cells to measure receptor activation after treatment with either ACh (Upper) or CNO (Lower). Data of accumulated radiolabeled inositol 1-phosphate ([3H]-IP₁) are normalized to maximal ACh-mediated response in HEK T cells expressing either wild-type rM₃Δi3 (A) or wild-type hM₃ (B) receptors. Values shown are mean ± SEM from representative assays performed in duplicate.

Fig. 3.

Functional characterization of HA-epitope tagged wild-type and DREADD hM₃ receptors in immortalized hPASMC. (A) Drug-induced PI hydrolysis in immortalized hPASMCs stably expressing wild-type (hM₃) or DREADD (hM₃D) receptors. Shown are mean ± SEM values of a representative experiment performed in duplicate comparing [3H]-IP₁ accumulation after ACh or CNO treatment of hM₃ (■ or ●, respectively) or hM₃D (□ and ○, respectively) cells. (B) Calcium mobilization resulting from delivery of ACh or CNO to hM₃ (■ or ●, respectively) or hM₃D (□ or ○, respectively) expressing immortalized hPASMCs. A representative experiment, performed in quadruplicate, with mean values of Ca²⁺ mobilization in relative fluorescent units (RFU), is shown. (C and D) A representative experiment determining change in ERK-1/2 phosphorylation compared with p90 ribosomal S6 kinase loading control after incubating 1 μM of the indicated drugs for 5 min with immortalized hPASMCs expressing either wild-type (C) or DREADD hM₃ (D) receptors by immunoblot (Upper) with quantification of ERK-1/2 phosphorylation (Lower) from three independent experiments with significant differences (∗, P < 0.001) between drug and vehicle treatment as determined by one-way ANOVA is shown.

Fig. 4.

Transformation of CNO response in mACh receptor family members. (A) Representative Western blots detecting either phosphorylated ERK-1/2 or, as a loading control, p90 ribosomal S6 kinase 1, after a 5-min application of a 1 μM final concentration of the indicated drugs to immortalized hPASMCs expressing wild-type or DREADD hM₁, hM₂, hM₄, and hM₅ receptors, as indicated. (B) Ca²⁺ mobilization response in immortalized hPASMCs expressing either the G_q/11-coupled hM₃ (black symbols; Left) or the G_i/o-coupled receptors hM₂ and hM₄ (blue and red symbols, respectively; Right) is shown. Cells were treated with ACh after overnight incubation with either vehicle control (solid symbols) or pertussis toxin or pertussis toxin (open symbols) on receptor-mediated Ca²⁺ release. Values shown are mean ± SEM from a representative experiment performed in triplicate.

Fig. 5.

Characterization of hM₄ DREADD on GIRK channel activation, membrane hyperpolarization, and neuronal silencing. (A) Sample traces of receptor-induced GIRK currents in HEK293 cells cotransfected with GIRK1/2 channel subunits and either hM₄ (Upper) or hM₄D (Lower) receptors and treated with either 10 μM CCh or CNO at a holding potential of −60 mV as described in SI Materials and Methods. (B) Comparison of induced GIRK channel currents at −60 mV when coexpressed with either hM₄ or hM₄D receptors. (C) Sample traces of CCh- and CNO-induced voltage changes in cultured hippocampal neurons infected with either hM₄ (Upper) or hM₄D (Lower). (D) Summary of the hM₄- and hM₄D-induced voltage changes by CCh and CNO. (E) Sample traces of hippocampal neurons spontaneously firing action potentials. In the presence of hM₄D receptors (lower trace), application of CNO induces hyperpolarization and neuronal silencing. (F) Comparison between the resting membrane potential of hM₄D receptor-expressing and control hippocampal neurons, indicating that the expression of hM₄D receptors does not change the resting membrane potential without activation of the receptor. Number of cells tested is indicated in parentheses with mean ± SEM shown.