Gs-DREADD Knock-In Mice for Tissue-Specific, Temporal Stimulation of Cyclic AMP Signaling

Abstract

Hundreds of hormones and ligands stimulate cyclic AMP (cAMP) signaling in different tissues through the activation of G-protein-coupled receptors (GPCRs). Although the functions and individual effectors of cAMP signaling are well characterized in many tissues, pleiotropic effects of GPCR agonists limit investigations of physiological functions of cAMP signaling in individual cell types at different developmental stages in vivo. To facilitate studies of cAMP signaling in specific cell populations in vivo, we harnessed the power of DREADD (designer receptors exclusively activated by designer drugs) technology by creating ROSA26-based knock-in mice for the conditional expression of a Gs-coupled DREADD (rM3Ds-green fluorescent protein [GFP], or “GsD”). After Cre recombinase expression, GsD is activated temporally by the administration of the ligand clozapine N-oxide (CNO). In the same allele, we engineered a CREB-luciferase reporter transgene for noninvasive bioluminescence monitoring of CREB activity. After viral delivery of Cre recombinase to hepatocytes in vivo, GsD is expressed and allows CNO-dependent cAMP signaling and glycogen breakdown. The long-term expression of GsD in the liver results in constitutive CREB activity and hyperglycemia. ROSA26-Gs-DREADD mice can be used to study the physiological effects of cAMP signaling, acute or chronic, in liver or any tissue or cell type for which transgenic or viral Cre drivers are available.

DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology

“…DREADD (designer receptors solely activated by designer drugs) technology overcomes these limitations by using a combined genetic/ chemical approach. [8][9][10][11][12] These genetically modified designer GPCRs are unable to bind endogenous ligands, but can be activated by otherwise inert small molecules. 13 The most commonly used DREADDs represent mutant muscarinic receptors that can be specifically activated by the DREADD agonist clozapine-N-oxide (CNO) but not by their natural ligand acetylcholine.…”

“…13 The most commonly used DREADDs represent mutant muscarinic receptors that can be specifically activated by the DREADD agonist clozapine-N-oxide (CNO) but not by their natural ligand acetylcholine. 14 DREADDs show only minimal constitutive activity even at high levels of expression and no noteworthy response to acetylcholine, 14,15 Up to now, DREADD technology has been mostly used by neuroscientists, but DREADDs have also been expressed in pancreatic b-cells 15,16 , hepatocytes, 12 and cancer cells. 17 Here, we present the first transgenic mouse line expressing a functional Gq-coupled designer receptor, M3Dq (Dq) 15 under the transcriptional control of the muscle creatine kinase (MCK) promoter.…”

Skeletal Muscle–Specific Activation of Gq Signaling Maintains Glucose Homeostasis

“…DREADDs represent mutant GPCRs that are no longer activated by any endogenous ligands but can be selectively activated by clozapine-N-oxide (CNO), a synthetic ligand that is otherwise pharmacologically inert (10,11). During the past few years, we and other investigators have successfully used DREADD technology to study the in vivo metabolic effects of activating distinct functional classes of GPCRs in several metabolically relevant cell types, including pancreatic b-cells (12,13), hepatocytes (14)(15)(16), and adipocytes (17).…”

Red blood cell β-adrenergic receptors contribute to diet-induced energy expenditure by increasing O2 supply

“…Mice lacking all 3 β-ARs (β-less) were on FVB background and provided by Bradford Lowell of Harvard Medical School (Boston, Massachusetts, USA) (13). Conditional Gs-DREADD mice (27) and EpoR-Cre mice (28,29) were obtained from Rebecca Berdeaux and Yang Xia of the University of Texas Medical School at Houston. The mice were crossed to generate rbc-GsD.…”

“…DREADDs are mutated muscarinic GPCRs with selectivity for individual G protein effector pathways (Gs, Gi, or Gq) and only respond to the otherwise inert ligand clozapine N-oxide (CNO) (26). ROSA26-LSL-Gs-DREADD animals enable temporal control of cAMP signaling in selective cell types using cell type-specific Cre drivers (27). EpoR-Cre mice were used to achieve rbc-specific expression of Cre (28,29).…”