Engineering receptors activated solely by synthetic ligands (RASSLs)

doi:10.1016/S0165-6147(00)01743-0

Trends in Pharmacological Sciences

Volume 22, Issue 8, 1 August 2001, Pages 414-420

https://doi.org/10.1016/S0165-6147(00)01743-0 Get rights and content

Abstract

The functional and molecular diversity of G-protein-coupled receptors presents a significant challenge to understanding the connection between a single receptor signaling pathway and a specific physiological or pathological response. To gain control over the timing and specificity of a G-protein signal, receptors activated solely by synthetic ligands (RASSLs) have been developed. These engineered receptors no longer respond to endogenous peptides, but can still be activated by a specific small-molecule drug. Further control over the location of the signal can be achieved by using RASSLs in conjunction with tissue-specific expression systems in vivo. Existing RASSLs have clarified the role of G_i signaling in cardiac physiology and are currently being used to study cardiomyopathy, muscle remodeling, sensory transduction and complex neurobehavioral responses.

Section snippets

RASSL construction

For our first RASSL, we modified the KOR. This receptor responds to endogenous peptides and signals via the well-characterized G_i pathway. Because of the importance of this receptor family for pain modulation, the pharmaceutical industry has developed many high-affinity opioid receptor agonists. Small-molecule ligands of the KOR are structurally distinct from the endogenous peptide ligands, including dynorphin 11, 12. Unlike mu or delta opioid receptor agonists, KOR agonists are nonaddictive ¹³.

Control of RASSL expression in vivo

The expression of RASSLs in specific tissues of whole animals is a potentially valuable tool for studying both normal and pathological signaling cascades. To take full advantage of the signaling control offered by a RASSL, it is also important to control the timing and location of RASSL expression in vivo. To achieve this, we have used the tetracycline-controlled inducible expression system (tet system) developed by Gossen and Bujard ²⁰. Briefly, transgene expression is driven by a minimal

Controlling RASSL signaling in vivo

When the receptor is expressed, it should be functionally silent until the administration of spiradoline stimulates the RASSL and activates signaling pathways rapidly and specifically. G_i signaling in the heart has been shown to reduce heart rate ²⁶. Therefore, when Ro1 is expressed in the heart (using the α-myosin heavy chain promoter), receptor activation can be studied by simply measuring changes in heart rate. Wild-type animals have few KORs in the heart and therefore show no change in

Detecting RASSLs in vivo

For many experiments that use RASSLs to study the role of G_i signaling in vivo, it is essential to pinpoint the receptor localization, sometimes down to the subcellular level. To this end, we have developed several tagged RASSLs^*.

Small epitope tags have been added to the N-terminus of the RASSL protein for subsequent detection with antibody staining. Common tags such as hemagglutinin (HA)

Applications of RASSL technology

Acute activation of RASSLs expressed in discrete tissues or cell types will allow researchers to probe the role of receptor-mediated G_i signaling in normal cell function. Correspondingly, extended activation of RASSLs can be used to explore disease models related to hyperactive G-protein signaling.

Tissue-specific expression and activation of RASSLs

To date, RASSLs have been expressed in multiple tissues of transgenic mice (Table 1), including heart, liver, salivary glands, smooth muscle, adipose tissue and specific brain regions. Ro1-mediated G_i activation has been shown to slow heart rate ⁵. RASSLs are being used to study the effects of G proteins on smooth muscle contractility, and mouse lines that express RASSLs in specific brain regions are being used to study how activation of G_i signaling in specific brain nuclei can affect

Future directions: new RASSLs

Existing RASSLs will allow study of the effects of G_i-mediated signaling in specific tissues under specific circumstances. In the future, RASSLs could be made to control all the major G-protein signaling pathways, individually or in combination (G_s, G_i, G_q, G₁₂, G₁₃). These new RASSLs can be developed using many of the same principles used to develop the existing G_i-coupled RASSLs. The ideal RASSL would have orally available, nanomolar affinity agonists and antagonists that are safe for

References (34)

D.M. Spencer
Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization
Curr. Biol.
(1996)
C.D. Strader
Allele-specific activation of genetically engineered receptors
J. Biol. Chem.
(1991)
M.J. Millan
κ-Opioid receptors and analgesia
Trends Pharmacol. Sci.
(1990)
J-C. Xue
The third extracellular loop of the μ opioid receptor is important for agonist selectivity
J. Biol. Chem.
(1995)
F. Meng
Mapping the receptor domains critical for the binding selectivity of δ-opioid receptor ligands
Eur. J. Pharmacol.
(1996)
P. Coward
Chimeric G proteins allow a high-throughput signaling assay of G_i-coupled receptors
Anal. Biochem.
(1999)
G. Wittert
Tissue distribution of opioid receptor gene expression in the rat
Biochem. Biophys. Res. Commun.
(1996)
N.G. Bowery
Pertussis toxin decreases absence seizures and GABA_B receptor binding in thalamus of a genetically prone rat (GAERS)
Neuropharmacology
(1999)
A. Cravchik
Functional analysis of the human D₂ dopamine receptor missense variants
J. Biol. Chem.
(1996)
H. Möhler et al.
GABA_B receptors make it to the top – as dimers
Trends Pharmacol. Sci.
(1999)

G.J. Kilpatrick

7TM receptors: the splicing on the cake

Trends Pharmacol. Sci.

(1999)

C.D. Strader

Structure and function of G protein-coupled receptors

Annu. Rev. Biochem.

(1994)

A.M. Spiegel

Receptor–effector coupling by G proteins: implications for normal and abnormal signal transduction

Endocrinol. Rev.

(1992)

D. Hoyer

VII. International union of pharmacology classification of receptors for 5-hydroxytryptamine (serotonin)

Pharmacol. Rev.

(1994)

P. Coward

Controlling signaling with a specifically designed G_i-coupled receptor

Proc. Natl. Acad. Sci. U. S. A.

(1998)

C.H. Redfern

Conditional expression and signaling of a specifically designed G_i-coupled receptor in transgenic mice

Nat. Biotechnol.

(1999)

C.H. Redfern

Conditional expression of a G_i-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy

Proc. Natl. Acad. Sci. U. S. A.

(2000)

Cited by (48)

CNO Administration Increases Dopamine and Glutamate in the Medial Prefrontal Cortex of Wistar Rats: Further Concerns for the Validity of the CNO-activated DREADD Procedure
2022, Neuroscience
Citation Excerpt :
In contrast, the ability to modulate G-coupled protein receptors (GCPRs) has lagged because of the lack of specificity of compounds for these targets. In 2000, a novel procedure created receptors activated solely by synthetic ligands (RASSLs) was developed to target select GCPRs (Bishop et al., 2000; Scearce-Levie et al., 2001). The RASSLs procedure involved the mutation of native receptors with the desired goal to create a receptor with functions identical to the native receptor.
The chemogenetic procedure DREADD (designer receptor exclusively activated by designer drugs) is an inventive way to selectively affect g-coupled protein receptors. In theory, DREADD receptors are only activated by administering inert compounds, primarily clozapine N-oxide (CNO). Research has shown that CNO does not cross the blood–brain barrier, and CNO is converted back to clozapine and N-desmethylclozapine (N-Des) in the brain. Clozapine and N-Des have many neurological effects including alterations in glutamate and dopamine (DA) levels in multiple brain regions. The current study examined the effects of peripheral administration of CNO on glutamate and DA levels in the medial prefrontal cortex (mPFC). Wistar rats were administered CNO, and microdialysis samples were collected from the mPFC. Administration of CNO significantly increased glutamate (31–87%) and DA (65–126%), CNO-induced increases in DA occurred for a longer duration than glutamate, and that for the two highest doses of CNO there was a significant correlation between the increase in glutamate and DA in the mPFC. In the mPFC, CNO-induced increases in DA occurred at 0.5 mg/kg, while increases in glutamate were observed at doses greater than 1.0 mg/kg. The source of the DA and glutamate could be caused by activation of projection neurons or local effects. The data replicate findings that CNO is not an inert compound and that interpretation of CNO-activated DREADD findings should be done with caution. The data indicate that low (‘safe’) doses of CNO still have neurochemical effects and that controlling for the actions of clozapine/N-Des in CNO-DREADD studies has many concerns.
Novel designer receptors to probe GPCR signaling and physiology
2013, Trends in Pharmacological Sciences
Citation Excerpt :
Thus, to better understand the physiological and, potentially, pathophysiological roles of specific GPCR signaling pathways, an experimental system that allows the activation (in vivo) of a particular GPCR in a cell type- or tissue-specific fashion would be highly desirable. In this regard, the development of designer GPCRs referred to as RASSLs (receptors activated solely by synthetic ligand) has provided important novel tools to study the in vivo relevance of distinct GPCR signaling pathways [3,4]. In most cases, these first-generation RASSLs represent engineered GPCRs that can be efficiently activated by a preexisting synthetic drug but are much less sensitive to activation by its endogenous ligand.
Muscarinic receptor-based designer receptors have emerged as powerful novel tools to study G-protein-coupled receptor (GPCR) signaling and physiology. These new designer GPCRs, which are most frequently referred to as DREADDs (designer receptors exclusively activated by designer drug), are unable to bind acetylcholine, the endogenous muscarinic receptor agonist, but can be activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound, with high potency and efficacy. The various DREADDs differ primarily in their G protein coupling preference. More recently, an arrestin-biased DREADD has also been developed. The expression of DREADDs in distinct tissues or cell types has enabled researchers to study the outcome of selective stimulation of distinct GPCR (or arrestin) signaling pathways in a temporally and spatially controlled fashion in vivo. In this review, we provide an up-to-date snapshot of where this field currently stands and which important novel insights have been gained using this new technology.
Pharmacological profile of engineered 5-HT<inf>4</inf> receptors and identification of 5-HT<inf>4</inf> receptor-biased ligands
2013, Brain Research
Citation Excerpt :
To overcome this problem, different GPCRs have been engineered over the last decade. First, receptors activated solely by synthetic ligands (RASSLs) were generated by site-directed mutagenesis and Gq-, Gs- and Gi-coupled RASSLs are now available (Conklin et al., 2008; Coward et al., 1998; Scearce-Levie et al., 2001). These GPCRs are insensitive to their endogenous ligand and can be activated only by specific synthetic ligands.
G protein-coupled receptors (GPCRs) can activate simultaneously multiple signaling pathways upon agonist binding. The combined use of engineered GPCRs, such as the receptors activated solely by synthetic ligands (RASSLs), and of biased ligands that activate only one pathway at a time might help deciphering the physiological role of each G protein signaling. In order to find serotonin type 4 receptor (5-HT₄R) biased ligands, we analyzed the ability of several compounds to activate the G_s and G_q/11 pathways in COS-7 cells that transiently express wild type 5-HT₄R, the 5-HT₄R-D¹⁰⁰A mutant (known also as 5-HT₄-RASSL, or Rs1) or the 5-HT₄R-T¹⁰⁴A mutant, which modifies agonist-induced 5-HT₄R activation. This analysis allowed completing the pharmacological profile of the two mutant 5-HT₄Rs, but we did not find any biased ligand for the mutant receptors. Conversely, we identified the first biased agonists for wild type 5-HT₄R. Indeed, RS 67333 and prucalopride acted as partial agonists to induce cAMP accumulation, but as antagonists on inositol phosphate production. Moreover, they showed very different antagonist potencies that could be exploited to study the activation of the G_s pathway, with or without concomitant block of G_q/11 signaling.
This article is part of a Special Issue entitled Optogenetics (7th BRES)
Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons
2005, Neuron
Citation Excerpt :
Silencing by expression of K+ or Cl− channels has been useful in invertebrate and cultured neurons (Burrone et al., 2002; Nitabach et al., 2002; Paradis et al., 2001; White et al., 2001), but has yielded mixed results in the mammalian brain in vivo (Nadeau et al., 2000; Sutherland et al., 1999). To introduce temporal control, K+ or Cl− channels have been coupled to ligands without endogenous receptors (Cully et al., 1994; Lechner et al., 2002; Li et al., 2002; Scearce-Levie et al., 2001; Slimko and Lester, 2003; Slimko et al., 2002), but these promising approaches have as yet only been demonstrated in vitro. An elegant alternative involves blocking sodium channels by expression of membrane-tethered toxins (Ibanez-Tallon et al., 2004).
Inducible and reversible silencing of selected neurons in vivo is critical to understanding the structure and dynamics of brain circuits. We have developed Molecules for Inactivation of Synaptic Transmission (MISTs) that can be genetically targeted to allow the reversible inactivation of neurotransmitter release. MISTs consist of modified presynaptic proteins that interfere with the synaptic vesicle cycle when crosslinked by small molecule “dimerizers.” MISTs based on the vesicle proteins VAMP2/Synaptobrevin and Synaptophysin induced rapid (∼10 min) and reversible block of synaptic transmission in cultured neurons and brain slices. In transgenic mice expressing MISTs selectively in Purkinje neurons, administration of dimerizer reduced learning and performance of the rotarod behavior. MISTs allow for specific, inducible, and reversible lesions in neuronal circuits and may provide treatment of disorders associated with neuronal hyperactivity.
DREADDs in Epilepsy Research: Network-Based Review
2022, Frontiers in Molecular Neuroscience
Parvalbumin Role in Epilepsy and Psychiatric Comorbidities: From Mechanism to Intervention
2022, Frontiers in Integrative Neuroscience

View all citing articles on Scopus

View full text

Trends in Pharmacological Sciences

ReviewEngineering receptors activated solely by synthetic ligands (RASSLs)

Abstract

Section snippets

RASSL construction

Control of RASSL expression in vivo

Controlling RASSL signaling in vivo

Detecting RASSLs in vivo

Applications of RASSL technology

Tissue-specific expression and activation of RASSLs

Future directions: new RASSLs

Curr. Biol.

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Biol. Chem.

Eur. J. Pharmacol.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Neuropharmacology

J. Biol. Chem.

Trends Pharmacol. Sci.

Trends Pharmacol. Sci.

Structure and function of G protein-coupled receptors

Annu. Rev. Biochem.

Receptor–effector coupling by G proteins: implications for normal and abnormal signal transduction

Endocrinol. Rev.

VII. International union of pharmacology classification of receptors for 5-hydroxytryptamine (serotonin)

Pharmacol. Rev.

Controlling signaling with a specifically designed Gi-coupled receptor

Proc. Natl. Acad. Sci. U. S. A.

Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice

Nat. Biotechnol.

Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy

Proc. Natl. Acad. Sci. U. S. A.

Review
Engineering receptors activated solely by synthetic ligands (RASSLs)

Controlling signaling with a specifically designed G_i-coupled receptor

Conditional expression and signaling of a specifically designed G_i-coupled receptor in transgenic mice

Conditional expression of a G_i-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy