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Generation of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) Using Directed Molecular Evolution

Ying Pei1,  Shuyun Dong1,  Bryan L. Roth1

1University of North Carolina, Chapel Hill, North Carolina

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
UNIT 4.33
DOI: 
10.1002/0471142301.ns0433s50
Online Posting Date: 
January, 2010
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Abstract

G protein–coupled receptors (GPCRs) and their signal transductions are important for both physiological and pathological processes in neuron systems. Neuronal GPCRs activated by synthetic ligands have been created by designed mutagenesis for studying their functions and signal pathways. However, these engineered GPCRs have problems, such as their high constitutive activity. To overcome this drawback, a new generation of receptors termed designer receptors exclusively activated by designer drugs (DREADDs), have been designed. DREADDs are exclusively activated by synthetic ligands, but are insensitive to their endogenous ligand and have no constitutive activity, which provides the ability to selectively modulate signal transduction of certain GPCRs in vitro and in vivo. This protocol provides detailed instructions for creating DREADDs using directed molecular evolution. The procedures to generate DREADDS include GPCR functional expression in yeast, mutant GPCR library generation, and high-throughput yeast screening. These methods are general and suitable for any GPCRs that can be functionally expressed in yeast. Curr. Protoc. Neurosci. 50:4.33.1-4.33.25. © 2010 by John Wiley & Sons, Inc.

Keywords: DREADD; RASSL; directed molecular evolution; synthetic biology; chemical biology

     
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Expression and Testing of the Function of the Target GPCR in Yeast
  • Basic Protocol 2: Generating Yeast Mutant Libraries Expressing Randomly Mutated Receptors
  • Basic Protocol 3: Yeast Mutant Library Screening and Liquid Yeast Growth Assays
  • Basic Protocol 4: Determine the Mutation Site(s) and Confirm the Pharmacological Profile of Selected Candidates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
  • Topics
    • Pharmacology and Drug Discovery
    • Cell Biology
    • Intermolecular Interactions
    • Neuroscience
     
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Materials

Basic Protocol 1: Expression and Testing of the Function of the Target GPCR in Yeast

 Materials
  • Target GPCR
  • Expression vector (e.g., p416GPD)
  • YAPD agar plates (see recipe)
  • Yeast strain (e.g., YB1)
  • YAPD (yeast extract/adenine/peptone/dextrose) medium (see recipe)
  • –URA agar plates: synthetic complete (SC) medium plate lacking uracil (see recipe)
  • Herring sperm DNA (e.g., Promega, cat. no. D181A)
  • 40% (w/v) polyethylene glycol (PEG) 4000 (e.g., Sigma, cat. no. 95904-1kg-F) made in 0.1 M lithium acetate (see recipe), filter sterilized
  • 0.1 M lithium acetate in TE buffer, pH 7.5 (see recipe), filter-sterilized
  • –URA medium: synthetic complete (SC) medium lacking uracil (see recipe)
  • 30% (w/v) glycerol, sterile
  • Liquid growth assay medium (see recipe)
  • Test (drug) compounds
  • 15- and 50-ml tubes, sterile
  • Humidified 25°C and 30°C cell culture incubators
  • Temperature-adjustable incubator shaker
  • 1.5-ml microcentrifuge tubes, sterile
  • 42°C water bath
  • 96-well flat-bottom plates with lids, low attachment (e.g., Costar, cat. no. 3937), sterile
  • Microplate reader
  • Parafilm
  • Plate shaker
  • Multi-channel pipettors (8-channel and 12-channel) and sterile pipet tips
  • Additional reagents and equipment for performing molecular biology techniques (e.g., see appendix 1A)

Basic Protocol 2: Generating Yeast Mutant Libraries Expressing Randomly Mutated Receptors

 Materials
  • GeneMorph II random mutagenesis kit (Stratagene, cat. no. 200550)
  • Yeast construct expressing the wild-type target GPCR (sequence and functions confirmed by Basic Protocol 1)
  • PCR primers (Fig. 4.33.4)
  • Agarose gel
  • QIAquick gel extraction kit (Qiagen, cat. no. 28704)
  • Restriction enzymes and 10× buffers
  • Calf intestine alkaline phosphatase (CIP)
  • 1-kb DNA ladder
  • Appropriate yeast reporter strains for transformation (e.g., YB1 for Gi-coupled receptors)
  • YAPD medium (see recipe)
  • –URA agar plates (see recipe)
  • Drug selection agar plates (see recipe)
  • 0.1 M lithium acetate in TE buffer (see recipe), sterile
  • Herring sperm DNA (e.g., Promega, cat. no. D181A)
  • 40% (w/v) PEG 4000 (e.g., Sigma, cat. no. 95904-1kg-F) made in 0.1 M lithium acetate (see recipe), sterile
  • Dimethyl sulfoxide (DMSO)
  • –URA medium (see recipe)
  • PCR thermal cycler
  • UV illuminator
  • 37°, 42°, and 55°C water baths
  • Temperature-adjustable incubator shaker
  • 30° and 37°C humidified cell culture incubator
  • Spectrophotometer
  • 50-ml centrifuge tubes, sterile
  • 1.5-ml microcentrifuge tubes, sterile
  • Sterile 14-ml conical tubes
  • Sterile 250-ml flasks
  • 100 × 15–mm sterile cell culture dishes

Basic Protocol 3: Yeast Mutant Library Screening and Liquid Yeast Growth Assays

 Materials
  • Vector plus insert transformants grown on drug selection agar plates (see Basic Protocol 2)
  • –URA medium (see recipe)
  • –URA agar plates (see recipe)
  • Liquid growth assay medium (see recipe)
  • Test compound
  • 96-well, flat-bottomed plate with lid, low attachment (e.g., Costar, cat. no. 3937)
  • Parafilm
  • Plate shaker
  • 25° and 30°C humidified cell culture incubator
  • 100 × 15–mm and 150 × 15–mm cell culture dishes, sterile
  • Multi-channel pipettors (8-channel and 12-channel) and sterile pipet tips
  • Centrifuge with 96-well plate adaptor
  • Microplate reader
  • 15- and 50-ml tubes, sterile

Basic Protocol 4: Determine the Mutation Site(s) and Confirm the Pharmacological Profile of Selected Candidates

 Materials
  • Yeast cultures (see Basic Protocol 3, step 21)
  • –URA medium (see recipe)
  • QIAprep spin miniprep kit (Qiagen, cat. no. 27104) containing:
    • Buffer P1
    • Buffer P2
    • Buffer N3
    • Spin columns
    • PB buffer
    • PE buffer
    • EB buffer
  • LB agar plates with appropriate antibiotic (see recipe)
  • One Shot TOP10 competent cells (Invitrogen, cat. no. C4040-10)
  • SOC medium (see recipe)
  • LB liquid with appropriate antibiotic (see recipe)
  • Restriction enzymes and 10× buffers
  • 1% agarose gel
  • Sterile 14-ml conical tubes
  • 25°, 30°, and 37°C humidified cell culture incubators with shakers
  • Sterile 1.5-ml microcentrifuge tubes
  • Acid-washed glass beads, 425- to 600-µm (e.g., Sigma, cat. no. G8772)
  • 42°C water bath
  • Electrophoresis apparatus and power source
  • Additional reagents and equipment for yeast transformations (see Basic Protocol 1)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 4.33.1
    An outline of experimental planning. Preliminary experiments that test whether the target GPCR can function in yeast cells (see Basic Protocol 1) are first performed, followed by screening compound selection (see Basic Protocol 1). Then, the mutant library is generated and screening cycles are done.

    View Image
  • Figure 4.33.2
    Liquid yeast growth assay for a small number of samples. See Basic Protocol 1, steps 9 to 23, for detailed descriptions.

    View Image
  • Figure 4.33.3
    Yeast cells show different growth curves on different days (EC50 is not a fixed number for yeast). Cells expressing hCB1 receptor (N-terminal truncated version) are utilized as an example in this figure. (A) Growth curves after 2-day incubation. The EC50 values with AEA and WIN55212-2 are 837 nM and 5691 nM, respectively. (B) Growth curves after 3-day incubation. The EC50 values with AEA and WIN55212-2 both decreased (223 nM and 676 nM, respectively) compared to that in A.

    View Image
  • Figure 4.33.4
    A diagram of the “gap repair” method. See Basic Protocol 2 for detailed description.

    View Image
  • Figure 4.33.5
    Liquid yeast growth assay designed for high-throughput screening. See Basic Protocol 3, steps 6 to 19, for detailed description.

    View Image
  • Figure 4.33.6
    An illustration of growth assay profiles of a potential mutant candidate for DREADD screening. (A) The selected pharmacologically inert drug cannot activate the wild-type receptor but can activate the mutant no. x. (B) The endogenous ligand shows significantly decreased potency on activating the mutant no. x compared to the wild-type receptor.

    View Image

Literature Cited

Literature Cited
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    Armbruster, B.N., Li, X., Pausch, M.H., Herlitze, S., and Roth, B.L. 2007. Evolving the lock to fit the key to create a family of G protein–coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. U.S.A. 104:5163-5168.
    Beukers, M.W. and Ijzerman, A.P. 2005. Techniques: How to boost GPCR mutagenesis studies using yeast. Trends Pharmacol. Sci. 26:533-539.
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    Ishii, J., Tanaka, T., Matsumura, S., Tatematsu, K., Kuroda, S., Ogino, C., Fukuda, H., and Kondo, A. 2008. Yeast-based fluorescence reporter assay of G protein-coupled receptor signaling for flow cytometric screening: FAR1-disruption recovers loss of episomal plasmid caused by signaling in yeast. J. Biochem. 143:667-674.
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