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

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2031

Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

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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 protocol 1 )
PCR primers (Fig. )
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 G_i ‐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 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 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 protocol 1 )

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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 ) are first performed, followed by screening compound selection (see ). Then, the mutant library is generated and screening cycles are done.

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Figure 4.33.2 Liquid yeast growth assay for a small number of samples. See , steps 9 to 23, for detailed descriptions.

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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.

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Figure 4.33.4 A diagram of the “gap repair” method. See for detailed description.

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Figure 4.33.5 Liquid yeast growth assay designed for high‐throughput screening. See , steps 6 to 19, for detailed description.

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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.

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Videos

Literature Cited

Literature Cited
	Alexander, G.M., Rogan, S.C., Abbas, A.I., Armbruster, B.N., Pei, Y., Allen, J.A., Nonneman, R.J., Hartmann, J., Moy, S.S., Nicolelis, M.A., McNamara, J.O., and Roth, B.L. 2009. Remote control of neuronal activity in transgenic mice expressing evolved G protein‐coupled receptors. Neuron 63:27‐39.
	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.
	Bockaert, J. and Pin, J.P. 1999. Molecular tinkering of G protein–coupled receptors: An evolutionary success. Embo. J. 18:1723‐1729.
	Chung, K.S., Won, M., Lee, J.J., Ahn, J., Hoe, K.L., Kim, D.U., Song, K.B., and Yoo, H.S. 2007. Yeast‐based screening to identify modulators of G‐protein signaling using uncontrolled cell division cycle by overexpression of Stm1. J. Biotechnol. 129:547‐554.
	Conklin, B.R., Hsiao, E.C., Claeysen, S., Dumuis, A., Srinivasan, S., Forsayeth, J.R., Guettier, J.M., Chang, W.C., Pei, Y., McCarthy, K.D., Nissenson, R.A., Wess, J., Bockaert, J., and Roth, B.L. 2008. Engineering GPCR signaling pathways with RASSLs. Nat. Methods 5:673‐678.
	Coward, P., Wada, H.G., Falk, M.S., Chan, S.D., Meng, F., Akil, H., and Conklin, B.R. 1998. Controlling signaling with a specifically designed Gi‐coupled receptor. Proc. Natl. Acad. Sci. U.S.A. 95:352‐357.
	Dowell, S.J. and Brown, A.J. 2002. Yeast assays for G‐protein‐coupled receptors. Receptor Channel 8:343‐352.
	Erlenbach, I., Kostenis, E., Schmidt, C., Hamdan, F.F., Pausch, M.H., and Wess, J. 2001. Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast. J. Neurochem. 77:1327‐1337.
	Gether, U. 2000. Uncovering molecular mechanisms involved in activation of G protein–coupled receptors. Endocr. Rev. 21:90‐113.
	Guettier, J.M., Gautam, D., Scarselli, M., de Azua, I.R., Li, J.H., Rosemond, E., Ma, X., Gonzalez, F.J., Armbruster, B.N., Lu, H., Roth, B.L., and Wess, J. 2009. A chemical‐genetic approach to study G protein regulation of beta cell function in vivo. Proc. Natl. Acad. Sci. U.S.A. 106:19197‐19202.
	Hermans, E. 2003. Biochemical and pharmacological control of the multiplicity of coupling at G‐protein‐coupled receptors. Pharmacol. Ther. 99:25‐44.
	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.
	Josefsson, L.G. 1999. Evidence for kinship between diverse G‐protein‐coupled receptors. Gene 239:333‐340.
	Ladds, G. and Davey, J. 2004. Analysis of human GPCRs in fission yeast. Curr. Opin. Drug Discov. Devel. 7:683‐691.
	Ladds, G., Goddard, A., and Davey, J. 2005. Functional analysis of heterologous GPCR signalling pathways in yeast. Trends Biotechnol. 23:367‐373.
	Minic, J., Sautel, M., Salesse, R., and Pajot‐Augy, E. 2005. Yeast system as a screening tool for pharmacological assessment of G protein–coupled receptors. Curr. Med. Chem. 12:961‐969.
	Mumberg, D., Muller, R., and Funk, M. 1995. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119‐122.
	Pei, Y., Rogan, S.C., Yan, F., and Roth, B.L. 2008. Engineered GPCRs as tools to modulate signal transduction. Physiology 23:313‐321.
	Scearce‐Levie, K., Coward, P., Redfern, C.H., and Conklin, B.R. 2001. Engineering receptors activated solely by synthetic ligands (RASSLs). Trends Pharmacol. Sci. 22:414‐420.

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited