以酵母为模型对人G蛋白偶联受体进行信号传导分析

Yasuyuki  Nakamura; Jun  Ishii; Akihiko  Kondo

doi:10.21769/BioProtoc.1206

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Peer-reviewed

Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast

以酵母为模型对人G蛋白偶联受体进行信号传导分析

Yasuyuki Nakamura

Jun Ishii

Akihiko Kondo email

发布: 2014年08月20日第4卷第16期 DOI: 10.21769/BioProtoc.1206 浏览次数: 10387

评审: Belen SanzMaureen WirschellAnonymous reviewer(s)

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Abstract

G-protein-coupled receptors (GPCRs) are the largest group of cell-surface proteins and are major molecular targets for drug development. The protocol described herein is for the detection of human GPCR signaling in the yeast Saccharomyces cerevisiae. Using Zoanthus sp. green fluorescent protein (ZsGreen) as the reporter, engineered yeast cells expressing human GPCRs emit strong fluorescence in response to stimuli leading to receptor signal activation. This assay method would allow screening for agonistic ligands and critical mutations required for human GPCR signaling.

Keywords: Signaling assay (信号检测)

G-protein-coupled receptor (G蛋白偶联受体)

Biosensor (生物传感器)

Yeast (酵母)

Human GPCR (人类GPCR)

Materials and Reagents

Engineered Saccharomyces cerevisiae strain in which the ZsGreen reporter genes were integrated into the genome (IMFD-72ZsD: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sst2Δ::AUR1-C ste2Δ::LEU2 fig1Δ::ZsGreen his3Δ::P_FIG1-ZsGreen far1Δ gpa1Δ::Gi3tp) (Nakamura et al., 2013)
Note: The expression of ZsGreen is controlled by the signal-responsive FIG1 promoter.
Multi-copy expression plasmid (pGK421 containing the PGK1 promoter, 2 μ origin and MET15 marker) (Togawa et al., 2010) encoding the GPCR of interest [e.g., somatostatin receptor subtype-5 (SSTR5); somatostatin receptor subtype-2 (SSTR2); or neurotensin receptor type-1 (NTSR1)] (Ishii et al., 2012; Ishii et al., 2014)
GPCR ligands [e.g., somatostatin (SST) (Merck KGaA, Calbiochem^®, catalog number: 51110-01-1 ) and neurotensin (NTS) (Merck KGaA, Calbiochem^®, catalog number: 39379-15-2)]
10 mg/ml carrier DNA (Takara Bio Company, Clontech, catalog number: 630440 )
Tris (hydroxymethyl) aminomethane (Tris^.HCl) (Nacalai Tesque, catalog number: 35409-45 )
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA 2Na∙2H₂O) (Nacalai Tesque, catalog number: 15111-45 )
1 M Hydrochloric acid (HCl) (Nacalai Tesque, catalog number: 37314-15 )
Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883 )
Acetic acid (Nacalai Tesque, catalog number: 00212-85 )
Polyethylene glycol (PEG) #4000 (Nacalai Tesque, catalog number: 28221-05 )
DMSO (Nacalai Tesque, catalog number: 13445-74 )
BD FACSFlow sheath fluid (BD, catalog number: 342003 )
Immersion oil (Olympus, catalog number: IMMOIL-F30CC )
Distilled water (dH₂O)
Yeast extract (Nacalai Tesque, catalog number: 15838-45 )
Peptone (BD, catalog number: 211677 )
D-Glucose (Nacalai Tesque, catalog number: 16806-25 )
Yeast nitrogen base without amino acids (YNB) (BD, catalog number: 291940 )
L-Histidine (Nacalai Tesque, catalog number: 18116-92 )
L-Leucine (Nacalai Tesque, catalog number: 20327-62 )
Uracil (Nacalai Tesque, catalog number: 35824-82 )
3-(N-Morpholino)-2-hydroxypropanesulfonic acid (Mopso) (Nacalai Tesque, catalog number: 23421-64 )
Sodium hydroxide (NaOH) (Nacalai Tesque, catalog number: 31511-05 )
Agar (Nacalai Tesque, catalog number: 01028-85 )
10 x TE buffer (see Recipes)
10 x LiAc (see Recipes)
50% PEG (see Recipes)
TE/LiAc solution (see Recipes)
LiAc/PEG solution (see Recipes)
YPD medium (see Recipes)
Synthetic dextrose (SD) selective medium supplemented with histidine, leucine and uracil (see Recipes)
SDM71 selective medium supplemented with histidine, leucine and uracil (see Recipes)

Equipment

16.5 x 105 mm test tubes (AGC Techno Glass, catalog number: 9820TST16.5-105NP )
10-ml conical flasks (AGC Techno Glass, catalog number: 4980FK10 )
2-ml microcentrifuge tubes (WATSON, catalog number: 332-720C )
1.5-ml microcentrifuge tubes (WATSON, catalog number: 131-815C )
96-well cell culture plate (Corning, catalog number: 3596 )
Microplate sealing tape (AS ONE Corporation, catalog number: 1-6774-05 )
12.5 x 75 mm test tubes with 2-position caps (B & M Equipment, catalog number: 222-2036-050 )
Microscope glass slides (Matsunami Glass, catalog number: S091150 )
18 x 18 mm coverslips (Matsunami Glass, catalog number: C218181 )
Centrifuge (Eppendorf, model: MiniSpin plus )
Block incubator (Astec Industries, model: BI-516C )
Shaking incubator for test tubes and conical flasks (TAITEC, model: BR-43FL )
Shaking incubator for a 96-well cell culture plate (TAITEC, model: M∙BR-022UP )
Spectrophotometer (Shimadzu, model: UVmini-1240 )
BD FACSCanto II flow cytometer (BD)
Fluorescence microscope (Keyence Corporation, model: BZ-9000 )
100x objective lens (Nikon Corporation, model: CFI Plan Apo VC 100x H )
BZ filter cube (excitation filter, absorption filter, dichroic mirror) (Keyence Corporation, model: OP-66836 GFP-BP )

Software

BD FACSDiva software (v5.0) (BD)

Procedure

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Nakamura, Y., Ishii, J. and Kondo, A. (2014). Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast. Bio-protocol 4(16): e1206. DOI: 10.21769/BioProtoc.1206.