Preface
Key Outcomes of This Edition
EditorsΒ΄ Final Remarks
Acknowledgments
Contents
Contributors
Chapter 1: Isolation of Lipid Rafts by the Detergent-Based and Non-detergent-Based Methods for Localization of GPCRs with Immu...
1 Introduction
1.1 GPCR Signaling and Trafficking
1.2 Perturbation of Raft Stability
1.3 Changing the Cholesterol Content
1.4 Fluorescence Imaging
2 Materials
2.1 Detergent-Based Method
2.2 Detergent-Free Method
2.3 Cell Handling
2.4 Localization of GPCRs in Lipid Rafts
3 Methods
3.1 Isolation of Lipid Rafts
3.2 Isolation of Lipid Rafts Using the Detergent-Based Method
3.3 Isolation of Lipid Rafts Using the Detergent-Free Method
3.4 Localization of GPCR in Lipid Rafts Using Laser Scanning Confocal Microscopy
4 Notes
References
Chapter 2: Detection of GPCR mRNA Expression in Primary Cells Via qPCR, Microarrays, and RNA-Sequencing
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Equipment
2.3 Reagents and Kits
2.4 Consumables
2.5 Software Tools
3 Methods
3.1 mRNA Isolation from Cultured Cells
3.2 cDNA Synthesis
3.3 qPCR Primer Design (Bioinformatics Protocol)
3.4 Primer Validation
3.5 Independent qPCR
3.6 qPCR-Based TaqMan Array
3.7 RNA-Sequencing Data Analysis for GPCR Expression (Bioinformatics Protocol)
4 Notes
References
Chapter 3: Construction of Recombinant Cell Lines for GPCR Expression
1 Introduction
2 Materials
2.1 Growth and Storage of HEK293S-GnTI--TetR Cells
2.2 Construction of HEK293S GnTI--TetR Cells Inducibly Expressing GPCRs
2.3 Identification of Cell Lines Expressing Rhodopsin
2.4 Determination of Rhodopsin Expression Levels in HEK293S GnTI--TetR-Cell Lines Stably Transfected with the Bovine Rhodopsin...
3 Methods
3.1 Growth and Long-Term Storage of HEK293S-GnTI--tetR Cells
3.2 Transfection of HEK293S-GnTI--TetR Cells Using Calcium Phosphate/N,N[Bis(2-Hydroxyethyl)-2-Aminoethanesulphonic Acid] Prec...
3.3 Isolation and Expansion of G418-Resistant Colonies
3.4 Identification of Cell Lines Exhibiting High-Level Inducible Expression of Rhodopsin by Dot Blot and Immunodetection
3.5 Determination of Rhodopsin Expression Levels in HEK293S GnTI--TetR-Rho Cell Lines
4 Notes
References
Chapter 4: Recombinant Expression and Purification of Cannabinoid Receptor CB2, a G Protein-Coupled Receptor
1 Introduction
2 Materials
2.1 GPCR Expression in E. coli Cells
2.2 GPCR Expression in Expi293F Cells
2.3 Solubilization of GPCR in Detergent Micelles
2.4 Purification
3 Methods
3.1 Expression of GPCR in E. coli
3.2 Expression of GPCR in Mammalian Suspension Cell Culture
3.3 Preparation of Membranes
3.4 Solubilization of Recombinant GPCR
3.5 Ni-NTA Chromatography
3.6 Removal of Fusion Partners by Tobacco Etch Virus (TEV) Protease
3.7 Purification Via StrepTactin Affinity Tag
4 Notes
References
Chapter 5: Screening for Serotonin Receptor 4 Agonists Using a GPCR-Based Sensor in Yeast
1 Introduction
2 Materials
2.1 Plasmids and Strains
2.2 Yeast Media and Transformation Supplies
2.3 Fluorescent Microscopy
2.4 Luminescent Plate Reader Assay
2.5 Software
3 Methods
3.1 Preparation of 5-HTR4B-Sensing, Control Sensing, and 5-HTR4B-GFP Fusion Strains
3.2 Fluorescent Microscopy
3.3 GPCR Luminescence Assay
3.4 Secondary Assay: dose-response Curves and Z-score Analysis
4 Notes
References
Chapter 6: Immobilization of Olfactory Receptors Carried by Nanosomes onto a Gold Sensor Surface
1 Introduction
2 Materials
2.1 Isolation of Membrane Fraction
2.2 Immobilization
3 Methods
3.1 Preparation of Membrane Fraction Carrying ORI7
3.2 Nanosomes Preparation
3.3 Immobilization of ORs onto the Gold Sensor Surface
4 Notes
References
Chapter 7: Screening Methods for Cell-Free Synthesized GPCR/Nanoparticle Samples
1 Introduction
2 Materials
2.1 S30 and Heat-Shocked (HS-S30) Lysate Preparation
2.2 Membrane Scaffold Protein (MSP) Expression, Purification, and ND Assembly
2.3 Cell-Free Expression
2.4 Radioligand-Binding Assay
3 Methods
3.1 E. coli Extract Preparation
3.2 MSP Expression and Purification
3.3 Nanodisc Assembly
3.4 Basic Cell-Free Expression Protocol
3.5 Tuning of Co-translational GPCR Folding
3.6 Screening Ligand Libraries
3.7 Characterization of GPCR Ligand-Binding Characteristics
4 Notes
References
Chapter 8: Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labe...
1 Introduction
2 Materials
3 Methods
3.1 Recombinant Baculovirus Construction
3.2 Virus Amplification and Virus Titer Determination
3.3 Production of GPCR Displaying Budded Baculovirus Particles
3.4 FA Measurements in Multiwell Microplates: Determination of Receptor Concentration
3.5 FA Measurements in Multiwell Microplates: Competition Binding Experiments
4 Notes
References
Chapter 9: Bioluminescence in G Protein-Coupled Receptors Drug Screening Using Nanoluciferase and Halo-Tag Technology
1 Introduction
1.1 Conformational GPCR BRET-Based Biosensors
1.2 HTS-Suitability
1.3 Advantages and Limitations of BRET-Based Assays
1.4 Transferability of the Sensor Design to Other GPCRs
2 Materials
2.1 Cell Culture
2.2 Transfection Materials
2.3 Counting and Plating
2.4 Measurement Materials and Devices
2.5 Data Analysis
3 Methods
3.1 Cell Culture, Counting, and Transfection
3.2 Labeling and Plating
3.3 BRET Measurement
3.4 Data Analysis and Graphing
4 Notes
References
Chapter 10: Nanolu0ciferase-Based Complementation Assay to Detect GPCR-G Protein Interaction
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Cell Preparation and Measurement for Nanoluciferase Complementation Assay
2.3 Design of GPCRs and GΞ± Subunit Expression Constructs
2.3.1 GPCR
2.3.2 GΞ± Subunit
3 Methods
3.1 Cell Culture and Transfection
3.2 Cell Preparation and Measurement for Nanoluciferase Complementation Assay
4 Notes
References
Chapter 11: Imaging of Genetically Encoded FRET-Based Biosensors to Detect GPCR Activity
1 Introduction
1.1 GPCR Signaling
1.2 Genetically Encoded FRET Biosensors
1.3 FRET Sensors for GPCR Activity
1.3.1 Heterotrimeric G Proteins
1.3.2 Second Messengers
1.3.3 Rho GTPases
1.4 Limitations
2 Materials
2.1 Plasmids and Storage
2.2 Insertion of FRET Sensors in piggyBac Plasmids
2.3 Cells
2.4 Generation of FRET Sensor Stable Cell Lines
2.5 FACS Sorting
2.6 Expression of G Protein-Coupled Receptors
2.7 Transient Expression of FRET Biosensor and GPCR
2.8 Imaging FRET Response to Stimuli
2.9 Ratio-FRET Data Analysis
3 Methods
3.1 Plasmids and Storage
3.2 Inserting FRET Sensors in piggyBac Plasmids
3.3 Making FRET Sensor Stable Cell Lines
3.4 FACS Sorting
3.5 Transient Expression of G Protein-Coupled Receptors
3.6 Transient Expression of FRET Biosensor and GPCR
3.7 Imaging FRET Response to Stimuli
3.8 Ratio-FRET Data Analysis
4 Notes
References
Chapter 12: cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activati...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmids and Generation of BacMam Virus
2.3 cAMP Biosensor Protein Expression
2.4 Fluorescence Microplate Reader for the cAMP Assay
2.5 Equipment for Dilution Series of Ligands
2.6 Software
3 Methods
3.1 Cloning, Generation, and Collection of BacMam Virus
3.2 Determination of Virus Titer with Cell Size Change-Based Assay
3.3 Epac-SH188 Biosensor Protein Expression
3.4 cAMP Assay
3.5 Data Analysis
3.6 Time Frame
4 Notes
References
Chapter 13: FLIPR Calcium Mobilization Assays in GPCR Drug Discovery
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Calcium Mobilization Assay
2.3 Running the Assay
3 Methods
3.1 Preparation of Cells and Loading Dye (Adherent Cells)
3.2 Preparation of Cells and Loading Dye (Non-Adherent Cells)
3.3 Loading Cells
3.4 Preparation of Compound Plate
3.5 Reading Assay Plate
3.6 Data Analysis
4 Notes
References
Chapter 14: Live Cell Imaging and Optogenetics-Based Assays for GPCR Activity
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Transfections
2.3 DNA Constructs: GΞ²Ξ³ Translocation Assay for Gi-Coupled Receptor Activity
2.4 DNA Constructs: GΞ²Ξ³ Translocation Assay for Gq-Coupled Receptor Activity
2.5 DNA Constructs: GΞ²Ξ³ Translocation Assay for Gs-Coupled Receptor Activity
2.6 DNA Constructs: PIP3 Assay for Gi-Coupled Receptor Activity
2.7 DNA Constructs: PIP2 Hydrolysis Assay for Gq-Coupled Receptor Activity
2.8 DNA Constructs: cAMP Assay for Gs-Coupled Receptor Activity
2.9 DNA Constructs: Cell Migration Assay for Gi-Coupled Receptor Activity
2.10 Imaging
2.11 Reagents
3 Methods
3.1 Cell Culture
3.2 Transfections (See Note 28)
3.3 Imaging: GΞ²Ξ³ Translocation Assay for Gi-Coupled Receptor Activity
3.4 Imaging: GΞ²Ξ³ Translocation Assay for Gq-Coupled Receptor Activity
3.5 Imaging: GΞ²Ξ³ Translocation Assay for Gs-Coupled Receptor Activity
3.6 Imaging: PIP3 Assay for Gi-Coupled Receptor Activity
3.7 Imaging PIP2 Hydrolysis Assay for Gq-Coupled Receptor Activity
3.8 Imaging: cAMP Assay for Gs-Coupled Receptor Activity
3.9 Imaging: Cell Migration Assay for Gi-Coupled Receptor Activity
4 Notes
References
Chapter 15: Split-Tobacco Etch Virus (Split-TEV) Method in G Protein-Coupled Receptor Interacting Proteins
1 Introduction
1.1 The Split-TEV Method
1.2 Interactions with GPCRs
2 Materials
2.1 Plasmids
2.2 Cell Culture
2.3 Split-TEV Assay
2.4 Equipment
3 Methods
3.1 HeLa Cell Culture and Transfection
4 Notes
References
Chapter 16: NanoLuc-Based Methods to Measure Ξ²-Arrestin2 Recruitment to G Protein-Coupled Receptors
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Assay and Detection
3 Methods
3.1 Day 1: Seeding HEK293T Cells for Transfection
3.2 Day 2: Transient Transfection HEK293T Cells
3.3 Day 3: Transfer Transfected HEK293T Cells into Assay Microplates
3.4 Day 4: Prepare Ligand Dilutions
3.4.1 Agonist Assay
3.4.2 Agonist in Competition with Antagonist Assay
3.5 Day 4: NanoBRET and NanoBiT Assay Procedure
3.6 Data Analysis
4 Notes
References
Chapter 17: Luciferase Complementation Approaches to Measure GPCR Signaling Kinetics and Bias
1 Introduction
1.1 Kinetic Context in G Protein-Coupled Receptor (GPCR) Compound Profiling
1.2 Luciferase Complementation to Detect Real-Time GPCR Signaling Events
1.3 Assay Optimization
2 Materials
3 Methods
3.1 Assay Optimization: Transient Transfection of NanoBiT-Tagged DNAs
3.2 Assay Optimization: Substrate Concentration
3.3 Assay Optimization: Staggered Kinetics with Endpoint Measurement
3.4 Assay Optimization: Luminescence Platereader Measurement Settings
3.5 NanoBiT Assays: Direct Analysis of Ligand Signaling and Pharmacology
3.6 NanoBiT Assays: Antagonist and Modulator Pretreatment
3.7 NanoBiT Assays: Stimulation with Agonist Followed by Antagonist
3.8 Analyses of Agonist and Antagonist Action: Normalizing Agonist Timecourse Data
3.9 Ligand Concentration Response Curves
3.10 Analysis of Antagonist Action
3.11 Agonist Pharmacology and Bias Across Timepoints Using the Operational Model: Calculating Transduction Coefficients for Us...
4 Notes
References
Chapter 18: Gradient Tracking by Yeast GPCRs in a Microfluidics Chamber
1 Introduction
2 Materials
3 Methods
3.1 Pouring Chambers
3.2 Fusing the Chamber
3.3 Setting Up Yeast Cultures
3.4 Prepare Syringes
3.5 Microscope Set Up
3.6 Image Acquisition
3.7 Clean Up
4 Notes
References
Chapter 19: Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microflu...
1 Introduction
2 Materials
2.1 Cell Assay
2.2 Microfluidics Fabrication
2.3 Chemicals and Reagents
2.4 Software
3 Methods
3.1 Preparation of Working Cell Banks
3.2 Microfluidics Fabrication
3.3 Stimulation Assays in Microtiter Plates
3.4 Stimulation Assays in Microfluidics
3.5 Data Analysis
4 Notes
References
Chapter 20: Homology Modeling Using GPCRM Web Service
1 Introduction
2 Materials
3 Methods
3.1 Input Methods
3.2 Entering Parameters/Selecting Options in Auto Mode
3.3 Running the Job
3.4 The Advanced Mode
3.5 Description of Output
3.6 Structural Viewers and the Table of Templates
4 Notes
References
Index