LG - Deorphanisation methods (ligand characterisation)

Question 1

Q: What are the three main categories of assays used to characterise GPCR-ligand interactions? (3)

Answer 1

• G-protein-based assays (e.g., Gq, Gi, Gs signalling)
• Arrestin-based assays (e.g., TANGO, PRESTO-TANGO)
• Electrophysiology and protein-protein interaction assays (e.g., Patch Clamp, BRET, FRET, NanoBiT)

Question 2

Q: What are the advantages of cell-based GPCR assays? (2)

Answer 2

• Quantitative and enable comparison of ligand potencies
• Allow high-throughput screening for rapid drug testing

Question 3

Q: What are alternative methods to measure Ca²⁺ signalling? (2)

Answer 3

• Fluorescence-based Ca²⁺ mobilisation assays
• IP3-based assays to detect IP3 generated downstream of PLCβ

Question 3

Q: How does the cAMP GloSensor assay measure Gs/Gi signalling? (3)

Answer 3

• Measures cAMP levels via luminescent biosensor
• Gs-coupled GPCRs → ↑ cAMP → ↑ luminescence
• Gi-coupled GPCRs → ↓ cAMP → ↓ luminescence
• Forskolin used as control to stimulate cAMP

Question 4

Q: How do Gq calcium mobilisation assays work using photoproteins? (3)

Answer 4

• Use Aequorin or Clytin that emit light upon binding Ca²⁺
• Ca²⁺ release triggered via GPCR → PLC → IP3 pathway
• Luminescence output is proportional to receptor activity

Question 5

Q: What is the principle of the TANGO assay for arrestin recruitment? (5 steps)

Answer 5

1. GPCR fused to a transcription factor via a protease cleavage site
2. Ligand binding activates GPCR
3. β-arrestin recruitment triggers protease cleavage
4. Transcription factor released, enters nucleus
5. Reporter gene (e.g., luciferase) is expressed and detected

Question 5

Q: What are the disadvantages of the TANGO assay? (2)

Answer 5

• Requires fusion constructs and stable cell lines
• Only detects β-arrestin recruitment, not G-protein signalling

Question 5

Q: How is Gβγ-mediated signalling detected? (2)

Answer 5

• Monitored through ion channel activation, especially GIRK channels
• Reflects GPCR activation and Gβγ release

Question 6

Q: What are the advantages of the TANGO assay? (2)

Answer 6

• High sensitivity due to signal amplification
• Effective for orphan GPCRs where signalling pathways are unknown

Question 7

Q: What is the PRESTO-TANGO assay? (1)

Answer 7

• A simplified version of the TANGO assay, easier to implement

Question 7

Q: What is the use of Xenopus oocyte electrophysiology in GPCR studies? (2)

Answer 7

• Two-electrode voltage-clamp technique
• Measures ion currents in response to heterologously expressed GPCRs

Question 7

Q: What does the Patch Clamp technique measure in GPCR assays? (2)

Answer 7

• Measures ion current across the membrane
• Detects GPCR-regulated ion channel activity (e.g., via Gβγ)

Question 8

Q: What is the principle behind FRET and BRET assays? (3)

Answer 8

• Measure protein-protein interactions by energy transfer
• FRET: Donor fluorophore excites acceptor fluorophore → fluorescence
• BRET: Uses Renilla luciferase as donor, avoids light excitation → lower background

Question 8

Q: How does the NanoBiT assay detect protein interactions? (3)

Answer 8

• Uses split luciferase: LgBiT (large fragment) and SmBiT (small fragment)
• Fragments are fused to interacting proteins (e.g., GPCR + β-arrestin)
• Interaction brings fragments together → reconstitutes luciferase → luminescence

Question 9

Q: What are the limitations of FRET and BRET? (3)

Answer 9

• Require proteins to be within 1–10 nm
• Low signal intensity
• Require careful fluorophore selection

Question 10

Q: What are the advantages of the NanoBiT system? (2)

Answer 10

• Live-cell compatible
• High sensitivity for detecting transient or weak interactions

Question 11

Q: Summarise the signalling pathways and detection methods used in GPCR deorphanisation. (6)

Answer 11

• Calcium mobilisation (Gq) – Aequorin or fluorescence-based luminescence
• IP3 assays (Gq) – NanoBiT-based detection
• cAMP GloSensor (Gs/Gi) – luminescence increase/decrease
• Gβγ signalling – ion channel activation
• TANGO (β-arrestin recruitment) – luciferase reporter
• Patch Clamp – direct ion current measurement

Question 12

Q: What are the key takeaways from this lecture? (5)

Answer 12

• Cell-based assays allow real-time, high-throughput GPCR analysis
• Calcium and cAMP assays monitor G-protein signalling
• TANGO assays detect β-arrestin recruitment, key for desensitisation
• Electrophysiology offers direct functional readouts
• BRET/FRET and NanoBiT reveal GPCR-protein interactions