LG - Orphan Receptors and Methods for Ligand Receptor Characterisation

Question 1

Q: What is biased signalling in GPCRs? (1)

Answer 1

• Occurs when different ligands or cellular contexts lead to distinct downstream signalling outcomes via the same receptor

Question 2

Q: What are four types of biased signalling? (4)

Answer 2

• Ligand-induced bias – different ligands preferentially activate certain pathways
• Receptor-induced bias – GPCR structure favours a specific pathway
• System-induced bias – cellular environment alters pathway preference
• Spatial/location-induced bias – receptor compartmentalisation affects signalling

Question 3

Q: What is an example of biased signalling in a GPCR? (1)

Answer 3

• μ-opioid receptor: Enkephalin is unbiased; morphine and other agonists cause analgesia + adverse effects via β-arrestin signalling
• G-protein-biased ligands (e.g. TRV130, PZM21) reduce side effects

Question 4

Q: Why is GPCR deorphanization important? (2)

Answer 4

• \~120 GPCRs remain orphan (ligand unknown)
• Deorphanization reveals new drug targets, especially for CNS and immune disorders

Question 5

Q: Name 5 examples of orphan GPCRs and their associated roles. (5)

Answer 5

• GPR37 – CNS expression; linked to Parkinson’s disease
• GPR139 – Habenula/striatum; neuropsychiatric disorders
• GPR61 – Feeding and mood regulation; obesity and depression
• GPR173 (SREB3) – Reproductive hormone signalling
• GPR35 – Immune/GI/nervous tissue; associated with inflammation and cancer

Question 6

Q: How does IUPHAR define an orphan GPCR? (2)

Answer 6

• A receptor is still orphan if its ligand:
  • Is a poor activator
  • Lacks physiological relevance (e.g. low endogenous concentration)
• Example: GPR35 and kynurenic acid

Question 7

Q: What are the IUPHAR criteria for removing orphan status from a GPCR? (5)

Answer 7

1. Independent validation – ≥2 studies confirming ligand activity
2. Physiological relevance – ligand present at effective concentrations in vivo
3. Selectivity/specificity – minimal off-target effects
4. Mechanistic insight – clear signalling pathway(s)
5. Pharmacological consistency – reproducible across assays and models

Question 8

Q: What are the advantages of cell-based assays for GPCR characterisation? (5)

Answer 8

• Maintain native physiological context
• Allow real-time signalling measurement
• Avoid need for purification or lysis
• Enable high-throughput screening
• Applicable to orphan GPCRs

Question 9

Q: What are the key elements of a cell-based GPCR assay? (3)

Answer 9

1. GPCR of interest – transient or stable expression
2. Reporter system – detects activation (e.g. luciferase, fluorescent proteins, calcium indicators)
3. Second messenger readouts – measures changes in Ca²⁺, cAMP, etc.

Question 10

Q: Which cell lines are commonly used for GPCR assays? (3)

Answer 10

• HEK293 – high transfection efficiency
• CHO – robust, stable expression
• COS-7 – fast-growing, good for transient assays

Question 11

Q: Why are controls important in cell-based GPCR assays? (4)

Answer 11

• Vehicle control – ensures effects are ligand-specific
• Endogenous ligand control – if receptor is not orphan
• Positive control – validates detection system
• Empty vector control – tests for background signal from transfection

Question 12

Q: What are key experimental considerations when designing a GPCR assay? (3)

Answer 12

• Ensure conditions prevent endogenous signalling interference
• Choose cell lines carefully (avoid background expression)
• Include appropriate controls for validity

Question 13

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

Answer 13

• Biased signalling shapes GPCR pharmacology and function
• Many orphan receptors remain with therapeutic potential
• IUPHAR criteria guide when receptors are no longer considered orphan
• Live-cell assays give the most physiologically relevant readouts
• Proper experimental controls are essential for reliable ligand-receptor characterisation