PTW - B-AR Flashcards
(19 cards)
Q: What residue is essential for noradrenaline binding in β-AR? (1)
- Asp113 in TM3 forms a salt bridge with the ligand; even a conservative change (e.g. Asp→Asn) abolishes binding
Q: Which other residues influence agonist binding in β-AR? (2)
- Asp79 and Asp318 reduce agonist binding but do not significantly affect antagonist binding
Q: How do α1 and α2-adrenergic receptors differ in binding requirements? (2)
- α1: Requires Ser212 for binding
- α2: Requires Ser202
- Phenylephrine (α1 agonist) only has one OH group
Q: What happens when an agonist binds to a β-AR? (2)
- Causes a conformational change from inactive (R) to active (R*), enabling G-protein binding
- Promotes downstream Gs signalling
Q: What is basal activity, and how does β₂-AR compare to β₁-AR? (2)
- Basal activity = receptor signalling in absence of agonist
- β₂-AR has \~5× higher basal activity than β₁-AR
Q: What is the clinical significance of the T164I polymorphism in β₂-AR? (1)
- Reduces basal activity to β₁ levels and is linked to heart disease
Q: What are the two classes of binding sites in β-AR? (2)
- High-affinity site = active R* state (detected at 10⁻⁹ M)
- Low-affinity site = inactive R state (seen at 10⁻³ M)
Q: What does the ternary model of GPCR activation describe? (3)
- Receptor exists in equilibrium: R ↔ R*
- AR ↔ ARG: Agonist stabilises active state for G-protein coupling
- Some R* can bind G-proteins even without an agonist
Q: What is the role of the D(E)RY motif in GPCRs? (2)
- Stabilises the inactive (R) state via ionic lock
- Mutation/disruption increases basal activity
Q: What are structural challenges in GPCR crystallography? (3)
- Requires lipids/detergents due to membrane environment
- GPCRs have intrinsic disorder and high flexibility (R/R*)
- Loops are hard to resolve; crystallisation needs stabilisation strategies
Q: What strategies help crystallise GPCRs? (2)
- T4 lysozyme fusion or Fab fragments to stabilise intracellular loops
- Cross-linking + camelid antibodies to lock GPCR–G-protein complexes
Q: What key interactions occur between β₂-AR and inverse agonist carazolol? (2)
- Polar interactions: Asp113, Ser203, Asn312, Tyr316
- Hydrophobic interactions: Val114, Phe290, Phe193
Q: What are the main steps in β₂AR–Gs complex purification? (5)
- Form stable complex in lipid or DDM + agonist (BI-167107)
- Remove GDP/GTP with apyrase
- Immunoaffinity chromatography purification
- Detergent exchange into MNG-3 for stability
- Use cross-linking and antibodies for crystallisation
Q: What structural changes allow GPCR activation? (3)
- TM6 moves outward by 14 Å, TM5 extends by 7 residues
- ICL2 becomes helical
- Opens hydrophobic cleft for α5 helix of G-protein to enter
Q: Describe G-protein binding to the active β₂-AR. (3)
- α5 helix of Gαs makes hydrophobic and polar contacts with TM3 and TM5
- IL2 bridges α5 to the conserved DRY motif
- Interaction is crucial for signal propagation
Q: What happens during G-protein activation by the receptor? (3)
- Large domain reorientation in Gαs
- Displacement of α5 helix, β6/α5 loop, and P-loop (β1–α1)
- Catalyses GDP → GTP exchange
Q: What disrupts the ionic lock in the active receptor state (R*)? (2)
- Asp130/Arg131 no longer form salt bridge with Glu268
- Arg131 instead interacts with Tyr391 of Gαs α5 helix
Q: Describe the mechanism of receptor-catalysed G-protein activation. (5)
- Agonist binds, receptor shifts from R to R*
- Receptor binds Gαβγ, promoting GDP release
- GTP binds Gα, activating it
- Gα-GTP dissociates from βγ, triggering downstream signalling
- GTP hydrolysis inactivates Gα, allowing reassociation with βγ
Q: What are the key features of the β₂-adrenergic receptor (β₂-AR)? (5)
- Ligand pocket in TM hydrophobic region
- Involves Asp113, Ser203/204/207 for polar interactions
- β₁/β₂ selectivity may involve TM7 (Thr164, Tyr308)
- GPCRs have basal activity and exist in R/R* states
- Activation involves IL3, DRY motif, and GTP exchange via Gα
