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Question 1

Define orthosteric & allosteric site

Answer 1

Orthosteric site:
- Same site for both ligand and endogenous ligand
- E.g. transmembrane bundles of the receptor

Allosteric site:
- Not the same site as endogenous ligand
- Binding to allosteric site can induce allosteric changes or conformational changes which can affect signaling or ligand binding.
- Allosteric ligand include sensing protein, signalling protein, G protein and other protein that interact with receptor
Example of allosteric site: intracellular loop 3, C terminus of receptor, etc.

Question 2

Define biased signalling

Answer 2

Different ligands can stabilised specific conformation of the receptor that favour coupling to G protein or G- independent signalling protein

Question 3

Define dimerisation

Answer 3

• Interaction of GCPR with a neighbouring receptor could can change the signalling or binding potential of the neighbouring receptor, leading to cooperativity or signalling bias.
  
  • Homodimer: interaction between identical receptor
  • Heterodimer: interaction between different receptor

Question 4

Decribe the role of calcium sensing receptor in homeostasis

Answer 4

CaSR role is to maintain calcium homeotstasis by inhibiting PTH synthesis and release and controlling bone reasorption and mineralisation.

Question 5

what drug can regulate the function of the CaSR?

Answer 5

Cinacalcet (calcimimetic) is a positive allosteric regulator and is administered to patients with chronic kidney disease, hyperparathyroidism or parathyroid carcinoma to reduce parathyroid level

Calcilytic regulator can reduce feedback mechanism on calcium and increase parathyroid hormone synthesis to increase calcium reabsorption into kidneys and bone which is useful in postmenopausal women.

Question 6

What are the mutation of CaSR?

Answer 6

• Mutation on calcium sensing receptor can either lead to gain or loss of the function of calcium sensing receptor. This can lead to:
• Autosomal dominant hypocalcemia (gain of function)
• Familial hypocalciuric hypercalcemia (loss of function)

Question 7

Decribe how herpesvirus-encode GPCR can be used as drug target

Answer 7

• Human cytomegalovirus infect human cells in which lead to the transcription of viral protein, GPCR called US28 on infected cell surface.
  
  • Chemokines bind to US28 to cause an increase in transcription of more viral protein. This can lead to cell proliferation and inflammation
  • US28 are also constitutively active in the absence of agonist.
  • Dose response curve:
    ○ Agonist increase basal signalling of receptor
    ○ Antagonist doesn’t change basal signalling of receptor
    Inverse agonist turn off the receptor, making it not constitutively active

Question 8

What are the HEGFR family?

Answer 8

EGFR (ErbB1, HER1)
HER2 (Erb2)
HER3 (ErbB3)
HER4 (ErbB4)

Question 9

Decribe what happen when agonsit binds to EGFR and HER4?

Answer 9

• Agonist bind to EGFR receptor causes the extracellular domain to open up, revealing the dimerisation domain.
  
  • Two receptor bind together to form a dimer complex (homodimer and heterodimer), leading to conformational changes and autophosphorylation of intracellular tyrosine kinase
    This lead to a cascade of signalling pathways leading to activation of, for example, cell proliferation and migration.

Question 10

What are the agonist for HER4?

Answer 10

Agonist: All the neuregulin (1-4) and HB-EGF

Question 11

Decribe what happen during the activation of HER2?

Answer 11

○ No known agonist for receptor
○ Constituitive active form of EGFR receptor as it have a open conformation (without presence of ligand binding to its orthosteric site) in which dimerisation domain is not hidden and can readily interact with neighbouring receptor.
Has intracellular tyrosine kinase hence it needed to bind to other receptor to cause autophosphorylation of tyrosine kinase domains

Question 12

Decribe what happen when agonist bind to HER3?

Answer 12

○ Agonist: Neuregulin 1 and neuregulin 2
○ Resting state: close conformation so dimerisation domain is hidden
○ Agonist need to activate HER3 to open up conformation and reveal dimerisation domain so that it can dimerise with neighbouring receptor
○ HER3 receptor don’t have intracellular tyrosine kinase so no intracellular tyrosine phosphorylation.
HER2 and HER3 can form dimeric complex and HER2 can provides the tyrosine kinase activity as a consequence of cross phosphorylation across the dimer interface.

Question 13

What are the inhibitors of HEGFR?

Answer 13

See lecture 5 slides

Question 14

What are three approaches for screening protein-protein interaction?

Answer 14

FRET, BRET and TR-FRET