GPCRs 2

Question 1

Describe the simple linear two-state model

Answer 1

Receptor is only in active and inactive states. Agonist-bound is active, agonist un-bound is inactive.

Based on an equilibrium between the R and R* state. Agonism causes R < R, inverse agonism causes R > R, and antagonist prevent agonist binding without affecting the equilibrium.

Question 2

Describe the ternary complex model

Answer 2

Seen as guanine nucleotides affect the binding affinity of agonists, but not antagonists. Identifies the G-protein role in GPCRs.

States:

A+R+G
AR+G
AR(active)+G
AR(active)G

With AR*G eliciting the response.

Sees G-proteins as allosteric proteins, increasing the affinity of the orthosteric site - ligand binding. This is seen vice versa, where interactions of the ligand with the receptor increase the G-protein binding.

Question 3

Historic and modern way of detecting ligand binding.

Answer 3

Traditionally drugs were labelled with radioisotopes (e.g., 3H, 125I). Saturation and competition binding assays can be done with them. It does require separation of the bound from the unbound ligand. Also is only an endpoint assay, with no real time info.

Fluorescent ligands can also be used. They are easier to separate between the bound and unbound forms. Can be used for real time info and visualisation. Are able to be done up to single cell resolution.

Question 4

Way to utilise fluorescent ligands

Answer 4

FRET/BRET

E.g., Time-resolved FRET (TR-FRET)

Would tag the ligand and the receptor and get the BRET/FRET ratio indicating binding.

Question 5

Description of extended ternary complex

Answer 5

GPCRs have also been seen to spontaneously (no ligand) form activated states capable of G-protein-mediated signalling. Constitutive activity.

Suggests states:

AR+G - ligand bound inactive
A+R+G
AR+G - ligand bound active but no G-protein
A+R+G - receptor active, no ligand
ARG - elicits response
A+RG - elicits response

Question 6

Inverse agonists mechanism of action

Answer 6

They are ligands that selectively bind to the inactive state of the receptor. They prevent the receptor from then signalling, reducing the constitutive activity. Act as antagonists that also reduce the constitutive activity of the receptor.

They have negative efficacy. An antagonist would not be able to prevent constitutive activity.