Receptor Pharmacology

Question 1

So came up with the ternary complex model?

Answer 1

De lean (1980)

Question 2

What is the ternary complex model?

Answer 2

A+R<=>AR+G
“ “
A+RG<=>ARG

RG = preformed receptor G protein complex

Question 3

What occurs to ligand affinity with out with the presence of GTP?

Answer 3

In the presence of GPP (GTP analog) will be low affinity because G proteins are activated therefore no preformed RG complex
Without GPP there is high affinity as RG complex is formed

Question 4

Why without GTP is the displacement curve hill slope change with increasing agonist?

Answer 4

Slope is less steep because affinity for agonist changes
Initially will bind to preformed RG complexes (high affinity) but there are no enough G proteins to form a preformed RG complex on every receptor (R = low affinity state)

Question 5

What can we infer from inverse agonists?

Answer 5

Some receptors have constitutive activity without a bound agonist
Therefore receptors can exist in an R (inactive) or R* (active) state

Question 6

On a graph measuring GTPase activity against Log(ligand) for a mew opioids receptor what does it look like on addition of :
DADLE (full agonist)
MR2266 (antagonist)
Naloxone (inverse agonist)

Answer 6

DADLE (full agonist) = dose responce curve
MR2266 (antagonist) - straight line
Naloxone (inverse agonist) = decrease in GTPase activity

Question 7

What does that fact naloxone decreases GTPase activity mean?

Answer 7

There must be a basal level of GTPase activity even without an agonist

Question 8

What percentage of anatagonists were actuall shown to be inverse agonists?

Answer 8

Approx 80%

Question 9

What residue is imporant to prevent constitutive activity of a1AR?

Answer 9

The alanine at 293 (WT) regulates constitutive activity as mutagenesis studies here increase constitutive activity
Alanine 293 keeps it in the R state and prevents conversion to R*

Question 10

What is the extended ternary complex model?

Answer 10

AR<=>AR<=>ARG
“ “ “
R <=> R* <=> R*G

ARG and RG = response

Question 11

What state does a full agonist stabilise?

Answer 11

R*»R

Question 12

What state does a partial agonist stabilise?

Answer 12

R*>R

Question 13

What state does an antagonist stabilise?

Answer 13

R*= R

Preserved equilibrium between R and R* therefore no net conformational change

Question 14

What state does an inverse agonist stabilise?

Answer 14

R

Question 15

In a system where there is no constitutive activity what is the difference between adding a antagonist and inverse agonist?

Answer 15

No effect as no baseline R* activity to being with

Question 16

What is a CAM?

Answer 16

Constitutively active mutant

Question 17

How do CAMs relate to disease?

Answer 17

A CAM can be a rare genetic mutation which increases baseline activity of a receptor in a ligand independent fashion

Question 18

Where might mutations which cause CAMs be?

Answer 18

D/ERY sequence i.e. Ionic lock of GPCR
Membrane proximal regions of ICL3 loop
TM6/e4 interface

Question 19

Name some CAMs?

Answer 19

Rare form of hyperthyroidism ~50 point mutations of TSH receptor can cause this
Retinitis pigmentosa
Male precocious puberty (signs of puberty before 8) (LH receptor)
Short limb dwarfism

Question 20

What might negative efficacy of agonist cause?

Answer 20

May change surface level expression of receptors shown by radioligand binding study for H2 receptor on chronic treatment of histamine (agonist) and cimetidine (inverse agonist)
Histamine = decreases in radioligand binding as decrease in receptors
Cimetidine = increase in radioligand binding as increase in receptors

Question 21

How can we observe conformational changes in GPCRs?

Answer 21

FRET signalling but using CFP and YFP

Either attach both to GPCR or one to GPCR and another to G protein subunit

Question 22

What wavelength of light cause CFP to fluoresce and what is emitted?

Answer 22

436nm stimulates

FRET and 480nm emitted

Question 23

What is FRET proportional to?

Answer 23

Distance between CFP and YFP

Question 24

What does a FRET signal cause YFP to do?

Answer 24

Fluoresce and emitted wavelength of 535nm

Question 25

Have can fret be used to observe conformational change?

Answer 25

If ligand binds and there is a conformational change in GPCR which moves CFP and YFP away from eachother CFP fluorescence will increase and YFP fluorescence will decrease and FRET signal from CFP to YFP is smaller

Question 26

What might different level of activation state e.g. R* and R** allow for?

Answer 26

Different coupling to G proteins e.g. Gq or Gs
May make they more or less susceptible to phosphorylation and desensitisation as confirmation stabilised by agonist may be different

Question 27

What are allosteric modulator?

Answer 27

Molecules which may modulate receptor activation positively to negatively independent of ligand binding of dependant on ligand binding bu binding to an allosteric site on receptor

Question 28

What is probe dependance?

Answer 28

Interaction between allosteric /orthosteric ligand depends on which orthosteric ligand is bound

Question 29

Why might allosteric modulation be clinically useful?

Answer 29

Some orthosteric sites are highly conserved across receptor subtypes. Therefore, allosteric modulation may provide subtype specificity which cannot be achieve through orthosteric site targeting as more variability exists between allosteric sites (less conserved)

Question 30

What is the advantage of a probe dependant allosteric modulator?

Answer 30

Orthosteric ligand needs to be bound for allosteric modulator to have an effect. Therefore, will only effect active receptors and maintain temporal and spacial aspect of endogenous physiological stimuli. Increase selectivity and mini uses ADRs

Question 31

How might allosteric modulators work?

Answer 31

Change in orthosteric site conformation
Change in affinity/ efficacy for ligand
Change effector protein coupling
May have allosteric agonism and induce signal pathway independent of orthosteric ligand
May favour an orthosteric site to bias a particular signal pathway

Question 32

Name the prime example of an allosteric ligand and its target?

Answer 32

Benzodiazepines acting on the GABAa receptors

Increases open probability therefore increase frequency of channel opening for a given concentration of GABA

Question 33

What is a GABAa positive allosteric modulator?

Answer 33

Benzodiazepines

Question 34

What is a GABAa neural allosteric modulator?

Answer 34

Flumazenil

Question 35

What is a GABAa negative allosteric modulator? (Inverse agonist)

Answer 35

Sapmazenil

Question 36

Name a GPCR family which the allosteric site is know?

Answer 36

Family C GPCRs e.g. GABAb and mGluRs

Question 37

Where is the orthosetic and allosteric site for family C GCPRs?

Answer 37

Orthosteric = extracellular N terminal 'Venus fly trap' domain
Allosteric = within the transmembrane domain

Question 38

What is the allosteric ternary complex model?

Answer 38

R <=> AR
“ “
RB <=> ARB

B = allosteric ligand 
AR = normal stimulus
ARB = modified stimulus