Receptor Pharmacology Flashcards
So came up with the ternary complex model?
De lean (1980)
What is the ternary complex model?
A+R<=>AR+G
“ “
A+RG<=>ARG
RG = preformed receptor G protein complex
What occurs to ligand affinity with out with the presence of GTP?
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
Why without GTP is the displacement curve hill slope change with increasing agonist?
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)
What can we infer from inverse agonists?
Some receptors have constitutive activity without a bound agonist
Therefore receptors can exist in an R (inactive) or R* (active) state
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)
DADLE (full agonist) = dose responce curve
MR2266 (antagonist) - straight line
Naloxone (inverse agonist) = decrease in GTPase activity
What does that fact naloxone decreases GTPase activity mean?
There must be a basal level of GTPase activity even without an agonist
What percentage of anatagonists were actuall shown to be inverse agonists?
Approx 80%
What residue is imporant to prevent constitutive activity of a1AR?
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*
What is the extended ternary complex model?
AR<=>AR<=>ARG
“ “ “
R <=> R* <=> R*G
ARG and RG = response
What state does a full agonist stabilise?
R*»R
What state does a partial agonist stabilise?
R*>R
What state does an antagonist stabilise?
R*= R
Preserved equilibrium between R and R* therefore no net conformational change
What state does an inverse agonist stabilise?
R
In a system where there is no constitutive activity what is the difference between adding a antagonist and inverse agonist?
No effect as no baseline R* activity to being with
What is a CAM?
Constitutively active mutant
How do CAMs relate to disease?
A CAM can be a rare genetic mutation which increases baseline activity of a receptor in a ligand independent fashion
Where might mutations which cause CAMs be?
D/ERY sequence i.e. Ionic lock of GPCR
Membrane proximal regions of ICL3 loop
TM6/e4 interface
Name some CAMs?
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
What might negative efficacy of agonist cause?
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
How can we observe conformational changes in GPCRs?
FRET signalling but using CFP and YFP
Either attach both to GPCR or one to GPCR and another to G protein subunit
What wavelength of light cause CFP to fluoresce and what is emitted?
436nm stimulates
FRET and 480nm emitted
What is FRET proportional to?
Distance between CFP and YFP
What does a FRET signal cause YFP to do?
Fluoresce and emitted wavelength of 535nm
Have can fret be used to observe conformational change?
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
What might different level of activation state e.g. R* and R** allow for?
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
What are allosteric modulator?
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
What is probe dependance?
Interaction between allosteric /orthosteric ligand depends on which orthosteric ligand is bound
Why might allosteric modulation be clinically useful?
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)
What is the advantage of a probe dependant allosteric modulator?
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
How might allosteric modulators work?
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
Name the prime example of an allosteric ligand and its target?
Benzodiazepines acting on the GABAa receptors
Increases open probability therefore increase frequency of channel opening for a given concentration of GABA
What is a GABAa positive allosteric modulator?
Benzodiazepines
What is a GABAa neural allosteric modulator?
Flumazenil
What is a GABAa negative allosteric modulator? (Inverse agonist)
Sapmazenil
Name a GPCR family which the allosteric site is know?
Family C GPCRs e.g. GABAb and mGluRs
Where is the orthosetic and allosteric site for family C GCPRs?
Orthosteric = extracellular N terminal 'Venus fly trap' domain Allosteric = within the transmembrane domain
What is the allosteric ternary complex model?
R <=> AR
“ “
RB <=> ARB
B = allosteric ligand AR = normal stimulus ARB = modified stimulus
