Receptor Pharmacology Flashcards
In 1980, De Lean et al came up with which model to explain ligand-receptor interactions?
The Ternary Complex Model (TCM)
What does the TCM (ternary complex model) say about the affinity of agonists for the different receptor states?
Agonist (but not antagonist) has higher affinity for RG than R
Are receptors always quiescent in the absence of agonist?
No. Some receptors appear to spontaneously transition between inactive (R) and active (R*) conformations - giving rise to constitutive activity
Give an example of a receptor that appears to require agonist to become active
Rhodopsin
After it was found that some receptors possess constitutive activity, the TCM was adapted by Samana et al (1993) and Weiss et al (1996). What were these new models?
The extended TCM
The cubic TCM
What did the extended and cubic TCMs allow for?
Receptor can spontaneously undergo R/R* conformational changes and for R* to initiate responses agonist-independently
Ligands exhibit differential affinities for the R and R* states. For example, full agonists have a selective affinity for the R* conformation (R*»_space; R). Give some examples of which conformation different ligand have a selective affinity for.
Full agonists: R*»_space; R
Partial agonists: R* > R
Antagonists: R* = R
Inverse agonists: R*
Wild-type GPCRs appear to vary considerably in their intrinsic constitutive activity. Give an example of a GPCR with: 1) VERY low 2) moderate 3) high constitutive activity
1) very low - rhodopsin
2) moderate - H3 histamine
3) high - virally-encoded GPCRs
The degree of constitutive activity for some GPCRs is cell/tissue-dependent. Why is this?
Because a variety of proteins can alter this activity through protein-protein interactions
The degree of constitutive activity for some GPCRs is cell/tissue-dependent due to the proteins present. Give an example of this.
Homer Proteins alter the constitutive activity of Metabotropic Glutamate Receptors
What is meant by a ‘CAM’ form of a GPCR? When might these be found?
Constitutively-Active Mutant
…found in some very rare genetic diseases
Constitutive activity can be induced experimentally by targeted mutation of GPCRs. Which are 3 key regions which can give rise to CAMs?
1) D/ERY sequence mutations (TM3/i2 interface)
2) membrane proximal regions of the i3 loop
3) the TM6/e4 interface
Rare, naturally-occurring mutations of GPCRs (eg CAMs) can lead to disease states characterised by increases in agonist-independent (constitutive) activity. Give some examples of such diseases associated with GPCR CAMs.
1) >50 point mutations have been found in the TSH (thyroid-stimulating hormone; thyotropin) receptor, many of which lead to HYPERTHYROIDISM
2) male precocious puberty: luteinising hormone (LH) receptor
3) retinitis pigmentosa and night blindness: rhodopsin
4) short-limb dwarfism: parathyroid (PTH) receptor
5) hypocalaemia/hypercalciuria: Ca2+-sensing receptor
Agents previously classified as antagonists actually represent what?
A heterogeneous pharmacological group exhibiting varying degrees of NEGATIVE EFFICACY (ie INVERSE AGONISM)
Whilst a neutral antagonist exhibits no efficacy, what do inverse agonists exhibit?
Varying degrees of NEGATIVE EFFICACY
With regard to receptors, what is a ‘quiescent’ system?
One with very low or no constitutive (ie agonist-independent) activity
In a quiescent system, neutral antagonists and inverse agonists are functionally indistinguishable. How does this differ from a constitutively active system?
Neutral antagonists and inverse agonists have very different effects: only an inverse agonist will suppress constitutive activity
Antagonists and inverse agonists cause pharmacologically distinct actions in constitutively active systems. How may this be helpful in the treatment of genetic diseases caused by CAM GPCRs?
Inverse agonists will suppress constitutive activity and thus may be useful in treatment
What type of experiments can be used to discern two distinct agonist binding affinities?
Radioligand binding studies
The negative efficacy of inverse agonists, in addition to suppressing constitutive activity, may cause further (long-term) changes to occur with respect to receptor regulation. Give an example of such a change.
An inverse agonist may cause a change in the cell-surface receptor expression level
Eg for the H2 Histamine receptor, long-term exposure to:
-histamine reduces receptor expression levels, whilst long-term exposure to
-cimetidine increases expression levels
The choice between a neutral antagonist and an inverse agonist is likely to be therapeutically important. Why?
Inverse agonists suppress constitutive activity whilst neutral antagonists do not.
The negative efficacy of inverse agonists may cause a change in the cell-surface receptor expression level.
Which two inverse agonists up-regulate H2 Histamine receptors?
Cimetidine and Ranitidine
Give an example of inverse agonists that, when used as a chronic treatment, regulate H2 Histamine receptor expression levels. Give an example of a neutral antagonist. What effect does this have on cell-surface H2 histamine receptor expression levels?
Up-regulate: cimetidine and ranitidine
Down-regulate: histamine
Neutral antagonist: burimamadine …has no effect on expression levels
Give an example of an experimental approach that may be used to investigate agonist-stimulated conformational changes in GPCRs.
GPCRs may be modified with different types of GFP (CFP and/or YFP) to report conformational change
…through the use of FRET
What does FRET stand for?
Fluorescence resonance energy transfer
Briefly outline FRET
Fluorescence resonance energy transfer occurs between a ‘donor’ and an ‘acceptor’ label; these labels can be attached to a single protein or to two different proteins.
FRET requires spectral overlap.
FRET generally occurs between cyan (CFP) and yellow (YFP) fluorescent proteins.
How can FRET be used to study GPCRs?
GPCRs can be modified with different types of GFP labels (CFP and YFP) through the attachment of a label to:
1) a single GPCR (in the third intracellular loop and the C-terminus)
or
2) the C-terminus of one GPCR and one of the subunits of a G-protein
…excitation of a CFP with light at 436nm causes CFP emission at 480nm plus FRET to YFP, which then emits at 535nm. The extent of FRET varies with sixth power of the distance and is thus an exquisitely sensitive indicator of conformational changes or protein-protein interactions
What frequency of light is required to excite CFP? What frequency light does CFP then emit, and what is the effect this might have on a close YFP molecule?
436nm
480nm
…this is absorbed by YFP, which emits light at 535nm
What changes the extent of FRET between CFP and YFP?
The extent of FRET varies with sixth power of the distance
What can FRET be used to study?
Conformational changes
Protein-protein interactions
How many the effect of addition of agonist to a GPCR be studied?
FRET: addition of an agonist presumably changes the distances between CFP and YFP; it can induce a rapid reduction in FRET in a single receptor labeled with CFP and YFP, and can promote the interaction between a YFP-labeled receptor and a G protein labeled at its gamma-subunit with CFP
What variation of FRET can be used to study GPCRs?
Bioluminescence resonance energy transfer (BRET)
How does BRET work?
It uses a light-emitting enzyme, luciferase, as a a donor, and a GFP variant as an acceptor.
What technique was used to generate the first three-dimensional structure of a 7TM protein? Which GPCR was this?
Electron microscopy
…Bacteriorhodopsin (a high expressed proton pump)
Which crystal structure was the first definitive study to provide information on the R* rather than the R state of the GPCR?
Scheerer et al (2008), on the 11-cis-retinal-free opsin-Galpha(t) protein fragment complex
What is the importance of the work carried out by Scheerer et al in 2008 on the 11-cis-retinal-free opsin-Gat protein fragment complex?
It is the first definitive study to provide information on the R* rather than the R state of the GPCR
When was the first GPCR crystallised?
1999?
Due to recent progresses in X-ray crystallography, the number of experimentally solved GPCRs rose from zero in 1999 to approximately what number in 2012?
Around 80
Which class of GPCRs have been characterised, and which remain to be experimentally solved?
The 'class A'/'family I'/'rhodopsin family' have over 80 characterised strucutures; 17 of which are in complex with ligand ...the remaining four classes remain to be characterised
Give some examples of GPCRs (all are Class A) that have been characterised?
Bacteriorhodopsin Squid rhodopsin 11-cis-retinal-free opsin 11-cis-retinal-free opsin BOUND TO Gat protein fragment B2-adrenoceptor B1-adrenoceptor A2A-adenosine receptor H1-histamine receptor
Describe, using an example, how solved crystal structures can be used to ‘see’ conformational changes in a GPCR
Comparison, e.g. of Gat fragment-bound opsin (R*) and dark-state rhodopsin (R) crystal structures reveals how activation caused conformational rearrangement of the TM domains
GPCR conformational complexity can give rise to what agonist-mediated phenomenon?
Agonist-directed trafficking of signal via distinct signalling pathways
Give an example of how different ‘active’ GPCR conformations may give rise to distinct signalling pathways
Different conformations may favour receptor coupling to different intracellular compartments. For example, different active conformational states of the receptor (R* and R**) may preferentially couple to distinct G protein subtypes (e.g. Gs and Gq/11) leading to distinct signalling readouts
In which two main ways might agonist-directed trafficking of GPCR-signalling occur?
1) Different active conformations may favour receptor coupling to different intracellular components (e.g. particular G protein subtypes)
2) Different active conformations may be more or less susceptible to PHOSPHORYLATION and DESENSITISATION (i.e conformational selection by the agonist may determine whether the receptor response is maintained or progressively attenuated)
GPCRs represent one of the largest classes of drug targets, with up to 40% of currently marketed drugs acting at these proteins. Give some examples of disorders for which GPCRs are therapeutic targets.
Cancer
Cardiac dysfunction
CNS disorders
Obesity
GPCRs can signal effectors independent of G-proteins to induce cellular behaviours. Give some examples of these cellular behaviours, and the effectors that mediate them
Cellular proliferation/apoptosis, which is mediated by the induction of B-arrestin-dependent signalling
What is ADTRS?
Agonist-directed trafficking of receptor signalling
Give an example of ADTRS (agonist-directed trafficking of receptor signalling)
Structurally unrelated orthosteric (competitive) agonists of serotonin 5-HT2A/2C receptors have demonstrated differential phospholipase C (PLC) and phospholipase A (PLA) activation
What is the binding site on a receptor for its natural ligand(s) called?
The ORTHOSTERIC binding site
Receptors may possess additional ligand/drug binding sites that can be pharmacologically distinguished from the orthosteric site. What are these called?
ALLOSTERIC binding sites
Why are allosteric interactions pharmacologically important?
They offer the possibility of POSITIVELY OR NEGATIVELY MODULATING RECEPTOR ACTIVATION, either dependently or independently of ligand binding at the orthosteric binding site
What is the orthosteric binding site?
The binding site on a receptor for its natural ligand(s)
What is the allosteric binding site?
The binding site(s) on a receptor that is pharmacologially distinct from the orthosteric binding site
What is a prototypic example of allosteric modulation of a receptor?
BENZODIAZEPINES acting at ligand-gated GABAa RECEPTOR/ION CHANNELS
What are GABA-A receptors?
Ligand-gated Cl- -conducting ion channels
What are GABA-A receptors responsible for?
The majority of inhibitory transmission in the CNS
What is the composition of GABA-A receptors?
They are functional pentamers made up of alpha, beta or gamma monomers. The usual stoichiometry is (alpha)2(beta)2(gamma)
What is the natural ligand for GABA-A receptors? Where does this ligand bind to activate the receptor?
GABA (gamma-aminobutyric acid)
It binds to orthosteric sites at the ALPHA-BETA interfaces
GABA-A receptor activation by GABA can be modulated by BENZODIAZEPINES (BZDs). How do BZDs do this?
They bind at the interface between an alpha (1, 2, 3, or 5) and a gamma (2) subunit
Give an example of a benzodiazepine agonist
Diazepam
What is the effect of a BDZ agonist, e.g. DIAZEPAM, on the activity of the GABA-A receptor?
It enhances the activity of the receptor: i.e. it enhances GABA-evoked Cl- conductance at sub-maximal GABA concentration
How can the enhancing actions of BDZ agonists (such as diazepam) at GABA-A receptors be blocked?
By BDZ ANTAGONISTS (eg FLUMAZENIL)
Give an example of a BDZ antagonist
Flumazenil
Whilst BDZ antagonists act to block the enhancing effects of BDZ agonists, which compounds DECREASE GABA-evoked Cl- conductance?
BDZ inverse agonists (eg SAPMAZENIL)
Give an example of a BDZ inverse agonist
Sapmazenil
What blocks the action of BDZ inverse agonists?
BDZ antagonists (eg Flumazenil)
What is the effect of BDZ inverse agonists, e.g. sapmazenil, binding to allosteric sites on the GABA-A receptor?
They DECREASE GABA-evoked Cl- conductance
What type of allosteric modulator are:
1) BDZ agonists
2) BDZ antagonists
3) BDZ inverse agonists
1) POSITIVE allosteric modulator (PAM)
2) NEUTRAL allosteric modulator
3) NEGATIVE allosteric modulator (NAM)
The Class C GPCRs include which 5 subfamilies?
1) Calcium-sensing receptor-related
2) GABA-B receptors (GB1, GB2)
3) Metabotropic glutamate (mGlu) receptors (mGlu1-8)
4) A subset of the taste receptor family
5) RAIG (retinoic acid-inducible orphan GPCRs) (RAIG1-4)
Where is the ORTHOSTERIC ligand binding site located in Class C GPCRs?
Within the extracellular N-terminal domain: in the Venus FlyTrap (VFT) module
What does ligand binding cause in Class C GPCRs?
A conformational change that is transmitted via the TM domains to the intracellular signal transduction/G protein-coupling domains (i2/i3 loops/C-terminal)
How many different families can the GPCRs be classified into based on the sequence phylogeny of a conserved heptahelical transmembrane domain?
Five
Which two distinct structural features distinguish Class C GPCRs from other GPCRs?
1) their unusually large extracellular domain that is responsible for orthosteric ligand recognition
…while the 7TM (which normally contains the orthosteric binding site) has gained many allosteric binding sites
2) they form dimers
Where in Class C GPCRs do the majority of allosteric modulation sites reside?
In the 7TM domain
In which three general ways can allosteric ligands affect receptor function?
1) allosteric modulation of orthosteric ligand BINDING AFFINITY
2) allosteric modulation of orthosteric ligand EFFICACY
3) DIRECT ALLOSTERIC AGONISM
Do allosteric modulators offer any advantage in the drug discovery process?
1) Orthosteric binding sites of receptors are often highly conserved and it is therefore difficult to generate subtype-selective orthosteric agonists/antagonists
2) Allosteric sites are like to be much less highly conserved and therefore more subtype-selective positive or negative allosteric modulators can be discovered
3) Allosteric modulators have the potential to increase (PAMs) or decrease (NAMs) the actions of the endogenous agonist WITHOUT altering the endogenous pattern of stimulation
4) Therefore, complex intermittent/phasic patterns of receptor stimulation can be preserved
Why is it difficult to generate subtype-selective orthosteric agonists/antagonists?
Because orthosteric binding sites of receptors are often highly conserved
Why is it easier to generate subtype-selective allosteric modulators than orthosteric agonists/antagonists?
Because allosteric sites are much less highly conserved than orthosteric sites
Why is it an advantage that allosteric modulators have the potential to alter the actions of the natural ligand WITHOUT altering the endogenous pattern of stimulation?
This means that complex intermittent/phasic patterns of phasic stimulation can be preserved
What is phospholipase C?
Membrane-bound enzyme that cleaves inositol phospholipids to produce IP3 and DAG in the inositol phospholipid signalling pathway.
What are the two main forms of phospholipase C (PLC), and how are they each activated?
PLC-beta: activated by GPCRs via specific G proteins
PLC-gamma: activated by RTKs
