Week 4 Flashcards
What is the two-state model of receptor activation?
Classical receptor theory:
- receptor is present in two states: resting and active which exist in equilibrium
- if no ligand is present, the equilibrium lies to the resting state
- an agonist binding to the receptor results in the equilibrium being shifted to the active state and the agonist stabilising the active state, producing a response
What are constitutively active receptors, inverse agonism, and inverse agonists?
Constitutive receptor activity is the ability of a receptor to activate downstream signalling pathways in the absence of bound agonist molecule
Inverse agonism is the process that inverse agonists bind to constitutively active receptors and shift the balance to the inactive conformational state
Inverse agonists are ligands that have negative efficacy and can reduce or abolish receptor spontaneity
What are the differences between inverse agonists and antagonists?
Inverse agonism depends on constitutive receptor activity which is only present when the receptor is constitutively active.
Inverse agonists act like competitive antagonists in the absence of constitutive activity of the receptor.
Antagonists only work when a ligand is present.
Types of inverse agonists:
1. orthostatic inverse agonist which binds to the agonist binding pocket and affects the receptor signalling domain
2. allosteric inverse agonist which binds to the allosteric site and affects the receptor signalling domain
3.pseudo inverse agonist which binds to the allosteric site, affects the agonist binding pocket but has no effect on the signalling domain
What is the molecular basis for the constitutive activity of GPCRs?
Using NMR it was found that beta2-ARs are not stable and it exists in different conformations both under basal state and when bound to different ligands.
Unliganded beta2-ARs and inverse agonist-bound beta2-ARs exist mainly in two inactive conformations that exchange very rapidly.
An agonist alone is not enough to fully stabilise and active beta2-ARs which require interaction with a G protein.
The inverse agonist binds differently to the ghrelin receptor compared to the agonist and antagonist (it changes the shape of the T<6 helix to close off the pocket).
A polar network and hydrophobic cluster are required for receptor activation and constitutive activity.
Constitutive activity of receptors depends on:
1. receptor expression level - the high the expression, the greater the observed constitutive activity
2. mutations - mutated receptors may be locked in an active state
3. changes in environment - pH, temp, ions, lipids
4. diseases - cancer, cardiovascular disease, neurological disorders
Constitutive Receptor Activity
- provides basal signalling allowing for continuous regulation of cellular processes
- enhances sensitivity of cells to low concentrations of ligand
- can vary between tissues, allowing for tissue-specific functions
- is associated with pathological conditions
Inverse Agonists
- used to study the role of receptors in various physiological processes
- treat conditions caused by excessive receptor activation
- used to compare effects of an agonist and its corresponding inverse agonist, potential side effects and development of safer drugs
What therapeutic applications of inverse agonists?
Inverse agonists may be safe and rational therapeutics in treating diseases associated with constitutively active receptors.
Asthma
- salbutamol has been the drug of choice for 40 years
- short and long lasting beta2-AR agonists are used extensively for the prophylactic management of asthma
- long term use of beta2-AR agonists increases the mortality of asthma patients which was thought to be associated with beta-agonists induced airway beta-2AR desensitisation
- inflammatory mediators can contract airway smooth muscle and stimulate mucus production
- managing inflammation and mucus production is more effective that beta2-AR agonists for asthma
Clinical effects of beta agonists and inverse agonists are highly dependent on the duration of therapy
- beta agonists are acutely beneficial but chronically can induce receptor desensitisation
- inverse agonists are acutely detrimental but chronically beneficial
What is an experiment that could differentiate between an antagonist and inverse agonist for a GPCR?
To determine if a drug is an inverse agonist or an antagonist:
- The GPCR is constitutively present in a confirmation that is already coupled to the downstream signalling pathway in the absence of any other ligand
- The drug must have a high affinity for the resting conformations of the GPCR
Most studies have been carried out in the overexpression cell system or transgenic animals .
What are ways that cellular signalling can be terminated?
Ligand Uptake/Degradation
- neurotransmission is rapidly terminated to maintain instant effect
- neurotransmitter returned to axon terminals for reuse
- enzymes inactive neurotransmitters
- neurotransmitters diffuse out of synaptic cleft
2nd Messenger Degradation
- phosphodiesterases are enzymes that break the phosphodiester bond in cAMP
- diacylgylcerol kinases convert DAG to phosphatidic acid
- IP3 kinases and phosphatases which add or remove phosphates
Intrinsic GTPase Activity
- helper proteins maintain efficiency in the GTP hydrolysis pathway to activate and stop GPCR activity
Desensitisation
- process where a receptor elicits a diminished response upon repeated ligand administration
- for GPCRs, this is caused by uncoupling of the receptor from its effectors (e.g. G proteins)
- arrestin is responsible for
Receptor Internalisation
- arrestin is responsible for
- carry receptors away from the cell surface into the cell on endosomes
Protein Degradation
- resensitisation and receptor downregulation
What is GTP hydrolysis in context of GEFs, GAPs, and GDIs?
Active G alpha turns itself off via its intrinsic GTPase activity, hydrolysis GTP to form GDP (not an efficient process so helper proteins needed)
GEFs (hurry up) promote the binding of GTP to the G protein by hastening the release of GDP.
- the active GPCR is a GEF and it increases the rate of GTP binding to G alpha
GDIs (pause) help keep the G protein in the inactive GDP-bound state.
- G beta gammas are GDIs and stabilised G alpha in the GDP bound form so that it waits in the inactive state, ready for action
GAPs (stop) help the G protein to use its intrinsic GTPase activity to hydrolyse GTP and returns the G protein to the GDP-bound state
- RGS (regulators of G protein signalling) are GAPs and they deform the GTP binding site to increase the efficiency of the G protein’s intrinsic GTPase activity which speeds up the G protein cycle
What are the steps involved in receptor internalisation?
Ligand binding -> conformational change of the receptor -> G protein signalling -> receptor phosphorylation -> tethering in the G protein binding site -> arrestin recruitment -> G protein blocked out from binding -> receptor internalisation -> reduced number of receptors at plasma membrane
Receptor can then be degraded or recycled.
Resensitisation
- receptor reset and returned to the cell surface for new round of activation via recycling residues
Receptor Downregulation
- receptors targeted to lysosome where they are degraded
What is homologous desensitisation and the enzymes involved?
Homologous desensitisation is when the activated receptor triggers its own desensitisation e.g. via GRKs
What is heterologous desensitisation and the enzymes involved?
Heterologous desensitisation is when the activated receptor triggers desensitisation of other receptors via second messenger-dependent kinases such as PKA or PKC
Negative feedback loops where phosphorylation prompts the demise
True heterologous desensitisation where PK phosphorylates and binds to an inactive receptor
PKA and PKC do not require active receptors
