7th and 9th Nov - Receptor Regulation Flashcards
In which organism was g-protein independent signalling first identified?
Slime mould
How was G-protein independent signalling identified?
Mutated Gbeta in slime mould and found that not all GPCR signalling was lost therefore the G protein must not be the sole source of signalling
Outline Turki’s 1995 experiment on beta adrenoceptors that showed desensitization.
Overdosed non-asthmatics with oxiprenaline over 24h
Collected their airway emipthelial cells and alveolar macrophages
–> Following drug application cAMP response was much lower
–> Forskolin application led to the same AC response in both treated and non-treated therefore reduction in response must be due to Beta adrenoceptor not AC
–> After application the number of beta 2 adrenoceptors decreased
What is homologous desensitisation?
Where the attenuation in the signalling response is specific to the receptor being stimulated
What is heterologous desensitisation?
Where collateral receptors signalling are also desensitised
How did Green and Clark (1981) show that desensitisation occurs at the level of the receptor?
Used cyc- (lack Beta adrenoceptor) S49 lymphoma cells —> no attenuation
Prepared their membranes and reconstituted with WT lysate containing Galpha S –> attenuation of cAMP levels on long application of agonist
How can you demonstrate that receptor phosphorylation correlates with desensitisation?
Label cells with [32-P]-orthophosphate –> 32P-ATP
Lyse cells
Immunoprecipitate receptor
Add agonist –> increase in receptor phosphorylation (shown on autoradiograph)
Which residue is a GPCR most commonly phosphorylated on?
Serine
Which kinase mediated heterologous receptor phosphorylation?
PKA/PKC
Is heterologous phosphorylation conformation dependent?
No
Is heterologous phosphorylation slow?
Yes takes minutes
What experiment showed that receptor desensitisation was not solely dependent on PKA/PKC?
Examined B2 adrenoceptor coupling in S49 lymphoma cells
WT - GPCR –> cAMP –> PKA
Cyc- = Lack GPCR
kin- = Lack PKA, however still showed receptor desensitisation therefore must not be entirely dependent on PKA
What is BARK?
Beta -adrenergic receptor kinase
Who discovered BARK?
Benovic 1986
How was it discovered that there was a missing link between BARK and receptor desensitisation?
Purified lung beta adrenoceptors were incubated with kin- S39 lymphoma cell lysate (lack PKA) and [32P]ATP/Mg2+. Expected desensitisation to correlate with receptor phosphorylation and increased [BARK] but it didn’t
Therefore receptor phosphorylation has little to do with desensitisation or something is missing
Who found that Beta-arrestin regulates beta adrenergic receptor function in 1990?
Lohse
How was arrestin identified as the missing link between BARK and receptor desensitisation?
Measured Receptor-gprotein coupling activity against increasing [GRK]. When GRK and Arrestin was present the predicted desensistisation was achieved.
Outline the current model of homologous GPCR desensitisation
GRK is recruited by the conformation of the receptor
GRK phosphorylates the receptor e.g BARK
Once phosphorylated the GRK recruits arrestin
Arrestin acts as a scaffold for clathrin mediated receptor endocytosis, interacting directly with AP2 and clathrin
What are the currently identified GRKs?
Visual: Rhodopsin Kinase (GRK1), GRK7 (in cone cells)
Non-Visual: BARK (GRK2), BARK2 (GRK3), GRK4, GRK5 and GRK6
What is an RH domain?
An RGS homology domain allowing G alpha interaction
How are GRK 2/3 recruited to the ligand occupied receptor?
- GRK2/3 recruitment to activated receptor via Gbeta gamma and PIP2
- RGS like domain of GRK2/3 can bind and sequester Galpha subunits
Can different arrestins bind the same source GRK?
Yes e.g.
ETa receptor - GRK2 –> Arrestin 3
P2Y2 receptor - GRK2 –> Arrestin 2
Outline an arrestin signalling complex (not for clathrin mediated endocytosis)
Phosphorylation and beta arrestin capped beta adrenoceptor recruits Src to activate MAPK signalling
What are the two forms of GPCR signalling?
Gprotein dependent –> AC, PLC, PI3K etc.
G protein independent –> MAPK (ERK/JNK), cAMP PDE, DAG Kinases
Outline the different signalling pathways that can be elicited using the AT1 receptor
Gprotein dependent - Gq –> Vasoconstriction and Fluid Retention
Gprotein independent - Arrestin –> Cardiac contractility and decrease cardiac cell apoptosis
What are the clinical benefits of the two forms of AT1 receptor signalling?
Currently Hypertension and heart failure is treated w/ ACE inhibitors or angiotensin receptor blockers, however the arrestin AT1 pathway elicits desirable effects in heart failure.
Thus using a biased agonist such as SII-AngII could block undesirable Gq effects and promote desirable Arrestin mediated effects –> greater therapeutic effects than Antagonists
What is tachyphylaxis?
Decrease in responsiveness of a drug with repeated dosing
What is drug tolerance?
A higher dose is required with repeated doses to create the desired effects
Outline the clinical benefits of using a biased agonist on B2 adrenergic receptors in obstructive lung disease
B2 adrenergic receptor regulates airway smooth muscle cell contraction and bronchial tone
Asthma treatment suffers from tachyphylaxis to Beta agonist stimulated bronchodilation, as repeated doses –> increased tolerance –> leads to higher doses –> increased health risks and reduced action of rescue inhalers
Desensitisation is mediated by Beta arrestin 2 sterically preventing g protein association by receptor internalisation thus a biased ligand which only evoked the G protein response would prevent desensitisation
