7th and 9th Nov - Receptor Regulation

Question 1

In which organism was g-protein independent signalling first identified?

Answer 1

Slime mould

Question 2

How was G-protein independent signalling identified?

Answer 2

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

Question 3

Outline Turki’s 1995 experiment on beta adrenoceptors that showed desensitization.

Answer 3

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

Question 4

What is homologous desensitisation?

Answer 4

Where the attenuation in the signalling response is specific to the receptor being stimulated

Question 5

What is heterologous desensitisation?

Answer 5

Where collateral receptors signalling are also desensitised

Question 6

How did Green and Clark (1981) show that desensitisation occurs at the level of the receptor?

Answer 6

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

Question 7

How can you demonstrate that receptor phosphorylation correlates with desensitisation?

Answer 7

Label cells with [32-P]-orthophosphate –> 32P-ATP
Lyse cells
Immunoprecipitate receptor
Add agonist –> increase in receptor phosphorylation (shown on autoradiograph)

Question 8

Which residue is a GPCR most commonly phosphorylated on?

Answer 8

Serine

Question 9

Which kinase mediated heterologous receptor phosphorylation?

Answer 9

PKA/PKC

Question 10

Is heterologous phosphorylation conformation dependent?

Answer 10

No

Question 11

Is heterologous phosphorylation slow?

Answer 11

Yes takes minutes

Question 12

What experiment showed that receptor desensitisation was not solely dependent on PKA/PKC?

Answer 12

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

Question 13

What is BARK?

Answer 13

Beta -adrenergic receptor kinase

Question 14

Who discovered BARK?

Answer 14

Benovic 1986

Question 15

How was it discovered that there was a missing link between BARK and receptor desensitisation?

Answer 15

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

Question 16

Who found that Beta-arrestin regulates beta adrenergic receptor function in 1990?

Answer 16

Lohse

Question 17

How was arrestin identified as the missing link between BARK and receptor desensitisation?

Answer 17

Measured Receptor-gprotein coupling activity against increasing [GRK]. When GRK and Arrestin was present the predicted desensistisation was achieved.

Question 18

Outline the current model of homologous GPCR desensitisation

Answer 18

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

Question 19

What are the currently identified GRKs?

Answer 19

Visual: Rhodopsin Kinase (GRK1), GRK7 (in cone cells)

Non-Visual: BARK (GRK2), BARK2 (GRK3), GRK4, GRK5 and GRK6

Question 20

What is an RH domain?

Answer 20

An RGS homology domain allowing G alpha interaction

Question 21

How are GRK 2/3 recruited to the ligand occupied receptor?

Answer 21

1. GRK2/3 recruitment to activated receptor via Gbeta gamma and PIP2
2. RGS like domain of GRK2/3 can bind and sequester Galpha subunits

Question 22

Can different arrestins bind the same source GRK?

Answer 22

Yes e.g.
ETa receptor - GRK2 –> Arrestin 3
P2Y2 receptor - GRK2 –> Arrestin 2

Question 23

Outline an arrestin signalling complex (not for clathrin mediated endocytosis)

Answer 23

Phosphorylation and beta arrestin capped beta adrenoceptor recruits Src to activate MAPK signalling

Question 24

What are the two forms of GPCR signalling?

Answer 24

Gprotein dependent –> AC, PLC, PI3K etc.

G protein independent –> MAPK (ERK/JNK), cAMP PDE, DAG Kinases

Question 25

Outline the different signalling pathways that can be elicited using the AT1 receptor

Answer 25

Gprotein dependent - Gq –> Vasoconstriction and Fluid Retention

Gprotein independent - Arrestin –> Cardiac contractility and decrease cardiac cell apoptosis

Question 26

What are the clinical benefits of the two forms of AT1 receptor signalling?

Answer 26

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

Question 27

What is tachyphylaxis?

Answer 27

Decrease in responsiveness of a drug with repeated dosing

Question 28

What is drug tolerance?

Answer 28

A higher dose is required with repeated doses to create the desired effects

Question 29

Outline the clinical benefits of using a biased agonist on B2 adrenergic receptors in obstructive lung disease

Answer 29

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