Receptor Regulation Flashcards by Peter Dudley

How was G protein independent signalling first suggested?

Work done in dictyostelium discoideum.
They only have 1 beta and 1 gamma subunit encoded fro in their genome. Therefore, one BY subunit couple to every alpha subunit.

D. Discoideum release cAMP which acts on neighbouring cAMP GPCR’s when they need to form fruiting bodies

On mutation of the beta subunit all G protein mediated signalling will be attenuated.

However, some aspects of fruiting body formation still exist which means there may be a G protein independent signalling occuring

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What is tachyphylaxis?

Adjustment sin the system due to over stimulation (drug tolerance) which leads to a decrease in response

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What did Turki et al 1995 show?

Patients who received a high dose of B2 agonist (metaproternol) showed a decreased amount of cAMP compared to non pretreated controls when given the B2 agonist isoprenaline. The same levels of cAMP were observed in the two groups on addition of forskulin (which stimulates AC)
Therefore, the decrease in cAMP was due to receptors not down stream effectors

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What is hypothesised to be the main mechanism of receptor desensitisation?

Internalisation

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What is a bias agonist favouring G protein signalling over Beta arrestin signalling useful?

Might prevent internalisation

Therefor, drugs will not develop tolerance

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What is homologous desensitisation?

Where the attenuation in the signalling reponce is specific for the receptor stimulated

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What is the mechanism of homologous desensitisation?

Phosphorylation of specific C terminal residues on GPCR with agonist still bound mediated by GRKs

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What is hetrologous desensitsation?

Collateral receptor signalling is also desensitised

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What is the mechanism for heterologous desensitisation?

Is not dependant on ligand binding therefore no confirmation dependant
PKC/PKA/CaMKII phospho of non specific residues of ICL3

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What are cyc- cells?

Cells which cannot activate AC as lack G protein to activate cAMP

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How can Cyc- cells be used to know desensitsation occurs at the level of receptor?

Add agonist to Cyc- cells. GPCRs bind to ligand but will not signal.

Add Gs and agonist again

These cells will show a decreased amount of cAMP compared to cells which did not receive the first round of agonist (no chance of desensitisation)

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In what order are residues phosphorylated?

Serine>threonine>tyrosine

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What process is essential for internalisation/desensitisation?

Phosphorylation
Occurs rapidly and through different kinases and at different sites which gives a unique phosphorylation profile
May mediate bias agonism

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What are Kin- cells?

Cells which lack PKA

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Why is PKA not essential for desensitisation?

Kin- cells still exhibit B2AR phosphorylation and desensitisation

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What does causes homologous desensitisation?

Lefkowitz group showed the importance eof a kinase called BARK. However, alone BARK does not cause the predicted amount of desensitisation.
B arrestin is also needed

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What are the different types of arrestin?

Visual arrestin = arrestin 1

Beta arrestin 1+2 = arrestin 2+3 (vast proportion of desensitisation)

Where does Beta arrestin bind?

Same site as G protein when GPCR is phosphorylated

Sterically inhibits G protein activation

What are a couple of important domains on beta arrestin?

Clathrin binding domain

AP2 binding domain (recruits clathrin to the membrane and regulates CME)

What do chimeric B2AR show?

Certain domains are important for clathrin binding

Inability to bind to clathrin decreases endocytosis

What other studies show B2AR, clathrin and AP2 localise?

Immunohistochemical studies

Interaction is agonist dependant

What does GRK stand for?

G protein coupled receptor kinase?

What does GRK phosphorylation do?

Causes G protein dissociation from the GPCR

Allows arrestin recruitment

When endocytosed through CME what can the vesicle do?

PH of endosome dephosphorylates GPCR
Can either be recycled to membrane and cause resensitisation
OR targeted to lysosome from degradation due to Beta arrestin ubquitination

What are the visual GRKs?

GRK1 | GRK7

What are the BAR GRKS

GRK2 (BARK1) GRK3 (BARK2) Can be recruited to membrane through plecktrin homology domain which allows phospholipid interaction

What other GRKs are widely expressed?

GRK4 GRK5 GRK6 These contain a lipidated of charged C terminus (GRK5) which mean they are membrane associated. Do not contain PH or beta-gamma domain

What domain do all GRKs have in common?

Catalytic domain which permits phosphorylation

What do different GRKs expression in cells mean?

Different phosphorylation patterns of GPCRs Same receptor may be phosphorylated differently by different GRKS Different down stream effects and arrestin recruitment

give an example where 2 different GRKs have different effects?

GRK2 phosphorylation of B2AR mediates the phosphorylation | GRK6 mainly regulates the rate of arrestin recruitment

How are GRKs recruited?

GRK2+3 contain a PH and beta-gamma domain which allows them to be recruited from the cytosol to the membrane. RGS-like (RH) domain on GRK2+3 can bind to a sequester Ga subunits (not through activating intrinsic GTPase activity)

What determines what GRKs are recruited?

``` Scaffolding proteins Type of receptor Expression of GRK in cell Whether it is the visual system The ligand that binds (different ligands may recruit different GRKs) ```

What govererns what arrestin is recruited?

Phosphorylation bar code. This may causes confirmational change in arrestin recruited which may or may not activate arrestin pathway. E.g. Interaction between rhodopsin and arrestin 1 depend on number/site of phosphorylated residues which may influence bias agnostic

Give an example of how phosphorylation bar code may influence bias agonism

Inhibition of GRK2/3 has no effect on arrestin mediated ERK1/2 activation for B2AR However, Inhibition of GRK5 and 6 does

What is arrestin mediated signalling?

Arrestins can act as adaptor proteins to assemble signal complex scaffolding upon GPCR internalisation E.g. Beta arrestin capped B@AR can recruited Src to complex which can activate the ERK pathway

Define Bias agonism

Different ligands may activate/ show preference for activating one signalling pathway over another

What may influence bias agonism?

Phosphorylation pattern on GPCR might causes conformational Change on arrestin which determines its downstream effects i.e which signalling molecules it scaffold which

How can different agonists causes bias agonism?

Different agonism may bind to same receptor But may favour different phosphorylation patterns on GPCR Different arrestins recruited/ conformational changes Different signaling cascades

Give an example of a bias agonist pathway?

Angiotensin 2 normally causes vasoconstriction, increased in fluid and sodium retention and increased aldosterone. Part of this of this arrestin mediated (increased cardiac contractility, decreased cardiac apoptosis and increased aldosterone) SII-AngII is similar to angiotensin 2 apart from 3 amino acids. This ligand favours arrestin mediated signalling and may be of use in heart failure

How can bias agonism be clinically useful?

1. Beta arrestin causes desentisation. If we can favours G protein over Beta arrestin signalling we can attenuate desensitisation and tolerance to drugs e.g. Dobutamine in HF is needed for ionotropic support but tachyphylaxis limits its use 2. Increases the effect of medication e.g. B arrestin 2 KO mice show improved morphine analgesia 3. Attenuation of side effects mediated by a particular pathway e.e.g beta arrestin 2signalling is responsible for respiratory depression in opioids use