GPCR 2 Flashcards by Rachael Christie

What types of post translational regulation of G protein dependent signalling exist?

Receptor phosphorylation
G protein phosphorylation
Effector phosphorylation

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How does post translational regulation alter the outcome?

By altering any of those aspects you alter the protein activity and the signalling pathway

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What happens when GPCR are phosphorylated by second messenger activated protein kinases?

Negative feedback mechanism on pathway activation
Heterologous desensitisation of neighbours
Influences affinity, efficacy of agonist receptor complex
Alters G protein selectivity
Can happen in the absence of an agonist

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What happens when GPCR are phosphorylated by GPCR kinases (GRKs)?

Homologous desensitisation -
Arrestins bind to phosphorylated receptors

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What is homologous desensitisation?

GPCR regulation phosphorylation by GRKs
only happens when activated by an agonist as the change in shape reveals the phosphorylation sites

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What happens when arrestins bind to phosphorylated receptors?

Prevent G protein interaction
Desensitisation/ internalisation via clathrin pits
Followed by dephosphorylation
Activate non G protein dependent signalling pathways

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Describe internal signalling

Persistent responses (every 30 mins)
Different sub cellular environment to cell surface
Will influence the effector and response
Receptor will remain active until late endosomal trafficking
Eventual resensitisation and return to the surface

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What are the targets of physiological regulation of G proteins at the mRNA level?

Receptor
G protein
Effector proteins

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What sort of pathways are affected by transcriptional regulation of G proteins?

Thyroid hormone
Glucocorticoids
Sex hormones
Cyclic AMP
Puberty
Menopause
Endocrine disorders

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What happens due to ‘gain of function’ mutations in G protein linked signalling during diseases at the receptor level?

Receptor proteins become constitutively active in absence of agonist
Some compensation by reduced receptor expression

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What happens due to ‘loss of function’ mutations in G protein linked signalling during diseases at the receptor level?

Loss of receptor transcription/ translation
Inappropriate sub cellular targeting
Abnormal receptor proteins
Altered affinity/ efficacy

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What happens due to ‘gain of function’ mutations in G protein linked signalling during diseases at the G protein level?

Causes a loss of GTPase activity
Constitutive activation in the absence of agonist receptor involvement

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What happens due to ‘loss of function’ mutations in G protein linked signalling during diseases at the G protein level?

Loss of G protein transcription/ translation
Inappropriate sub cellular targeting
Abnormal forms of G protein

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What are the receptor gain of function mutations?

LH - familial make precocious puberty
TSH - toxic thyroid hyperplasia/ adenoma
PTH - Jansen’s chondrodysplasia

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What are the receptor loss of function mutations?

V2 - nephrogenic disbetes insipidus
ACTH - glucocorticoid deficiency
TP - congenital bleeding
ET;B - aganglionic megacolon

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What are the G protein gain of function mutation?

G alpha s - thyroid adenoma, cardiomyopathy, McCune-Albright syndrome
G alpha q - cardiac hypertrophy decompensation to failure
G alpha t - stationary night blindness

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What are the G protein loss of function mutation?

G alpha s - pseudohypoparathyroidism
G alpha i2 - ulcerative colitis and adenocarcinoma of the colon, type ll diabetes
G alpha q - obesity, impaired lipolysis

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What are RGS proteins?

Multifunctional GTPase accelerating protein that promote GTP hydrolysis by the alpha sub unit of heterotrimeric G proteins

How does phosphorylation affect RGS proteins?

Can increase or decrease RGS activity or influence translocation to / interaction with membrane
Influences RGS stability / rate of degradation

How do is RGS activity affected by palmitoylation?

Reversible palmitoylation or lipid modification promotes membrane association
Increases RGS activity

How are RGS proteins orientated?

Sequences in receptor proteins help localise and orientate RGS proteins towards G proteins

What influences RGS protein expression?

2nd messenger pathways
Transcription factors (PKA, PKC)
Disease
Drugs of addiction

Give some examples of recent development in GPCR research

Agonist trafficking / functional selectivity
Allosteric regulation
GPCR oligomerisation
Other accessory proteins
Orphan GPCRs - approaches to identify

What are the factors of agonist trafficking and functional selectivity?

Multiple receptor activation states possible
Different agonists cause different conformational changes
Some antagonists only block subsets of agonist induced signalling events at same receptor

Describe the multiple receptor activation state factor

Agonist increase the proportion of receptors in various active conformations / associated with various signalling pathways (pluri-dimensional) Natural agonist ligands have potential to activate all possible pathways depending on context

Describe the different agonists cause different conformations factor

Increase or decrease favourable interactions with specific G proteins and extent of activation of various signalling pathways Conformations differ in susceptibility to desensitisation TRV130 biased agonist against mu opioid receptor - retains analgesia but reduced side effects than morphine

Explain the some antagonist only block subsets of agonist pathways factor

Increases drug specificity Decreases side effects

What is an orthosteric site?

The ligand / agonist binding site

What is an Allosteric site and why do molecules bind here?

Allosteric sites can be intracellular or extracellular Any molecule of appropriate size to influence the orthosteric site without actually binding there

Describe some of the novel drugs that may act at Allosteric sites

They will modify responses to various orthosteric ligands They will be small and less complex ligands Less complex binding interactions Smaller, saturable effects May display bias for some signalling events Greater distinction between receptor subtypes that orthosteric ligands

Name some of the benefits of novel drugs acting at Allosteric sites

Fewer side effects Safer in the case of overdose Positive allosteric regulators display less desensitisation phenomena vs orthosteric agonists Potential for pathway dependant allosteric modulation

What is maraviroc?

Novel antiretroviral for HIV A negative allosteric regulator of CCR5 chemokine receptors on the surface of CD4+ cells Prevents binding and entry of HIV

What is palonosetron?

A novel Allosteric antagonist at serotonin 5-HT3 receptors Used for anti-emetic therapy

What is oligomerisation?

A chemical process that converts monomers to macro molecular complexes through a finite degree of polymerisation

How does GPCR oligomerisation afford greater diversity?

There are doing hot 900 GPCRs?

What are the implications for GPCR oligomerisation?

Transport to the cell surface Ligand binding Receptor activation G protein coupling Assymetrical binding of RGS proteins Receptor desensitisation Disease

Explain the implication of transport to the cell surface membrane

Masks sequences for ER retention by gate keeper proteins Needed to enable receptors to pass quality control

Explain the implication of ligand binding

Both partners contribute to novel binding site Binding of agonist/ antagonist to one partner influences binding profile of other E.G binding of D3 agonist increases affinity of agonists for D1

Explain the implication of G protein coupling

2 receptors per G protein - do both need to be activated? Cis or trans activation depending on which receptor G protein is physically closest to

Give an example of how heteromer specific signalling is characterised

Monoclonal antibodies can be generated that specifically recognise receptor heteromers but not homomers

Describe the GPCR recent development including accessory proteins

Similar consequences to dimerisation but the protein is not a GPCR

What are orphan GPCRs?

100-200 GPCRs with no known ligand or function identified by sequencing human genomes

How can orphan GOCRs be identified?

Using bioinformatics to predict candidate ligands based on comparisons with known sequences and structures