GPCR 2 Flashcards
What types of post translational regulation of G protein dependent signalling exist?
Receptor phosphorylation
G protein phosphorylation
Effector phosphorylation
How does post translational regulation alter the outcome?
By altering any of those aspects you alter the protein activity and the signalling pathway
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
What happens when GPCR are phosphorylated by GPCR kinases (GRKs)?
Homologous desensitisation -
Arrestins bind to phosphorylated receptors
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
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
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
What are the targets of physiological regulation of G proteins at the mRNA level?
Receptor
G protein
Effector proteins
What sort of pathways are affected by transcriptional regulation of G proteins?
Thyroid hormone
Glucocorticoids
Sex hormones
Cyclic AMP
Puberty
Menopause
Endocrine disorders
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
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
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
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
What are the receptor gain of function mutations?
LH - familial make precocious puberty
TSH - toxic thyroid hyperplasia/ adenoma
PTH - Jansen’s chondrodysplasia
What are the receptor loss of function mutations?
V2 - nephrogenic disbetes insipidus
ACTH - glucocorticoid deficiency
TP - congenital bleeding
ET;B - aganglionic megacolon
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
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
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
