Week 5 Flashcards
What are the steps involved in GPCR endocytosis, trafficking, recycling and degradation?
Endocytosis (clathrin-mediated)
- ligand binding induces G protein recruitment and signalling
- GRKs phosphorylate the GPCR C-terminal tail and cause beta-arrestin recruitment
- beta-arrestin bound GPCRs interact with clathrin via direct interaction of the adaptor protein AP-2 and clathrin
- GPCR interaction with clathrin leads to induction of endocytosis with the receptor incorporated into the clathrin-coated pit
- dynamin catalyses pinching off the clathrin-coated pit resulting in receptor internalisation
- clathrin uncoats from the endosome and the GPCR is delivered to the sorting endosome
- the slightly acidic environment leads to dissociation of receptor and ligand
- the receptor is either degraded in the lysosome or recycled to the plasma membrane
Trafficking
- follows endocytosis
- trafficked into sort endosomes which are demarked by GTPase Rab5
- bind of GTPase Rab5 to an endosomal membrane can effect a change in receptor trafficking
Recycling
- receptors targeted for recycling are removed from the Rab5 sorting endosome
- Rab4 (rapid) or Rab11 (slow) localise to microdomains on the sorting endosomal membrane
- receptors localised to Rab4 vesicles are recycled directly to the plasma membrane
- receptors localised to Rab11 vesicles are first delivered to the endocytic recycling compartment, and are then recycled to the plasma membrane
- recycling allows further rounds of ligand binding and receptor signalling
Degradation
- receptors targeted for degradation remain in the Rab5 sorting endosome
- sorting endosome matures into a late endosome which is marked by the loss of Rab5 and acquisition of Rab7 on the endosomal membrane
- receptors cargoes bound by ubiquitin are incorporated into the lumen of the late endosome by ESCRT proteins which forms a multivesicular body (MVB)
- this MVB matures into a lysosome and receptors are exposed to degradative proteases and are broken down
- degradation terminates signalling by reducing receptor levels at the membrane
How do different endocytic mechanisms lead to differing signalling outcomes?
Dynamin dependent
- clathrin: predominant form and regulates GPCRs
- endophillin: requires dyamin and regulates GPCRs
Clathrin independent
- CLIC/GEEC: rapid carbohydrate and receptor uptake
- phagocytosis: bacterial/dead cell uptake
- micropinocytosis: cell drinking, nutrient uptake
What are the roles of beta-arrestin and G proteins in endosomal signalling?
When GPCR, beta-arrestin and G proteins are coexpressed, all three are clearly identifiable on endosomes following ligand addition
Given the strong colocalisation of the receptor, beta-arrestin and G protein on the same endosome, it is likely the receptor Gs-alpha and beta-arrestin form a complex together on the endosome
G proteins
- present on endosomes
- G protein binds after endocytosis
- blockade of endocytosis reduces cAMP production by beta2AR which confirms G protein contributing to signal
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beta-arrestion
- not found on the active beta2AR endosomes
- beta-arrestin mediated endocytosis is indispensable for endosomal signalling
- beta-arreston localisation to endosomes is required for endosomal signalling even f the signalling is G protein mediated
- beta-arrestin binding affinity tunes the magnitude of endosomal signalling
Why does endosomal signalling occur and how is it regulated?
A signal e.g. cAMP is in reality a small, diffusible molecule
cAMP can only diffuse a short distance to exert its effect before it is degraded
An endosome localised receptor is much closer to the nucleus
cAMP produced by endosomal receptors can diffuse to the nucleus, activating PKA and transcription factor
cAMP must diffuse from the plasma membrane or endosome to activate PKA
Phosphodiesterases (PDEs) are cytosolic enzymes that hydrolyse cAMP, degrading it and limiting its action to a nanometre scale
PDE inhibition restores endosomal signalling following beta2AR endocytosis inhibition or redirection to the plasma membrane
Endosomal signalling delivers the signal to specific subcellular locations to diversify the signalling response, evading the cells regulatory mechanisms
How is efficacy defined by the theory of biased signalling?
Efficacy is an intrinsic property of the receptor/ligand pair for all responses evoked.
What are experimental examples that support biased signalling?
Propanolol and ICI118551 are defined as beta1-AR receptor antagonists/inverse agonists when measuring cAMP (under threshold) but they are agonists when measuring ERK phosphorylation and reporter gene activity (above threshold).
D-Trp-OPE is a CCk receptor antagonist when measuring cAMP, CCK receptor phosphorylation and Ca2+ inhibition but it s an CCK receptor agonist when measuring internalisation.
Opioids such as morphine are strong analgesics but dose-limiting adverse effects often prevent adequate pain relief.
Theory behind TRV130:
- G alpha i coupling at the MOR = more powerful analgesia
- beta arrestin 2 recruitment = respiratory/GI side effects and inhibition of the analgesic effect
- TRV130 stabilises MOR conformations that promote G protein coupling by not beta arrestin recruitment
- TRV130 causes less constipation and respiratory depression than morphine dose are equi-analgesic doses
What factors contribute to the molecular basis of signalling basis?
Bias signalling describes the propensity of a ligand to signal via a subset of pathways, or even new pathways, compared to those activated by a reference ligand, usually the endogenous ligand.
Fluorescein maleimide is an environmentally sensitive fluorescent probe, meaning that it will report changes in its local microenvironment as changes in fluorescence intensity over time/fluorescence lifetimes
By placing a probe at the base of the dynamic TM6 it is possible to monitor conformational changes in the beta 2 AR
Agonist treatment leads to the formation of new conformations in the beta 2 AR and loss of other conformations that were found in the intreated condition
Binding of the neutral antagonist alprenolol to beta 2 AR narrows the distribution of lifetimes, suggesting that ALP stabilises the receptor and reduces conformational fluctuations. These conformations are largely shared by the no drug state of the receptor
The receptor bound to the agonist isoprenaline forms a subset of the conformations found with the unbound receptor (no drug) and alprenolol as well as forming its own very different new conformations
Binding of the full agonist ISO and the partial agonists SAL and DOB to beta 2 AR narrows distribution of the lifetimes >3 ns suggesting that the ligands stabilise a subset of conformations present in the unbound receptor
The differences in the short lifetimes (<3 ns) for the full agonist and the partial agonists indicate different agonist-specific active states for these ligands
How does ligand binding kinetics influence biased signalling?
Residence time refers to the temporal duration of a binary receptor-ligand complex
Advocates of the concept of residence time argue that Koff is actually more important than ligand affinity for the effects of drugs in vivo
For example:
- there are a variety of biased dopamine receptor agonists
- they each have different efficacies at 5 minutes (evidence of biased signalling)
- bifeprunox, aripiprazole and cariprazine are biased towards G protein-dependent pathways
- biased dopamine receptor agonists display slower dissociation rates and their bias profile changes over time
- bifeprunox, aripiprazole and cariprazine have longer residence time
Hypotheses proposed:
- a give ligand stabilises a given conformational state, which leads to specific signalling outcomes due to selective engagement of different subsets of effectors
- the duration of a ligand-receptor complex determines the different effector and regulatory proteins that can be engaged over time
What are the therapeutic consequences of ligand-specific receptor confirmations with clinical examples?
Consequences of ligand-specific receptor confirmations are:
- more than one signalling pathways needs to be examined in determining the function of a drug (increased $$$ and time)
- side effects could arise from the activation or inhibition of alternative signalling events
- structure activity relationships (SAR) need to be developed for each signalling pathway
Mu opioid receptor
- originally biased signalling supported by beta-arrestin II mice
- in knock-in mice that cannot recruit beta-arrestin as no phosphorylation sites were present, arrestin effects were still present
- different source of mice that didn’t support the bias hypothesis as there were still effects when arrestin was removed in a different way
- techniques support G protein-dependent adverse effects
