GPCRs

• General mechanism by which GPCRs are activated by their ligand:

  
  

  Agonist binding leads to a conformational change in the GPCR, which stimulates GDP/GTP exchange in the G protein.

  Active, GTP-bound G protein dissociates into alpha subunit and beta-gamma dimer (both are membrane-bound due to lipid modifications)

  The alpha subunit and beta-gamma subunit regulate effector proteins (e.g. adenylyl cyclase)

  GTP hydrolysis deactivates the alpha-subunit which reassembles the beta-gamma dimer
• An agonist is an effector/ligand molecule that binds to the extracellular side of the receptor
• Full agonist - causes 100% activity of the GPCR

  Partial agonist - causes partial activity of GPCR
• An inverse agonist turns the level of GPCR activity from its basal, constitutive level to zero, therefore the GPCR is completely inactivated
• A neutral agonist has no effect on the GPCR activity levels
• Constitutive activity of the GPCR refers to the fact the GPCR tends to usually have a low level of activity at all times, even when not bound to an agonist
• The two main classes of GPCR are:

  Class A - rhodopsin-like

  Class B - binds larger peptides e.g. glucagon
• Downstream effects of GPCR activation:

  
  

  ATP converted to cAMP

  Regulation of ion channels
• G protein subunits are found bound to the inner leaflet of the lipid membrane, close to the membrane-bound GPCR
• GPCRs tend to activate downstream signalling effects - inhibition is very rare
• Adenylyl cyclase structure has two membrane-spanning domains and two intracellular catalytic domains
• Describe the function of adenylyl cyclase in signal transduction:

  
  

  Converts ATP to cAMP via a hydrolysis reaction that releases Pi

  cAMP is prevalent in the whole human body and mediates many hormone-induced cell responses
• What enzyme converts cAMP back to ATP? cAMP phosphodiesterase - converts via AMP
• Describe how cAMP activates protein kinase A. What is this an example of? cAMP binds to regulatory subunitsInduces conformational changeCauses dissociation of regulatory subunits and catalytic domainsExample of a double-negative - inhibition + inhibition leads to activation
• Give an example of a slow response mediated by protein kinase A

  
  

  Somatostatin expression

  Slow response as activated protein kinase A must be transported into the nucleus via the NPC, where it binds to inactive CREB (transcription factor)

  CREB is phosphorylated and becomes active

  CREB binds to the cAMP response element (CRB) in the genome, causing transcription of the somatostatin gene
• Somatostatin is an inhibitory hormone in the digestive system
• Give an example of a fast response mediated by protein kinase A

  Phosphorylates protein typically responsible for keeping calcium ion channel shut

  So calcium ion channels open causing influx of calcium ions
• Summarise the downstream signalling process associated with adenylyl cyclase

  
  

  G-alpha protein

  Adenylyl cyclase

  cAMP (second messenger)

  Protein kinase A

  Transcription factors (slow) or ion channels (fast)
• Phospholipase C-beta is another important G-alpha protein effector

  Binding of G-alpha causes phospholipase activation

  Cuts bond in phosplipid, releases inocetyl phosphate (IP3) as a secondary messenger

  Diacylglycerol left over in the phospholipid bilayer
• IP3 mediates the influx of calcium ions from the ER into the cytoplasm
• Explain how intracellular calcium ion oscillations are generated

  
  

  Combined effect of positive and negative feedback loops

  IP3 mediates influx of calcium ions into the cytosol

  Positive feedback - influx of calcium ions induces adjacent calcium ion channels to open

  High calcium ion concentration in the cytosol causes channels to close

  Calcium pumps in ER membrane restore the low calcium concentration in the cytosol
• Give an example of a GPCR whose downstream signalling occurs via the PLC-beta-IP3 pathway

  
  

  Vasopressin - induces liver cells to release glucose from glycogen in response to stress

  Therefore during stress, vasopressin receptor activation induces calcium ion oscillations in the liver
• Describe how calmodulin-dependent kinases (CaMKs) decode the calcium level

  
  

  Calcium ions bind to calmodulin, activating it

  Activated calmodulin binds to regulatory domain of CaMKII

  Binding causes conformational change that leads to release of CaMKII autoinhibitory domain

  Specific threonine residues in the regulatory domain of CaMKII are autophosphorylated, enhancing activity

  Activated CaMKII phosphorylates substrate proteins
• Give some examples of substrate proteins phosphorylated by CaMKII

  
  

  Synaptic transmission

  Gene expression

  Cell signalling