L3 - G-protein coupled receptors

• Many different GPCR (very diverse):

  • Respond to hormones, neurotransmitters, endocrine, paracrine, signals, odours & light (>1,000 types in human)
  • Receptor (gives specificity)
  • G-protein (transduces signal)
  • 3 components = ɑ & 𝛽𝑦, dissociate to have an effect
  • Effector (ɑ & 𝛽𝑦 subunits may provide different signals)
  • Both do things
• The intracellular signals generated by the effectors often regulate protein kinases:

  • Adenylate cyclase activates protein kinase A (PKA)
  • Activated by ɑ (alpha) subunit
  • Phospholipase C activates protein kinase C (PKC) & calcium / calmodulin-dependent protein kinase
  • Acting on different effector, signals different pathway in cell
  • Ca2+, activated at same time
• • Many additional signalling pathways also exist
  • A single GPCR can regulate multiple pathways
• 
  

  Protein kinases provide the major mechanism for changing the activity of an existing protein

  • Increase or reduce the activity of an enzyme
  • Turn a signalling protein on or off
  • Change the location of a protein
  • Alter the interaction of a protein w/ other molecules
  • Proteins have specific shape, made up of amino acids; that shape controls what it does
• Post-translational modification of a protein:

  • The regulated addition of a small molecule
  • Most often the addition of a phosphate
  • Adds a large negative charge
  • Will cause the folded protein to adjust its shape
• Protein phosphorylation is carried out by protein kinases (/phosphate):

  • Transfer a phosphate from ATP to the target protein
  • Protein kinases add phosphates
  • Protein phosphatases remove them
  • Regulate them so don’t undo all your proteins
• Protein kinases are categorised after their amino acid target
• Often named by what activates them:

  • Protein kinase A - requires cAMP
  • Protein kinase C - requires Calcium
  • Cyclin-dependent protein kinases
  • What makes cells go round (cell division)
  • Receptor tyrosine kinases
  • Note: protein kinases must be regulated
  • As are changing shape of proteins
  • Changing activity & location
  • Changes how cell is behaving
• Protein kinase A activation:

  • PKA exists as an inactive complex with its regulatory subunits
  • Kinetic activity (enzyme)
  • Regulators, stopping them
  • cAMP binds to the regulatory subunits
  • Changes to shape, kinase falls off
  • Causes them to dissociate
  • Releasing the active kinase
• PKC activation (something bind, changing its shape):

  • Inactive PKC located at cell membrane
  • Interacts with lipid produced following receptor activation
  • PKC complex disassociates & releases active (catalytic) subunit of PKC
• • There are many different protein kinases
  • (2 % of human genome)
  • Some are “promiscuous” having many protein “targets”
  • Promiscuous: put phosphate’s on multiple proteins
  • Others highly specialised to a single protein
  • Grouped into families based on their sequence
• Protein kinases may increase the expression / amount of a protein via transcription factors

  • Changes the amount of a specific protein in a cell
• Protein kinases may also change the activity of existing protein

  • Increases the activity of an enzyme
  • Change by putting phosphate on protein
  • Illustrate the latter w/ reference to the enzyme tyrosine hydroxylase
• Tyrosine hydroxylase (TH) - major metabolic pathway:

  • Catecholamines have multiple functions
  • Dopamine, noradrenaline & adrenaline (neurotransmitters)
  • Dopamine → emotions
  • Noradrenaline → sympathetic system
  • Adrenaline → stress pathway
  • All made by the same pathway
  • First & rate limiting step carried out by TH
  • Dopamine provides a neg feedback
  • Also related to parkinson's disease
• Tyrosine hydroxylase (TH) → Dopamine (DA):

  • Dopamine synthesised / made in nerve terminals
  • Released in response to nerve stimulation
  • Now need more dopamine (feedback is now not helpful)
  • Increase TH activity via phosphorylation
  • Phosphorylation reduces the negative feedback by dopamine
  • Acted by signalling pathways
  • Inhibitory mechanism
• • TH can be phosphorylated 4 different sites
  • Different kinases act at each site
  • Different sites have different effects on activity
  • The order of phosphorylation matters
  • Lots of signals feeding in 1 enzyme ser40