3. Old stuff
          3.1. Old pharm stuff (pre 2009)
              3.1.3. Pharmacology
                  3.1.3.1. Pharmacology principles
                      3.1.3.1.2. Pharmacodynamics
                          3.1.3.1.2.2. Mechanism of drug actions
                              3.1.3.1.2.2.1. Receptors
 3.1.3.1.2.2.1.2. G-protein system 

G-protein system

[WG21:p34-35; RD5:p26-45]

  • G-protein system allows the transduction and amplication of the original signal

Structure

  • G protein coupled receptors (GPCR) has a single polypeptide that has 7 transmembrane alpha-helical segments
    * Ligand-binding site is extracellular
  • G proteins are heterotrimers
    * 3 subunits (alpha, beta, gamma)
  • Gs = Activate adenylate cyclase --> mostly stimulatory effects
  • Gi = Inhibit adenylate cyclase --> mostly inhibitory effect
  • Gt = Photoreception in the eyes
  • Gq = Phospholipase C regulation

Mechanism

  1. A ligand binds to GPCR in the cell membrane
  2. Binding causes G-protein to become activated
  3. Alpha subunit release GDP and binds to GTP instead, and GTP-alpha complex separate from beta-gamma complex
  4. GTP-alpha complex interacts with effector proteins
  5. GTP becomes hydrolysed (by the intrinsic GTPase activity of the alpha subunit)
  6. GDP-alpha re-unite with beta-gamma complex

NB:

  • Beta-gamma complex may exert actions on their own
    * But require higher receptor occupancy for this effect to manifest
    * [RD5:p34]

Examples

  • Adrenergic receptors
  • Dopamine receptors
  • Adenosine receptors
  • Most 5HT receptors
    * Except for 5HT3
  • Muscarinic ACh receptors
  • Cholera toxin acts on Gs protein
    --> Persistent activation
    --> Excessive secretion of fluids

Targets for G-proteins

[RD5:p36-39]

Main targets for G-proteins:

  • Adenylate cyclase
    --> Responsible for cAMP formation
  • Phospholipase C
    --> Responsible for inositol phosphate (IP) and diacylglycerol (DAG) formation
  • Ion channels
    * Especially Ca2+ and K+ channels
  • Phospholipase A [RD5:p41]
    * Arachidonic acid and eicosanoids formation

Adenylate cyclase

  • Adenylate cyclase is
    * Membrane bound
    * Synthesize cAMP (a nucleotide) from ATP
    * Activated by Gs, inhibited by Gi protein
  • Examples [WG21:p99,p112; RD5:p139]
    * Dopamine receptors (D1,D5 = Gs; D2-4=Gi)
    * Alpha2 adrenergic receptors (Gi)
    * Beta adrenergic receptors (Gs)
    * Adenosine receptors (A1=Gi; A2=Gs)
    * Muscarinic ACh receptors M2, M4 (Gi)
    * 5-HT1 and 5-HT4 receptors (5-HT1 = Gi; 5-HT4 = Gs)
    * GABAb receptor (Gi)

cAMP

  • cAMP activates protein kinases
    --> Protein kinases catalyse phosphorylation of enzymes or ion channels
    --> Activation or inhibition of these enzymes or ion channels
    --> Various effects
  • Examples of cAMP actions (via protein kinases)
    * Phosphorylation of voltage-activated calcium channel in heart --> Increase Ca2+ flow --> Increased contractility
    * Phosphorylation of myosin-light-chain kinase in smooth muscles --> Inactivation --> Smooth muscle relaxation
  • cAMP is hydrolysed by phosphodiesterase
  • Phosphodiesterase can be inhibited by drugs such as
    * Methylxanthines (theophylline, caffeine)
    * Sildenafil (aka Viagra)

Phospholipase C / inositol phosphate system

  • Activation of G-protein-coupled receptor
    --> Phospholipase C is activated
  • Phospholipase C
    * Membrane-bound
    * Catalyses phosphatidylinositol-4,5-bisphosphate (PIP2) ===> diacylglycerol (DAG) + inositol-1,4,5-trisphosphate (IP3)
  • Examples [WG21:p99; RD5:p139]
    * Muscarinic ACh receptors M1, M3, M5
    * Alpha1 adrenergic receptors
    * 5-HT2 receptors
    * GABAb receptors

Diacylglycerol (DAG)

  • Activate a membrane-bound protein kinase, protein kinase C (PKC)
  • Highly lipophilic and remains within the membrane (unlike IP3)
  • DAG is later phosphorylated
    --> Phosphatidic acid
Protein kinase C (PKC)
  • There are 12 subtypes of PKC
  • Most are activated by DAG and raised intracellular Ca2+ level
  • Also activated by phorbol esters (a highly irritant and carcinogenic compound)
  • One of the subtypes are activated by arachidonic acid
  • PKC catalyses the phosphorylation of various intracellular proteins
    --> Various effects

Inositol-1,4,5-trisphosphate (IP3)

  • Water-soluble
    --> Released into the cytosol
  • Acts on the IP3 receptor
    * Which is a ligand-gated calcium channel on the membrane of endoplasmic reticulum
    --> Control the release of Ca2+ from intracellular store
    --> Increases free intracellular Ca2+
    --> Initiate other events (secretion, contraction, enzyme activation, membrane hyperpolarisation)
  • IP3 is later dephosphorylated
    --> Inositol
    --> Bind with phosphatidic acid (i.e. phosphorylated DAG)
    --> PIP2 is formed once again
  • Lithium blocks the formation of inositol

Ion channels (as a target of G-protein)

  • G-proteins can directly act on ion channels without a secondary message
    * Via the action of the free alpha-subunit, or the beta-gamma-subunit complex
  • Examples:
    * Opiate receptors opening K+ channels
    * Muscarinic ACh receptors enhancing K+ permeability

Desensitisation of GPCR

Homologous (agonist-specific) desensitisation

  1. Agonist binds to receptor --> Activation of the receptor
  2. Activated receptor becomes phosphorylated by a specific kinase (GRK)
  3. Phosphorylated receptor binds to arrestin
    --> Loss of ability to associate with G-protein (loss of G-protein coupling)
    --> Undergoes endocytosis

Heterologous desensitisation

  • Phosphorylation of the receptor is by protein kinases which were activated by another type of receptors