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