Second Messenger Signalling

What are G-protein coupled receptors (GPCRs)

• Play a crucial role in neuronal signalling, vision, smell and taste

• The GPCR binds a ligand and transmits a signal to the inside of the cell

• This activates a G protein bound to the inner surface of the GPCR

• This triggers an intercellular signalling cascade

Difference between 7TM receptors and GPCRs

• 7 TransMembrane receptors are a broad class of receptors that has 7 transmembrane segments

• They activate different intracellular signaling pathways, but not all of them use G proteins.

• GPCRs are specific type of 7TM receptor that always signals through G proteins to trigger intracellular pathways

How many subunits does a G-protein have?

3

• Alpha

• Beta

• Gamma

What are the effects of different GPCRs determined by?

Which G-protein alpha subunit they activate

Mechanism of GPCR signalling

1. Ligand e.g. NE, E binds to 7TMR / GPCR

2. GPCR undergoes a conformational change

3. G protein is activated, leading to GTP replacing GDP on the α-subunit of the G protein.

4. G protein dissociates from the GPCR in 2 parts: Gα-GTP (active form) and Gβγ complex

5. These components interact with downstream effectors (e.g., proteins, ion channels). One activated, the effectors can activate other proteins leading to an intracellular signalling cascade.

6. GTP is hydrolysed to GDP and Gα. The Gα rebinds to Gβγ complex, returning the G protein to the inactive state.

3 examples of G-protein coupled receptors used in the sympathetic nervous system

• α₁ noradrenaline adrenergic receptor

• α₂ noradrenaline adrenergic receptor

• β₁/β₂ noradrenaline adrenergic receptor

Explain the intracellular signalling mechanism of α₁ noradrenaline adrenergic receptor

• NE/E binds to receptor coupled with G_q/11 protein

• This causes a conformational change in the receptor

• G_q/11 protein is activated. GTP replaces GDP on the Gαq subunit. Gαq-GTP dissociates from the Gβγ complex

• Gαq-GTP activates Phospholipase C

• This produces IP₃ and DAG » these are the second messengers

• IP₃ causes Ca2+ to be released

• Ca2+ and DAG activate protein kinase C

• Causing smooth muscle contraction

• Effect: pupil dilation, constriction of sphincters, contraction of blood vessels

Explain the intracellular signalling mechanism of α₂ noradrenaline adrenergic receptor

• NE/E binds to receptor coupled with Gi protein

• This causes a conformational change in the receptor

• G_i protein is activated.

• Inhibits adenylate cyclase, decreasing cAMP levels » this is the second messenger

• So less activation of protein kinase A = smooth muscle contraction

• The Gβγ subunit dissociates and inhibits VG Ca2+ channels, reducing Ca2+ influx = prevents release of neurotransmitter

• Effect: decreases release of noradrenaline

Explain the intracellular signalling mechanism of β₁/β₂ noradrenaline adrenergic receptor

• NE/E binds to receptor coupled with Gs protein

• This causes a conformational change in the receptor

• G_s protein is activated. GTP replaces GDP on the Gαq subunit. Gαq-GTP dissociates from the Gβγ complex.

• Gαq-GTP activates adenylate cyclase which converts ATP to cAMP

• Increases cAMP levels » this is the second messenger

• So more activation of protein kinase A

• Effect: increases heart rate, relaxes bronchial smooth muscle, glycogenolysis

β₁ adrenergic receptor

After protein kinase A is activated…

In cardiac muscle:

• Protein kinase A phosphorylates L-type Ca2+ VG channels

• Increased influx of Ca2+ through VG Ca2+ channels

• More Ca2+ released from sarcoplasmic reticulum

  = stronger contractions

• Protein kinase A phosphorylates PLB

• So more Ca2+ pumped back into sarcoplasmic reticulum

  = relaxation of heart muscle

β₂ adrenergic receptor

After protein kinase A is activated…

In bronchial smooth muscle:

• Protein kinase A phosphorylates and inhibits MLCK

• This prevents actin-myosin cross bridges from forming

  = relaxation of bronchial smooth muscle

• Protein kinase A also activates phosphorylase kinase

  = This increases the breakdown of glycogen to glucose (glycogenolysis)

GPCR vs ligand-gated ion channels

G-protein coupled receptors (metabotropic) are a lot slower than normal ligand-gated channels (ionotropic)

What do we mean by ‘activating’ a GPCR

• GPCRs may appear to be active even when no ligand is present » this is called basal activity

• This means ligands can either increase or decrease basal activity

NOTE:

Muscarinic receptors are…

A type of GPCR that binds Ach!!

Role of GPCRs in pharmacy

• Important drug targets » >30% of drugs target GPCRs

• Partial and inverse agonists are being used to regulate signalling e.g. by only partially activating or suppressing the GPCR

Why is it difficult to develop selective drug agonists/antagonists for GPCRs

• Many 7TMRs form heterodimers (2 different GPCRs which create a new drug target)

• Drug development must consider how GPCRs interact with each other, making drug design more complex.

What are Receptor Tyrosine Kinases (RTKs)?

• Single transmembrane proteins (have 1 segment that crosses the membrane)

• When a ligand binds to the extracellular part of the RTK, it dimerises (2 RTK molecules come together)

• Each tyrosine is phosphorylated, forming an activated RTK

• The phosphorylated tyrosines activate relay proteins which then produce a response

What does it mean by RTKs have constitutive activity?

Have the capacity to autophosphorylate themselves

What conditions have treatments that target RTKs?

Cancer

Examples of TRKs

• TrkA and TrkB which bind nerve growth factors

• Ephrins