L6 - G protein coupled receptors (GPCRs) signalling

what are effectors?

• proteins that create the actual cellular response once they are activated by signals like G proteins

what are examples of effectors?

• enzymes that create 2nd messengers

• ion channels whose gating is regulated

what are the two ways in which ion channel gating is regulated?

• directly (beta-gamma subunits)

• indirectly by 2nd messengers and their effectors

what are examples of 2nd messengers?

• hydrophobic lipids

• cAMP/cGMP

• calcium ions

what produces cAMP (cyclic AMP)?

• adenylyl cyclase (enzyme) - regulates and controls levels and activity of cAMP

what is adenylyl cyclase?

• membrane anchored enzyme

• has 10 different isoforms

what is the structure of adenylyl cyclase?

• 2 homologous domains

• each homologous domain contains 6 TM domains/segments (12 TMs total)

what is adenylyl cyclase activated by?

• activated by Gas (G alpha stimulatory)

what is adenylyl cyclase inhibited by?

• inhibited by Gai (G alpha inhibitory)

steps in cAMP production

1. Ligand (like adrenaline or glucagon) binds to a Gαs-coupled GPCR

2. The receptor activates Gαs

3. Gαs activates adenylyl cyclase

4. Adenylyl cyclase converts ATP → cAMP

5. cAMP acts as a second messenger, activating targets like Protein Kinase A (PKA)

6. PKA activates any protein within the cell that has a binding site for PKA

7. this initiates a response within the cell

what can the activation of cAMP be stimulated by?

• the addition of

• neurotransmitters like serotonin

• chemicals like forskolin

what is an example of cAMP activation in the body?

• the regulation of glucose metabolism in the liver and skeletal muscle

• protein kinase A (PKA) phosphorylates calcium and other proteins to change glycogen → glucose

what are 3 ways to switch off cAMP activation in cell signalling?

1. Gas inactivation

   Gas hydrolyses its GTP → GDP (stops activation of adenylyl cyclase)

2. breakdown of cAMP

   phosphodiesterase degrade cAMP → AMP

   removes second messenger (enzyme)

3. agonist dissociates from receptor

   no ligand = no receptor activation

what is cGMP?

cyclic guanosine monophosphate

what makes cGMP?

• guanylate cyclase (enzyme) regulates and controls the level and activity of cGMP

how does guanylate cyclase make cGMP?

• the guanylate cyclase enzyme converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP)

what are second messenger levels of cAMP and cGMP controlled by?

1. rate of production

2. rate of diffusion

3. rate of removal

rate of production of cAMP and cGMP

• how fast adenylyl cyclase works to make cAMP from ATP

• how fast guanylate cyclase works to make cGMP from GTP

rate of diffusion of cAMP and cGMP

• the rate at which cAMP or cGMP spread away from where they were made

rate of removal of cAMP or cGMP

• the rate of how fast cAMP or cGMP is broken down/cleared from the cell

• removal by phosphodiesterase

• cAMP → AMP (inactive form)

• cGMP → GMP (inactive form)

what is phospholipase Cb (beta)?

• second messenger

• targets hydrophobic lipids in the membrane (specifically lipid called PIP2)

what does phospholipase C do?

• phospholipase C cleaves the inositol head from the PIP2 molecule

• this cleaving produces 2 important 2nd messengers

  1. IP3 (hydrophilic) - water soluble part that diffuses through the cytoplasm

  2. DAG (hydrophobic) - part that remains in the membrane

what regulates phospholipase C?

• lipid kinases

• they add phosphate to make PIP2 (substrate that phospholipase C cuts)

• so lipid kinases control how much PIP2 is available = indirectly regulating phospholipase C activity

  more PIP2 = more fuel for phospholipase C to work on = more active = more PI3 and DAG produced

steps of how GPCRs can activate phospholipase C to generate IP3 and DAG

1. G proteins activate phospholipase C (PLC)

2. PLC cleaves PIP2 into either

   DAG - remains within membrane and activates PKC (protein kinase C)

   IP3 - diffuses through cytoplasm and binds IP3 receptors

what do isoforms of PLC (phospholipase C) have?

• x and y domains which make up the catalytic domain

what do isoforms of PKC (protein kinase C) have?

• regulatory domains (C1 activated by DAG)

• kinase domain (C2 activated by Ca2+)

what is PMA phorbol ester?

• analogue of DAG

• used to activate PKCs

what happens after phospholipase C cleaves PIP2 into DAG?

• DAG binds to protein kinase C (PKC)

• PKC has a pseudosubstrate domain - when DAG binds to PKC it causes the pseudosubstrate domain to dissociate from the active site

• this frees the active site allowing other substrates to bind to PKC and activate it

what happens once PKC is activated?

• it can provide either positive or negative feedback in the signalling pathway

what happens when phospholipase C is phosphorylated?

• it reduces its activity creating a negative feedback which helps turn off or limit GPCR signalling

• this phosphorylation of GPCRs can cause desensitisation meaning the receptor becomes less responsive over time

what is calcium?

• second messenger

• regulates diverse cell functions

what are 5 processes regulated by calcium signalling?

• synaptic transmission

• hormone secretion + synthesis

• fertilisation

• muscle contraction

• cytokinesis

cytoplasmic levels of Ca2+ in resting cells

• kept low

• within cell (100nM)

• outside cell (1-2mM)

• calcium concentration in ER (400um)

what do calcium receptors on extracellular membrane and on ER do?

they regulate activity of Ca2+ channels to produce transcient rises in Ca2+

what are ways that calcium can enter into the cell?

1. calcium enters into cytosol by channels in the extracellular membrane + ligand gated ion channels on ER

2. store operated channels (made of ORAI and gated by STIM) responsible for refilling + maintaining ER calcium

what do mutations in store operated channels lead to?

a loss of function mutation in these channels (like ORAI1 mutation) can lead to severe immunodeficiency

what does fluorescent calcium imaging allow?

allows to track calcium signals in live cells and animals which helps to understand cell signalling, disease and drug development

what can overstimulation of GPCRs cause?

desensitisation and lead to tachyphylaxis and cancer

what are two ways to control/stop overstimulation of GPCRs?

1. GRKs (G protein receptor kinases) - stops pathway and stops G protein from binding

2. B-arrestin - internalises receptor so it is degraded and recycled

if there’s a mutation in the retina that causes the receptor kinase not to work what can this lead to?

• rod apoptosis or prolonged photon responses