Signal Transduction Flashcards
Describe the structure of GPCRs
Single polypeptide chain
7-transmembrane domains
Extracellular N-terminal, intracellular C-terminal
What are the 3 superfamilies of receptors?
Ligand gated ion channels
Receptors with intrinsic enzyme activity
G-protein coupled receptors (GPCRs)
Where on GPCRs can ligands bind?
To the N-terminal region (eg peptides, glutamate)
To the 3rd cytoplasmic loop- between the 2nd and 3rd transmembrane domains (eg ACh, adrenaline)
Give an example of a Beta adrenoceptor agonist and its use
Salbutamol, anti-asthmatic
Give an example of a u-opioid receptor agonist and its use
Morphine, anaesthesia
Give an example of a beta adrenoceptor antagonist and its use
Propranolol/atenolol, hypertensive drugs
Give an example of a D2 dopamine receptor antagonist and its use
Haloperidol, anti-schizophrenic
Give some examples of mutations to GPCRs
Loss of function mutation to rhodopsin causes Retinitis Pigmentosa.
Loss of function mutation to V2 vasopressin receptor causes Nephrogenic Diabetes Incipidus
Gain of function mutation to LH receptor causes Familial Male Precocious Puberty
What stimuli can GPCRs respond to?
Sensory GPCRs sense light, odours, taste
Other GPCRs sense to changes in extracellular ions, neurotransmitters, hormones, large glycoproteins
How do GPCRs change cellular activity?
Agonist binding results in a conformational change, which allows interaction with a Guanine-nucleotide Binding Protein, a G-protein.
Describe the structure of a G-protein
They are heterotrimeric, with alpha, beta and gamma subunits.
GDP is bound to the alpha subunit
Describe the events that follow GPCR G-protein interaction
This activates the G-protein.
The GDP on that alpha subunit is exchanged for GTP by Guanine Nucleotide Exchange Factors (GEF).
The complex dissociates into alpha-GTP and free beta-gamma subunits which interact with effector proteins
How is the signal terminated?
Interaction with effector proteins lasts until the alpha subunit GTPase activity hydrolyses the GTP to GDP.
The alpha-GDP and beta-gamma subunits reform a heterotrimeric complex which is inactive.
How can magnitude of the signal be regulated?
By regulation of the GTPase activity by GTPase Activating Protein (GAP).
This alters the time that the subunits interact with effector proteins before reforming the heterotrimeric complex.
How can G-protein diversity lead to a specific cellular response?
Activated GPCRs preferentially act with specific types of G protein (mainly determined by alpha subunit type) and the G-protein subunits interact with specific effector proteins.
Extracellular signals work via a specific GPCR, activating a single/sub population of G-proteins and effectors, bringing about a specific response.
What are the different types of G-protein and what do they stimulate/inhibit?
- Gs, stimulates adenylyl cyclase to produce cAMP
- Gi, inhibits adenylyl cyclase to reduce cAMP levels
- Gq, stimulates phospholipase C, increased PIP2
- Gt, stimulates cyclic GMP phosphodiesterase
Which G-protein is activated by adrenaline/noradrenaline on beta-adrenoceptors and what physiological response is brought about?
Gs proteins, stimulates adenylyl cyclase, increases cAMP, leading to increased glycogenolysis/lipolysis
Which G-protein is activated by adrenaline/noradrenaline on alpha2-adrenoceptors and what physiological response is brought about?
Gi proteins, inhibit adenylyl cyclase, reduce cAMP levels, reduced glycogenolysis/lypolysis
Which G-protein is activated by adrenaline/noradrenaline on alpha1-adrenoceptors and what physiological response is brought about?
Gq proteins, stimulates phospholipase C, smooth muscle contraction
Which G-protein is activated by light on rhodopsin receptors and what physiological response is brought about?
Gt proteins, stimulate cyclic GMP phosphodiesterase, leading to visual excitation
Which G-protein is activated by ACh on M2/M4 muscarinic receptors and what physiological response is brought about?
Gi, inhibits adenylyl cyclase but stimulates K channels, leading to slowing of cardiac pacemaker
Which G-protein is activated by ACh on M1/M3 muscarinic receptors and what physiological response is brought about?
Gq, stimulates phospholipase C, leading to smooth muscle contraction
Explain the use of cholera and pertussis toxins in experimental manipulation of the G-protein cycle
The toxins are enzymes that ADP-ribosylate specific G-proteins
Cholera toxin eliminates the GTPase activity of the G-alpha subunit, making it irreversibly activated.
Pertussis interferes with the GDP/GTP exchange on the G-alpha subunit, making it irreversibly inactivated.
How does Adenylyl Cyclase activation bring about a cellular response?
Adenylyl cyclase hydrolyses ATP to give cyclic AMP, this activates cAMP-dependent protein kinase (PKA) which phosphorylates other proteins within the cell to affect activity.
What physiological responses can activation of adenylyl cyclase bring about in the cell?
Increased glycogenolysis and gluconeogenesis in liver cells.
Increased lypolysis in adipocytes.
Relaxation of various smooth muscle.
Positive inotropic and chronotropic effects in the heart.
Describe the activation of PKA
cAMP binds to the 2 R (regulatory) subunits, causing them to dissociate from the 2 C (catalytic) subunits.
The protein kinase active site on the C subunits is now available to phosphorylate proteins (specific target proteins with serine and threonine amino acids)
How does Phospholipase C activation bring about a cellular response?
Phospholipase C hydrolyses the membrane phospholipid (PIP2) to IP3 and DAG (diacylglycerol).
IP3 binds to IP3 receptors on the ER/SR to allow Ca to leave the ER lumen and enter the cytoplasm.
DAG activates protein kinase C (PKC) which phosphorylates target proteins in the cell.
How does activation of cyclic-GMP Phosphodiesterase bring about a cellular response?
It hydrolyses cyclic-GMP to 5’-GMP in the photoreceptive cells of the retina.
How may GPCR pathways be deactivated?
- Once the receptor has interacted with the G-protein, agonist binding is weakened and dissociation is likely.
- Protein kinases phosphorylate the activated receptor, preventing activation of further G-proteins.
- The lifetime of a alpha-GTP is limited by cellular factors that stimulate GTPase activity.
- High activity enzymes can metabolise 2nd messengers, returning their levels to a preferred basal state.
- Enzymatic cascades can be activated downstream of the 2nd messengers to oppose their effects.
Describe the signalling pathways used in regulation of inotropy in the heart.
Sympathetic innervation can influence force of contraction.
Adrenaline/NA bind to ventricular B1-adrenoceptors.
These are Gs coupled, so activate adenylyl cyclase leading to increased cAMP and PKA.
PKA phosphorylates voltage operated Ca channels, meaning more Ca enters per depolarisation, leading to increased CICR via ryanodine receptors.
=>increased contractility of the ventricles = POSITIVE INOTROPY
Describe the signalling pathways used in regulation of chronotropy in the heart
Parasympathetic innervation can effect the rate at which APs are fired.
ACh binds to M2 cholinoceptors.
These are Gi coupled, inhibiting adenylyl cyclase, cAMP and PKA, leading to an increased open probability of K channels.
Increased plasma membrane permeability to K causes hyperpolarisation, slowing the intrinsic firing rate = NEGATIVE CHRONOTROPY
Describe the signalling pathways used Arteriolar Vasoconstriction
Sympathetically released NA binds to vascular a1-adrenoceptors.
These are Gq coupled so stimulate phospholipase C and IP3 production. IP3 causes Ca release from the SR in the SMCs, causing increased contractility, leading to VASOCONSTRICTION
Describe the signalling pathways used in bronchconstriction
Parasympathetically released ACh binds to bronchiolar M3 muscarinic receptors.
These are Gq coupled so stimulate phospholipase C and IP3 production. IP3 causes Ca release from the SR in the SMCs causing increased contractility leading to BRONCHICONSTRICTION.
Describe the signalling pathways used to modulate neurotransmitter release
Presynaptic GPCRs can influence neurotransmitter release.
Endogenous opioids or Morphine stimulates presynaptic u-opioid receptors which are Gi coupled.
The beta-gamma subunit inhibits specific voltage operated calcium channels, reduces Ca entry upon depolarisation, therefore decreasing neurotransmitter release.
