M&R session 7: signal transduction receptors and effectors Flashcards
What is signal transduction?
The transmission of molecular signals from a cell’s exterior to its interior, responding to external ones t alter its activity in some way.
All cells need receptors to respond to signalling molecules: intracellular for steroids and thyroid hormones, cell-surface for the majority of molecules:
-some receptors can directly alter cellular activity
-many need transduction of initial ligand-binding event by other intracellular signalling components to generate a response
What are the three cell-surface receptor superfamilies?
- LIGAND GATED ION CHANNELS: binding of the ligand ‘gates’ the channel to allow ions to move into or out of the cell. E.g. nAChR
- RECEPTOR TYROSINE KINASES: receptors with intrinsic enzyme activity. Ligand binds, activates enzyme activity that phosphorylates the receptor itself and other substrates. E.g. insulin receptor (though this is an atypical example)
- GPCRs: 7 transmembrane domain receptors. E.g. mAChR. 40% of current prescription drugs exert their effects at GPCRs
Describe some therapeutics which target GPCRs
Agonists: bind to receptor and mimic natural activity of hormone/neurotransmitter to activate receptor, causing intracellular signal transduction. E.g. B2 adrencoceptor agonists such as salbutamol and salmeterol; mu-opioid receptor agonists such as morphine and fentanyl
Antagonists: bind to receptor but have no efficacy so block the effects of agonists. E.g. beta adrenoceptor antagonists such as propranolol and atenolol, D2 dopamine receptor antagonists such as sulpiride and haloperidol
Give some diseases which are caused by defective GPCRs
Retinitis pigmentosa: loss of function to rhodopsin
Nephrogenic diabetes insipidus: loss of function to V2 vasopressin receptor
Familial male precocious puberty: gain of function to LH receptor
Name some stimuli of GPCRs
Light, odours, tastes Ions: H+, Ca2+ Neurotransmitters: ACh, glutamate Hormones: glucagon, adrenaline, TSH Large glycoproteins
There are >800 types of GPCR in the human genome. Describe the common basic structure
Single polypeptide chain of 300-1200 amino acids
7 transmembrane-spanning regions
Intracellular C-terminal and extracellular N-terminal
Where do ligands bind?
To 2-3 of the transmembrane domains, or the N-terminal region/other extracellular domains
Binding site has high affinity and specificity
Describe the effect of GPCR activation
- Must interact with a G-protein
- GPCR-G protein interaction activates the G protein by causing GTP to exchange for GDP on the alpha subunit
- alpha-GTP and betagamma subunits dissociate
- each subunit can interact with effector proteins (second messenger-generating enzymes or ion channels)
How is G protein signalling terminated?
alpha-GTP or betagamma interaction with receptors lasts until the alpha subunit GTPase activity hydrolyses GTP back to GDP. The subunits then reform an inactive heterotrimeric complex
Describe the structure of a G protein
3 distinct subunits by structure, but functionally is a dimer (alpha and beta-gamma subunits).
Alpha subunit has guanine nucleotide binding site to bind GTP, and intrinsic GTPase activity
What causes the G protein to be switched “on” and “off”?
On switch: receptor-facilitated GDP/GTP exchange
Timer/off switch: length of the time taken for GTP hydrolysis on Galpha subunit. Regulated by other cellular proteins
State the receptors adrenaline/noradrenaline act on, the G protein and the effector that are activated
Beta-adrenoceptor-Gs alpha-adenylyl cyclase (stimulates)
Alpha 1-adrenoceptor-Gq alpha-phospholipase C (stimulatory)
Alpha 2-adrenoceptor-Gi alpha-adenylyl cyclase (inhibitory)
At which receptor does light act, and which G protein and effector is activated?
Rhodopsin
Gt (transducin)
Cyclic GMP phosphodiesterase (stimulatory)
At which receptors can acetylcholine act, and which G protein and effector will this activate?
M2/M4 muscarinic: Gi. Adenylyl cyclase (inhibitory)
M1/M3 muscarinic: Gq. Phospholipase C (stimulatory)
How were cholera toxin (CTx) and pertussis toxin (PTx) used to study GPCR-G protein signalling?
Both have similar mechanisms of action: the toxin complex binds to the cell and an enzyme is injected into the cell, causing its harmful effects.
The toxin enzymes are enzymes that make ADP-ribosylate G proteins:
-CTx eliminates GTPase activaste of Gs alpha, so it is irreversibly activated
-PTx interest with GDP/GTP exchange on Gi alpha, so it is irreversibly inactivated
-ADP-ribosylation of Gs alpha by CTx prevents deactivation of Gs protein-mediated signalling, so signalling is not switched off and alpha subunit loses ability to hydrolyse the GTP, increases cAMP, water channels open, symptoms!
-similar mechanism by PTx
What can effectors be?
Enzymes:
- phospholipase C: catalyses PIP2–>IP3 + DAG
- adenylyl cyclase: catalyses ATp–>cAMP
- PI3K: catalyses PIP2–>PIP3
- cGMP phosphodiesterase: catalyses cGMP–>5’-GMP
Ion channels:
- voltage operated calcium channels
- G-protein regulated inwardly-rectifying K+ channels (GIRK)
Describe the action of the second messenger adenylyl cyclase
Mechanism: hydrolyses cellular ATP to generate cAMP, which then activates PKA so that it can phosphorylate other proteins to either stimulate or inhibit their activity.
Receptors which activate adenylyl cyclase can cause:
- increased glycogenolysis & gluconeogenesis in liver
- increased lipolysis in adipose
- relaxation of smooth muscle
- positive inotropic and chronotropic effects in heart
Increased cAMP:
- causes dissociation between regulative and catalytic subunits on PKA
- regulatory subunits inactivate catalytic, so when released the catalytic are free to phosphorylate their substrates on a serine or threonine residue
Describe agonist-stimulated regulation of adenylyl cyclase
Gs-coupled receptors: beta-adrenoceptros, D1-dopamine receptors, H2-histamine receptors
Gi-coupled receptors: alpha 2-adrenoceptors, D2-dopamine receptors, mu-opioid receptors
Describe the activity of phospholipase C
Catalyses the cleavage of PIP2 (membrane phospholipid) into 2 second messengers:
- IP3: interacts with specific intracellular receptors on the ER to allow Ca2+ to leave and enter cytoplasm, which then activates Ca2+-sensitive protein kinases
- DAG: also interacts with a family of protein kinases (protein kinase C)
Which G protein and receptors regulate phospholipase C?
Gq-coupled receptors
E.g. alpha 1-adrenoceptors, M1 muscarinic receptors, H1 histamine receptors, 5-HT2 receptors
What cellular processes does phospholipase C regulate?
Vascular, GI and airway smooth muscle contraction
Mast cell degranulation
Platelet aggregation
Describe cyclic GMP phosphodiesterase activity
In the rod and cone cells of the retina
Breakdown of cyclic GMP is regulated by this enzyme by Gt (transducin) following excitation of rhodopsin by a photon of light.
In the dark, [cGMP] sufficient to open the second messenger-activated ion channel allowing calcium and sodium ions to enter cytoplasm. In the light, activation of the enzyme causes decreased cGMP, so channel closure and hyper polarisation result–>alters signal output to the CNS
What are the different levels of signal amplification?
- Activated receptor can cause GTP/GDP exchange on >1 G protein
- Activated G alpha-GTP / free G beta gamma can activate multiple effector molecules
- Effector molecules act catalytically; e.g. opening of an ion channel by alpha-GTP allows 100-1000s of ions to move across the plasma membrane
Amplification of adrenaline signal
A binds to beta-adrenoceptors
Activates Gs protein, so adenylyl cyclase causes a small amount of amplification
Adenylyl cyclase also generates many molecules of cAMP–>activates PKA
How is deactivation of signalling pathways facilitated?
- Once receptor and G protein interact, dissociation between them is more likely
- Protein kinase phosphorylates receptor and prevents it activating more G proteins
- Lifetime of alpha-GTP limited by favourable GTPase activity
- Enzymes favour basal state
- Opposition of cascades by protein phosphatase activity
Describe regulation of chronotropy in the heart
SA node firing rate is affected by ACh from parasympathetic nerves. Mainly acts on M2 musarinic ACh receptors.
Activation increases open K+ channels (direct regulation by Gi subunit), and also inhibits adenylyl cyclase. More K+=hyperpolarisation, so firing slows and prevents intrinsic firing rate, so NEGATIVE CHRONOTROPY
Describe regulation of inotropy in the heart
Blood-borne adrenaline and sympathetically-released noradrenaline interact with ventricular beta-1 receptors to INCREASE force of contraction, by making Ca2+ more available for contraction by:
- Beta 1 adrenoceptors activate adenylyl cyclase via Gs, cAMP activates PKA, this phosphorylates and opens the VOCCs
- Alpha s-GTP can also directly interact with VOCCs
Describe GPCR-mediated regulation of smooth muscle contraction
All utilise the Gq-phopsholipase C pathways
Sympathetic noradrenaline and some blood-borne adrenaline interact with vascular smooth muscle alpha 1 adrenoceptors to cause vasoconstriction
Parasympathetic acetylcholine can interact with bronchiolar smooth muscle M3-muscarinic receptors to cause bronchoconstriction
How can GPCRs modulate neurotransmitter release?
Pre-synaptic mu opioid receptors can be stimulated by endogenous opioids or analgesics, e.g. morphine to couple to Gi proteins. Liberated G beta gamma subunits can interact with VOCCs to reduce Ca2+ entry and therefore INHIBIT neurotransmitter release from the pre-synaptic terminal
