Lecture 7 Flashcards
Receptors can be i_________ or e___________
Steroid hromones are l_________
intracellular or extracellular
lipophillic
What is signal transduction?
Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events, most commonly protein phosphorylation catalyzed by protein kinases, which ultimately results in a cellular response
Give examples of drugs that are agonists and work on beta-2 adrenoreceptors
Salbutomol and salmeterol
Give examples of drugs that are agonists on u-opioid receptors
Morphine, heroin, fentanyl
What are G proteins made up of?
They are heterotrimeric. They are made up of 3 subunits:
- alpha (attached to a GDP)
- beta
- gamma (y)
(beta and gamma don’t seperate)
Beta-2 adrenoreceptor structure - show it’s 3 subunits
Recap G protein-coupled recptors structure
- Single polypeptide chain (300-1200 amino acids)
- 7-transmembrane (7TM)- spanning regions
- Extracellular N-terminal
- Intracellular C-terminal
Binding domains: there are 2
- For some receptors the ligand binding site is formed by (2-3 of) the transmembrane (TM) domains
- In other cases the N-terminal region (and other extracellular domains) form the ligand binding site
How GPCR work as a receptor?
- Ligand binds to the binding domain
- Causes a conformational change
- This leads to the activation of G proteins by causing the GDP to exchange for GTP on the G protein alpha subunit
- The alpha-beta-gamma complex immediately dissociates into alpha GTP and free beta-gamma subunits. Each can then interact with effector proteins (second-messenger generating enzymes or ion channels)
Note - this is actually happening much closer to the plasma membrane, this is becuase the alpha-GTP and beta-gamma are always associated with the cell membrane.
Termination of G-protein signalling
The α-GTP and/or βγ interaction with effectors lasts until the α subunit GTPase activity hydrolyses GTP back to GDP. α-GDP and βγ subunits then reform an inactive heterotrimeric complex.
‘I’ for inactive
Summary of the G-protein cycle (activated -> inactivated -> activated… etc)
G protein diversity
The human genome encodes 18 Gα (alpha), 5 Gβ (beta) and 12 Gγ (gamma) proteins Therefore, there are >1000 possible Gα-βγ protein combinations.
Activated GPCRs preferentially interact with specific types of G protein. The Gα subunit is a primary determinant.
In turn, Gα subunits and Gβγ subunits interact with specific effector proteins.
WE NEED TO KNOW ABOUT 3 SPECIFIC TYPES OF G PROTEINS
3 types of G proteins we need to know about?
Gαs – stimulates adenylyl cyclase
Gαi – inhibits adenylyl cyclase
Gαq – stimulates phospholipase C
THIS RELATES TO QISS QIQ (cover later!)
The GPCR receptors in the autonomic nervous system -
Sympathetic and parasympathetic
list receptor, Gα protein type and effector
QISS QIQ
Adrenergic Sympathetic receptors:
α1 Gαq Stimulate Phosholipase C
α2 Gα<span>i </span> Inhibit Adenylyl Cyclase
β1 Gα<span>S</span> Stimulate Adenylyl Cyclase
β2 Gα<span>S</span> Stimulate Adenylyl Cyclase
Cholinergic Parasympathetic receptors:
M1 Gαq Stimulate Phospholipase C
M2 Gα<span>i </span>Inhibit Adenylyl Cyclase
M3 Gαq Stimulate Phospholipase C
Whooping cough - another name for this
What happens?
Pertussis
- interferes and modifies Gαi
- This means that the Gαi can no longer undergo GTP to GDP exchange, therefore the G protein can’t be activated, nothing after this step can then occur
“A component of the toxin (known as pertussis toxin) produced by this bacterium acts to ADP-ribosylate Gαi proteins locking them in their inactive GDP bound form.”
Cholera
Cholera toxin (CTx) prevents termination of signalling by Gs -preferring GPCRs leading to longlasting activation of downstream pathways
● Is does this as the bacterium acts to ADP-ribosylate Gαs proteins locking them in their active form
● Since there is more alpha(S)-GTP this will go to phosphorylate adenylyl cyclase which converts ATP→ cAMP
● The increase in cAMP in enterocytes (small intestine epithelia) causes the stimulation of CFTR transporter which pumps more Cl- ions into the lumen causing the loss of water down the osmotic gradient
Where is 99% of the human bodies 1kg of calcium found?
What is the serum levels of Ca2+ in the body?
How is whole body Ca2+ homeostasis regulated?
- 99%: In bone
- Blood serium: 1.9-2.3mM of which 50% is free Ca2+
- Regulated: by intestinal Ca2+ uptake (from diet), Ca2+ reabsorption in kidneys and bone calcium regulation
- these are under endocrine control.
What kind of processes are changes in IC Ca2+ responsible for?
Muscle contraction, neurotransmission, fertilisation, cell death, learning and memory.
By what mechanisms are Ca2+ gradients set up and maintained?
- Relative permeability of plasma membrane to Ca2+ (Ca2+ doesn’t leak across the membrane)
- Pumps and transporters that move Ca2+ out of the cytoplasm
- Ca2+ buffer proteins.
- -> PMCA (plasma membrane Ca2+-ATPase) and SERCA (endoplasmic reticulum Ca2+-ATPase) move Ca2+ out of the cytoplasm (this is against Ca2+ conc gradient, so uses ATP)
- -> NCX moves Ca2+ out of the cell in exchange for Na+ (this is along Na+ conc gradient)
Which mechanisms increase IC Ca2+?
Two ways to increase intracellular [Ca2+]:
- Ca2+ movement across the plasma membrane (‘influx’)
- Ligand-gated ion channel (LGIC)
- Voltage-operated Ca2+ channels (VOCC) - Ca2+ movement out of the ER/SR (‘release’):
- Inositol 1,4,5-trisphosphate receptors (IP3R)
- Ca2+-induced Ca2+ release (CICR) = ryanodine receptors
In summary - three parts for a GPCR response
GPCR -> G protein -> Effector
Describe the signal pathway that occurs when Gs is activated.
- Gs activated, GDP swapped for GTP
- alpha and beta/gamma subunits dissociate
- Adenylyl cyclase activated, converts ATP into cAMP
- cAMP activates PKA which phosphorylates target proteins within cell.
Give 3 examples of receptors that are coupled to Gs and their associated ligands.
1) β-adrenoreceptors (adrenaline/noradrenaline) *think QISS QIQ*
2) D1-dopamine receptors (dopamine)
3) H2-histamine receptors (histamine)
Describe the signal pathway that occurs when Gi is activated.
- Gi activated, GDP swapped for GTP
- Inhibits the activation of adenylyl cyclase
- Less/no cAMP synthesised
- Less/no activation of PKA, and therefore intracellular effects lacking.
AC in pic is adenyl cyclase
Give 3 examples of receptors coupled to Gi and their associated ligands.
1) a2-adrenoreceptors (adrenaline) *think QISS QIQ*
2) D2-dopamine receptors (dopamine)
3) H1-histamine receptors (histamine)
4) M2/M4 mAChR’s (ACh) *think QISS QIQ*
Describe the structure of PKA and how cAMP levels regulate its activity.
- 2 regulatory subunits (R subunit) attached to 2 catalytic sub-units (S subunit)
PKA - cyclic AMP-dependent protein kinase
- When cAMP is low, regulatory subunits and catalytic subunits have high affinity for each other, stay bound and inhibit activity of catalytic subunits
- When cAMP is high, cAMP binds to R sub-units and catalytic subunits dissociate, leading to phosphorylation of target proteins.
Describe the signal pathway that occurs when Gq is activated.
Phospholipase C (PLC): PIP2 -> IP3 + DAG
- Gq activated, GDP swapped for GTP
- Activated phospholipase C (PLC), causing phosphorylation of PIP2 into DAG and IP3
- IP3 acts on IP3 receptors (IP3R) in ER, activating Ca2+ channel and entry of Ca2+ into cytoplasm
- DAG remains in membrane, binds to PKC which phosphorylate’s key substrate proteins (different to the ones PKA phosphorylates
Give 3 examples of receptors coupled to Gq and their associated ligands
1) a1-adrenoreceptors (adrenaline) *think QISS QIQ*
2) M1/M3/M5 mAChR’s (ACh) *think QISS QIQ*
3) H1 histamine receptors (histamine)
Why is only a few molecules of ligand required to create a massive cellular response in GPCR’s?
SIGNAL AMPLIFICATION IS A KEY FEATURE OF MANY (BASCIALLY ALL) OF CELL SIGNALLING PATHWAYS -> Due to the large amplification effect that occurs
- the step from AC to cAMP is large amplification step, that means we don’t need to waste cellular energy providing large amounts of ligand for receptors.
What is the effect of adrenaline binding to B1-adrenoreceptors in the heart on inotropy?
- Activated Gs, activates adenyl cyclase, changes ATP into cAMP, increase cAMP activates more PKA (cyclic AMP-dependent protein kinase), PKA phosphorylates VOCC.
- When VOCC are phosphoylated, it allows more Ca2+ to enter when the membrane next depolarises, therefore increases Ca2+ entry through cascade, increased contractility, and therefore a positive inotropic effect.
(Positive inotropes strengthen the force of the heartbeat)
What is the effect of noradrenaline binding to a1-adrenoreceptors in smooth muscle
What is the effect of acetylcoholine binding to M3-muscarinic receptors in smooth muscle
- Vasocontriction via the Gq signalling pathway (as this pathway results in a increasing Ca2+ concentration in the cytoplasm, so increasing contractility
• Contraction of blood vessels:
Sympathetically released noradrenaline (and to some extent blood-borne adrenaline) can interact with vascular smooth muscle α1 -adrenoceptors to cause vasoconstriction
• Contaction of airways:
Parasympathetically released acetylcholine can interact with bronchiolar smooth muscle M3 -muscarinic receptors to cause bronchoconstriction
Regulation of neurotransmitter release using μ-opioid receptors
1- reminder of how calcium enters pre-synatpic neurone in an action potential
2- GPCR’S can be expressed closely with VOCC
3- GPCR’s modulating neurotransmitter release
- Depolarization of the nerve terminal activates voltage-operated Ca2+ channels (VOCC) that result in Ca2+ influx. This leads to synaptic vesicle fusion and neurotransmitter release.
- Particular subtypes of GPCR (e.g. µ-opioid receptors) are expressed in close proximity to the VOCCs and can regulate channel activity via a G protein-dependent mechanism
- In both the central (CNS) and peripheral (PNS) nervous systems neurotransmitter release is often modulated by presynaptic G protein-coupled receptors, they are lose to the specific VOCC found in the neurone.
* *Gβγ** subunits inhibit specific types of voltage-operated Ca2+ channels (VOCCs) reducing Ca2+ -influx and neurotransmitter release (next time the VOCC is regulated by a change in membrane voltage, in the presence of βγ subunits, less calcium enters the nerve terminal so less vesicle fusion occurs, so less neurotransmitter is released into the synapse)
Summary of GPCRs:
- Diversity
- Specificity
- Amplification
- DIVERSITY: Multiple subtypes of receptors, G proteins and effectors (this diversity allows different effects)
- SPECIFICITY: Specific ligand-receptor interactions, specific G protein α-subunits (βγ) recruited, which are coupled to specific effector pathways. The same receptor results in the same transducing pathway, so the same physiological outcome is achieved.
- AMPLIFICATION: “Gain” control on all signalling pathways, allowing relatively small changes in extracellular signal to elicit physiologically significant changes in cellular behaviour.
