Lecture 7 Flashcards by Hannah Strafford

Receptors can be i_________ or e___________

Steroid hromones are l_________

intracellular or extracellular

lipophillic

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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

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Give examples of drugs that are agonists and work on beta-2 adrenoreceptors

Salbutomol and salmeterol

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Give examples of drugs that are agonists on u-opioid receptors

Morphine, heroin, fentanyl

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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)

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Beta-2 adrenoreceptor structure - show it’s 3 subunits

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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

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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.

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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

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Summary of the G-protein cycle (activated -> inactivated -> activated… etc)

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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

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3 types of G proteins we need to know about?

Gα_s – stimulates adenylyl cyclase
Gα_i – inhibits adenylyl cyclase
Gα_q – stimulates phospholipase C

THIS RELATES TO QISS QIQ (cover later!)

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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_{i } Inhibit Adenylyl Cyclase
β1 Gα_{S} Stimulate Adenylyl Cyclase
β2 Gα_{S}Stimulate Adenylyl Cyclase

Cholinergic Parasympathetic receptors:

M1 Gαq Stimulate Phospholipase C
M2 Gα_{i }Inhibit Adenylyl Cyclase
M3 Gαq Stimulate Phospholipase C

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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.”

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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

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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

1. 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. 2. 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 3. 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

1. DIVERSITY: Multiple subtypes of receptors, G proteins and effectors (this diversity allows different effects) 2. 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. 3. AMPLIFICATION: “Gain” control on all signalling pathways, allowing relatively small changes in extracellular signal to elicit physiologically significant changes in cellular behaviour.