Intracellular Signalling Pathways

Question 1

What is signal transduction?

Answer 1

The process by which a physical or chemical signal is transmitted through a cell as a series of molecular events which results in a cellular response.

Signal transduction is when, after the initial ligand binding event, receptors require transduction vis other intracellular signalling components to generate a response. Eg contraction, secretion, proliferation, differentiation.

Question 2

What are the two different places a receptor can be located?

Answer 2

A receptor can be intracellular (eg receptors for steroid and thyroid hormones).
Or, receptors are more commonly located on the cell surface because the majority of extracellular signalling molecules do not readily cross the cell membrane.

Question 3

What are the three “super families” of cell urbane receptor?

Answer 3

1. G protein-coupled (7TM) receptors - eg muscarinic acetylecholine receptors.
2. Ligand gated (receptor operated) ion channels - eg nicotine’s acetylcholine receptors.
3. Receptors with intrinsic enzymatic activity - eg receptor tyrosine kinases like the insulin receptor.

Each receptor subtype of specific for one (or a very limited number of) chemical ligands. Ligand binding then activates the receptor which in turn directly or indirectly brings about a change in cellular activity.

Question 4

What percentage of prescription drugs exert their therapeutic effect directly (as agonists or antagonists) or indirectly at GPCRs?

Answer 4

40%.

Although, this number is slowly falling because the pharmaceutical industry is heading in different directions.

Question 5

What is the difference between agonists and antagonists?

Answer 5

Agonists bind the the receptor and activate it (leading to intracellular signal transduction events).

Antagonists bind to the receptor but do not activate it (block the effects of the agonist at the receptor by preventing the ligand from binding)

Question 6

Give some examples of agonists

Answer 6

Salbutamol and salmeterol (anti asthma drugs) are agonists for the beta 2 adrenoreceptor.

Morphine and fentanyl (analgesics/ anaesthetics) agonise the gamma opioid receptor.

Question 7

Give some examples of antagonists.

Answer 7

Propranolol and atenolol (cardiovascular drugs for hypertension) are antagonists for the beta adrenoreceptor.

Haloperidol and sulpiride (neuroleptic, ain’t-schizophrenic drugs) are antagonists for the D2 dopamine receptor.

Question 8

Give some examples of things GCPRs can respond to (5).

Answer 8

A third of GPCRs are sensory and respond to light, odours and taste.

Other GCPRs respond to:
Ions (H+, Ca2+)
Neurotransmitters (acetylcholine, glutamate)
Hormones both peptide and non peptide (glucagon, adrenaline)
Large glycoproteins (thyroid-stimulating protein) - for these ones, the receptor and ligand are the same size.

Question 9

Describe the basic structure of a G protein coupled receptor.

Answer 9

There are over 800 GCPRs identified in the human genome (over 2% of all identified genes) and they all share the same basic structure:

All made of a single polypeptide chain of variable length (between 300 and 1200 amino acids)
They all have 7 transmembrane (7TM) spanning regions
They also all have an extracellular N-terminal (with a methionine) and an Intracellular C-terminal.

Question 10

What are the two different regions on GCPRs that can be responsible for ligand binding?

Answer 10

1. Ligand binding can be formed by (2-3) of the transmembrane (TM) domains. (can form a binding pocket).
   Eg acetylcholine or adrenaline.
2. In other cases, the N-terminal region (and other extracellular domains) form the ligand binding site. (It binds to residues near the N terminal)
   Eg glutamate.

Question 11

How to GCPRs respond to ligand binding?

Answer 11

Ligand binding causes a conformational change of the GCPRs which then activates the intracellular G proteins (Guanine nucleotide binding protein).

Question 12

What is the structure of a G protein?

Answer 12

G proteins are made up of 3 subunits (They are heterotrimeric), alpha, beta and gamma.when activated, the alpha subunit is separated but the beta and gamma remain as a dimer.

Question 13

What happens when a G protein is activated?

Answer 13

It causes the GDP which is attached to the alpha subunit to be exchanged for a GTP. This GTP binding causes the alpha-GTP subunit to dissociate with the free BY (beta-gamma) so that then can both interact with their effector proteins.

Question 14

What two two types of effector proteins could be activated and then what happens ?

Answer 14

Either ions or enzymes are activated and they both generate a second messenger.

Question 15

How do you terminate G protein signalling?

Answer 15

Alpha subunit GTP-ase activity hydrolyses GTP back to GDP (with loss of a phosphate). The a-GDP and BY subunits then reform to form an inactive hetrotrimeric complex.

Question 16

Discuss specificity of G proteins

Answer 16

The human genome encodes for 20 alpha, 5 beta and over 12 gamma G proteins. This means that there are over 1000 possible Ga-By protein combinations.
However, GPCRs preferentially interact with specific types of G protein (the Althea subunit is the primary determinant)
Also, Galpha and GBeta-Gamma subunits interact sugar specific effector proteins.

So, an extracellular signal working via a specific GPCR will activate a single or small sub-population of G proteins and effects in the cell to bring about a specific cellular response. (Which ensures the same thing happens every time)

Question 17

How does Cholera interfere with G protein function?

Answer 17

Prevents GTPase activity so, the G protein can be activated but not switched off (They prevent termination of signalling by Gs). This means that there is more cAMP in cells so more water secretion. This leads to dehydration and diarrhoea.

Question 18

What is pertussis and ho pow does it interfere with G protein function?

Answer 18

Pertussis is the toxin that causes Whooping cough. It prevents GDP to GTP exchange by covalently bonding to the G alpha i subunit. This means that the G protein cannot be activated and effector proteins are not stimulated.

Question 19

Give examples of what enzymes can be effectors and what second messenger molecule they activate. (4)

Answer 19

Adenylyl Cyclase which converts ATP to Cyclic AMP (the second messenger)

Phospholipase C which converts PIP2 to IP3 + DAG

Phosphoinositide 3-Kinase (PI3K) converts PIP2 TO PIP3

cGMP phosphodiesterase which converts cyclic GMP to 5’-GMP

Question 20

Give examples of what ion channels can be effectors

Answer 20

Voltage operated Ca ion channels (VICCs)

G Protein-Regulated inwardly rectifying potassium ion channels (GIRKs)

Question 21

Explain the agonist stimulated regulation of adenylyl cyclase

Answer 21

The agonist binds to the GPCR which causes a conformational change.
This then causes the G-alpha S subunit to undergo GDP to GTP exchange and then dissociate from the beta-gamma dimer
The G-alpha S bind to adenylyl cyclase (the enzyme) which converts ATP to cyclic AMP.
Cyclic AMP activates:
Cyclic AMP dependant kinases (PKA)
EPACs (guanine neucleotide exchange factors)
Cyclic nucleotide-gated exchange factors (CNGs)

Question 22

Explain the antagonist regulation of adenylyl cyclase.

Answer 22

The antagonist binds to the GPCR which causes a conformational change.
This then causes the G-alpha i subunit to undergo GDP to GTP exchange and then dissociate from the beta-gamma dimer
The G-alpha S bind to adenylyl cyclase (the enzyme) which inhibits the conversion of ATP to cyclic AMP so cyclic AMP cannot activate:
Cyclic AMP dependant kinases (PKA)
EPACs (guanine neucleotide exchange factors)
Cyclic nucleotide-gated exchange factors (CNGs)

Question 23

What is the main way in which cAMP works?

Answer 23

Cyclic AMP exerts the majority of its actions through cyclic AMP-dependant protein kinase (PKA is A-kinase).

Question 24

How does cyclic AMP dependant protein kinase work?

Answer 24

PKA is make of two regulatory and two catalytic subunits. CAMP binds to the regulatory subunits. Once enough cAMP is bound the the regulatory subunits release the catalytic ones.
This then creates a catalytic domain that can covalently modify particular protein substrates in the cell.
cAMP phosphorylates serine (and sometimes threonine) residues in proteins.

Question 25

Why is phosphorylation of serine or threonine residues by cAMP important?

Answer 25

Because, phosphorylation alerts the activity of the protein by modifying it (activating it) eg
activate enzyme,
Move protein (from cytoplasm to nucleus as transcription factor).

Question 26

Explain the agonist stimulated regulation of phospholipase C

Answer 26

The agonist binds to GPCR which undergoes a conformational change. This causes GDP to GTP exchange which causes the G-alpha Q GTP to separate from the beta-gamma dimer.
The alpha-Q GTP complex to bind to phospholipase C. This then activates PIP2 causing it to cleave into DAG and IP3 (both second messengers).
IP3 binds to the IP3 receptor in the endoplasmic reticulum which can cause the cytoplasmic calcium concentration to increase 5-10 fold within a few seconds of agonist addition.

Question 27

Other than Gq coupled receptors, what other receptors activate the agonist-stimulated phospholipase C pathway?

Answer 27

A1- adrenoreceptors
M1- muscarinic receptors (acetylcholine)
H1- histamine receptors

Question 28

Explain what parts of the signalling pathway involves lots of signal amplification

Answer 28

The first part of the signalling pathway (the receptor binding to the G protein which then binds to the effector (eg adenylyl cyclase) has very little amplification. But, the activation of the effector generates many molecules of the second messenger which activates many more enzymes.

Question 29

Explain Inotropy of the heart

Answer 29

Intropy is the force by which the heart contracts.
Both blood-borne adrenaline and sympathetically released noradrenaline can interact with ventricular beta1-adrenoreceptors to increase the force of contraction (positive intropy)

This occurs by increasing amount if cAMP (by Gs protein pathway) which increases the activity of cyclic dependant protein kinase. This causes the voltage gates Calcium ion channels to open every time the membrane depolarises increasing the concentration of calcium in the cells. This calcium triggers the contraction of this ventricular cell (using a mechanism called calcium induced calcium released).

If voltage gated calcium channels are phosphorylated, then each time membrane depolarises, allow a bigger influx if calcium into the cell (bigger trigger pulse) so, get greater contractility.

Question 30

Explain how smooth muscle contracts.

Answer 30

Sympathetically released noradrenaline (and to some extent blood borne adrenaline) can interact with vascular smooth muscle alpha1-adrenoreceptors to cause vasoconstriction.

Parasympathetically released acetylcholine can interact with bronchioles smooth muscle M3-muscarinic receptors to cause bronchoconstriction.

A variety of agents acting at G protein-coupled receptors can contract gastrointestinal and Genito-urinary smooth muscle.

They all utilise the Gq - phospholipase C - IP3/Ca, DAG/ protein kinase C pathways.

Question 31

How is vasoconstriction of the arterioles mediated?

Answer 31

Alpha1-adrenoreceptors mediate vasoconstriction of the arterioles through Ca/PKC dependant mechanisms (Same as intropy of the heart)

Question 32

How is neurotransmitter release modulated?

Answer 32

In both the CNS and the PNS neurotransmitter release is often modulated by oresynaptic G protein coupled receptors.

Eg morphine binds to GPCR. The BY subunit (after G protein activation) then binds and inhibits a voltage-operated Calcium channel. This reduces the influx of calcium ions and therefore neurotransmitter release.