Block A Lecture 3 - Modern Approaches to Enhance Our Understanding of Receptors

Question 1

What are multiple approaches we used to try to understand GPCRs (and other receptors) better?

Answer 1

Efforts to look at them directly - e.g radioligand binding

Look at GPCRs immediate coupling to second messengers such as cAMP or IP3 / Ca2+ ions

Look at labelling ligand receptors in more sophisticated ways

Structure function studies using X-ray crystallography
(Slide 5)

Question 2

What is a radioligand?

Answer 2

A radioactively labelled drug that can associate with a receptor, transporter, enzyme or any other site of interest
(Slide 7)

Question 3

What are some examples of radioligand drugs?

Answer 3

Examples include:
[3H]-Histamine
[3H]-Mepyramine
[3H]-Adrenaline
[3H]-Salbutamol
(Slide 7)

Question 4

What does the [3H]- designation mean?

Answer 4

It indicates that a molecule has been labelled with tritium / hydrogen-3 (T/3H)
(Slide 7)

Question 5

What is tritium / hydrogen-3 (T/3H)?

Answer 5

It is a radioactive isotope of hydrogen
(Slide 7)

Question 6

What is a radiolabelled ligand added to?

Answer 6

A tissue homogenate containing the target receptor, enzyme etc
(Slide 7)

Question 7

What is a tissue homogenate?

Answer 7

A preparation made by physically breaking down tissue samples into a uniform suspension of cells and cellular components
(Slide 7)

Question 8

What is radioligand binding used for?

Answer 8

It is used to study the interactions between a ligand and its receptor
(Slide 7 and 8)

Question 9

How do you find out the rate and extent of a agonist / antagonists binding using radioligand binding?

Answer 9

As higher and higher concentrations of an agonist / antagonist, more and more of the radioligand bound to the receptor becomes dislodged, so by measuring the amount of radioligand still bound to the receptor, you can work out the rate and extent of the agonists / antagonists binding
(Slide 8)

Question 10

What 3 things does the rate and extent of a agonist or antagonists binding found from radioligand binding tell you?

Answer 10

The number of binding sites (receptors), their affinity, and where the receptors are
(Slide 9)

Question 11

What insights did radioligand binding give scientists when it was first discovered in the late 60s / early 70s?

Answer 11

Inisights into the molecular identity and binding properties of GPCRs with their ligands
(Slide 10)

Question 12

What are 3 things that radioligand binding doesn’t tell you?

Answer 12

It doesn’t tell you what is happening early on in the pathway

It’s hard to compare binding

It’s hard to correlate binding with function (can’t really tell function from this alone)

It requires parallel functional studies (due to the above point)
(Slide 10)

Question 13

What 4 things did measuring cell signalling pathways (measuring 2nd messengers) help scientists do?

Answer 13

Test agonists and antagonists

Link the receptor to functional responses

Identify G-protein and Beta Arrestin pathways

Identify biased agonists and antagonists

(Slide 14)

Question 14

What is probe dependence?

Answer 14

How the measurement of receptor signalling or function can depend on the specific experimental probe (e.g., a particular ligand, agonist, or antagonist) being used
(Slide 15)

Question 15

What is an example of a biased ligand and what is it biased for?

Answer 15

TRV120027 (TRV 027) is a synthetic analogue of endogenous angiotensin II peptide which binds to the AngII receptor (AT1).
It is biased towards the ß-Arrestin pathway
(Slide 16)

Question 16

Who cloned the first receptor, what receptor was it, when did they do it and where did they clone it from?

Answer 16

In 1986, Kobilka and Lefkowitz cloned the ß2-adrenoceptor from a
genomic library
(Slide 18)

Question 17

What did the breakthrough of receptor cloning mean for science?

Answer 17

That receptors could be expressed in cells - allowing us to see which pathways its linked to, test agonists and antagonists and make mutations in the receptor at different points - to see what happens

It also means we have more direct biochemical and biophysical measures as we can tag the receptor.

We can also Immunoprecipitate the receptor to see what it is bound to, and this can be used to make a hydrophobicity plot.
(Slide 18)

Question 18

What is a hydrophobicity plot?

Answer 18

Also known as hydropathy plots, they are graphical representations used to predict regions of proteins that are hydrophobic or hydrophilic
(Slide 18)

Question 19

What are 4 examples of things which mutagenesis techniques helped scientists understand?

Answer 19

Ligand binding sites
G protein coupling regions
Arrestin recruitment
Cell signalling
(Slide 19)

Question 20

How can fluorescence and bioluminescence approaches be used to measure receptor mediated events?

Answer 20

Fluorescence approach: FRET - This technique measures the interaction between two fluorescently labeled molecules (often proteins). When a donor fluorophore is excited, energy can transfer to an acceptor fluorophore if they are in close proximity.

FRET can be used to measure receptor dimerization or conformational changes upon ligand binding. E.g you could tag a receptor with one fluorophore and its interacting partner (such as a G protein or arrestin) with another. A change in FRET efficiency upon ligand binding indicates that the receptor and its partner are coming together.
(Slide 22)

Question 21

How can TRUPATH biosensors be used to measure heterotrimeric G protein dissociation?

Answer 21

Uses bioluminescence resonance energy transfer (BRET)

1. Once the drug binds the alpha and beta subunits dissociate, which results in a decreased BRET signal.
2. With increasing concentrations of the drug, the BRET2 signal decreases

Changes in signal can be plotted on a concentration response curve graph (signal on y axis, -log of drug concentration on x axis) and then analysed
(Slide 22)

Question 22

What are 4 things a TRUPATH assay can tell us?

Answer 22

It can distinguish between agonist properties

It can reveal differences in the efficacy of between drugs

Can show antagonism

Can show inverse agonism (the BRET signal should increase rather than decrease)

(Slide 23)

Question 23

What kind of crystallography was used to determine structures originally?

Answer 23

X-ray crystallography
(Slide 25)

Question 24

What was the first high resolution structure created using X-ray crystallography?

Answer 24

A structure of rhodopsin
(Slide 25)

Question 25

What states were the first structures of GPCR receptors made in?

Answer 25

Antagonist-bound inactive states or agonist-bound intermediate states
(Slide 26)

Question 26

What do inactive and active GPCR crystal structures reveal?

Answer 26

Common conformational changes which occur upon receptor activation
(Slide 28)

Question 27

What is the largest conformational change which happens when a GPCR is activated?

Answer 27

The TM6 (trans-membrane 6) domain moving inwards on the extracellular side. This also creates a reciprocal intracellular large outwards movement
(Slide 28)

Question 28

What is a bitopic ligand?

Answer 28

A ligand which is able to interact with both an orthosteric site and an allosteric site
(Slide 33)

Question 29

What 3 GPCRs have reported bitopic ligands?

Answer 29

Muscarinic, adenosine and dopamine receptors
(Slide 33)

Question 30

What is McN-A-343?

Answer 30

A bitopic muscarinic mAChR partial agonist which has functional selectivity as it engages with both an orthosteric and allosteric site at the M2 mAChR (muscarinic acetylcholine receptor).
It also displays mixed modes of orthosteric or allosteric pharmacology depending on the experimental assay conditions
(Slide 33)

Question 31

What is structure based drug design?

Answer 31

Using the three-dimensional (3D) structure of a biological target, typically a protein or receptor
(Slide 35)