Receptor Characterisation

Question 0

Why are there different receptors for the same agonist?

Answer 0

1. Same agonist can elicit multiple effects, so has a number of functions
2. Fine control of a response-depending on concentration of agonist (a low concentration may have a different function from a higher concentration)

Question 1

Why is receptor characterisation important?

Answer 1

Understand receptor function
Understand pathogenic basis of disease
To aid drug discovery

Question 2

Where is 5-HT receptors found?

Answer 2

In the wall of the intestine in enterochromaffin cells

Question 3

Where is 5-HT formed from?

Answer 3

Dietary tryptophan

Question 4

What is 5-HT broke down by?

Answer 4

MAO and excreted in the urine

Question 5

What is the function of 5-HT in the intestine?

Answer 5

Increase gastrointestinal motility (peristalsis)

Question 6

What is the function of 5-HT in the blood?

Answer 6

Vascular construction and dilation.

Platelet aggregation

Question 7

What is the function of 5-HT in the CNS?

Answer 7

Neurotransmission ( appetite, sleep , mood)

Question 8

What are clinical conditions associated with disturbed 5-HT?

Answer 8

Migraine and mood disorders

Question 9

What main two reasons where there to why the international Union of pharmacological sciences was established (IUPHAR)?

Answer 9

1. molecular classifications (receptors in isolation) were incompatible with established pharmacologically defined receptor classes (receptors in tissue)
2. In a number of tissues, agonist effects were not entirely blocked by known antagonists

Question 10

How are receptors characterised?

Answer 10

By ligand binding experiments

Question 11

Briefly explain ligand binding experiments?

Answer 11

1. allow equilibrium to be reached
2. remove tissue by filtration
3. dissolve fluid in scintillation fluid
4. measure radioactive content

Question 12

Radioactivity tells us that our drug binds to receptor in the tissue, but what does it not explain?

Answer 12

Not distinguish agonist from antagonist or receptor function

Question 13

What is saturation binding?

Answer 13

(A single ligand)
Vary the concentration of drug then measure the concentration of drug that binds to 50% of receptors at equilibrium which is the Ka value (equilibrium constant)

Question 14

What does a drug with high affinity have?

Answer 14

A low Ka

Question 15

What is competition binding?

Answer 15

Experiments to measure the binding of a single concentration of labelled ligand in the presence of various concentrations of antagonist.

Question 16

For competition binding experiments is the pki value?

Answer 16

The concentration of the antagonist that inhibits radioligand binding by 50%

Question 17

What two functional response can characterise receptors?

Answer 17

In vitro and in vivo

Question 18

What may identification of the G protein coupled to receptor utilise?

Answer 18

Pertussis toxin- inhibits Gi
Assays for cAMP,cGMP,I3
Forskolin- activated adenylate cyclase

Question 19

What can receptors be classified based on?

Answer 19

Molecular cloning

Question 20

What can molecular cloning identity?

Answer 20

mRNA for a receptor subtype can be identified in a particular tissue

Question 21

How can molecular cloning take place?

Answer 21

Quantitatively- in situ hybridisation

Quantitative- real time - PCR

Question 22

What can explain functional similarities/differences between receptors?

Answer 22

Identification of DNA sequence of a receptor enables protein sequence to be deduced.
Differences in certain domains between receptors affect ligand binding, receptor trafficking (mutagenesis)

Question 23

What does cell-based expression systems allow?

Answer 23

DNA coding for a receptors is transferred into an immortal cell line.
Resulting in re error protein being expressed at high levels so can investigate ligand binding or downstream pathways.
Can study receptors in isolation.
Can mutate the receptor and study functional domains.
Enables high throughput drug screening.

Question 24

How can knockout mice be used?

Answer 24

1. Target a specific gene within a genome and destroys it

2. Targeting vectors are inserted precisely in the position of the desired gene to be targeted in embryonic stem cell

Question 25

What do knock out mice enable?

Answer 25

Physiological role of gene (i.e. Receptor subtype) to be determined.

Question 26

What is a problem with knock out mice?

Answer 26

Natural compensation

Question 27

What is pharmacogenomics?

Answer 27

The science of understanding the correlation between an individual patients genetic make-up (genotype) and their response to drug treatment

Question 28

What makes individual variations in response to receptor-targeted drugs?

Answer 28

Levels of expression
Subtype expression
Splice variants- different mRNA molecule transcribed from one gene. Several forms of receptor subtype with differ properties.

Question 29

What can biochemical assays determine?

Answer 29

Pathways linking receptor activation with cellular response

Question 30

Name one advantage of in vivo?

Answer 30

essential for proof of concept of final drug

Question 31

Name one disadvantage of in vivo?

Answer 31

Complex interaction of responses, variability

Question 32

Name advantage dog in vitro

Answer 32

More powerful statistically , shows effects of downstream signalling.
Differentiates agonist from antagonist

Question 33

What is an advantage of radiolabelled ligand binding?

Answer 33

Most sensitive for showing binding affinities of ligands

Question 34

What is an disadvantage of radiolabelled ligand binding?

Answer 34

Doesn’t distinguish agonist from antagonist

Question 35

What are an advantage if molecular techniques?

Answer 35

Definitive identification of receptor subtype with other tissues

Question 36

What is an disadvantage of molecular techniques?

Answer 36

Receptors may not be functional