Molecular Mechanisms Of Protein-Coupled Receptors

Question 1

What are the four major targets of drugs?

Answer 1

1. Enzymes
2. Transporters
3. Ion Channels
4. Receptors

Question 2

What is a receptor?

Answer 2

Receptors are target molecules through which soluble physiological mediators can produce their desired biological effects.
Example: agonist attaches onto the receptor and this causes a signal transduction reaction that causes arteriolar dilation.

Question 3

What are the four different types of receptors that exist?

Answer 3

1. Ligand Gated ion Channels (Ionotropic Receptors)- e.g. nicotinic acetylcholine receptors
2. G Protein coupled Receptors (metabotropic receptors) e.g. adrenoceptors, opioid receptors (for pain relief)
3. Enzyme Coupled Receptors- e.g. growth factor and insulin receptors
4. Nuclear Receptors- e.g. Oestrogen and bile acid receptors

Question 4

What is a G Protein Coupled Receptor?

Answer 4

1. Seven transmembrane domains- found normally on cell surface (bundled together)- form the pore
2. Couple to G proteins to initiate signal transduction
3. GPCRS currently represent more than half the current drug targets

Question 5

What are Class A: Rhodopsin-like?

Answer 5

1. Seven transmembrane domains- Alfactory
2. Short N-terminus
3. Agonists bind with extracellular hoops and intracellular hoops and transmembrane domains
4. Majority of prescribed GPCR drugs target class A- agonists are small so can target certain receptor molecules
5. Beta-adrenoceptors, histamine receptors, dopamine receptors

Question 6

What are Class B: Secretin-like?

Answer 6

1. Seven transmembrane domain
2. Large, globular N terminus- plays role in agonist binding
3. Currently no small molecule drugs on market to target these receptors
4. Secretin, calcitonin and glucagon (role in metabolism of glucose) receptors

Question 7

What are Class C: Metabotropic glutamate?

Answer 7

1. Seven transmembrane domain
2. Very large N-terminal domain binds agonists and form obligatory dimers
3. Few small molecule drugs on market
4. Metabotropic glutamate receptors, GABAb receptors
5. Ready made dimers

Question 8

How are GPCRS activated? How does the biological signal cross the membrane after bound by a ligand?

Answer 8

1. Agonist attaches to the GPCR which changes the shape of receptor- different shape charge
2. Forms a Heterotrimeric G protein unit formed of alpha, beta and gamma types- can now bind the G proteins
3. The Heterotrimeric G Protein alpha unit binds to the tail that displaces GDP (GTPase activity) which phosphorylates it to GTP
4. The G protein changes it’s location and interacts with the enzyme which activates a cellular response
5. When the G protein is deactivated the G protein takes the steps backwards

Question 9

What are the signalling events triggered by G proteins in the stimulatory G protein?

Answer 9

1. Activated GPCR
2. G subunit protein (stimulatory) promotes adenylate cyclase activitiy
3. Causes ATP to turn into cAMP
   to activate Protein kinase A
4. Example: Beta1- adrenoceptors

Question 10

How are G protein receptors activated?

Answer 10

Activated by diverse signals- photons (eyes), hormone, peptides, peptidases

Question 11

What is the olfactory and non-olfactory in relation to G proteins?

Answer 11

Olfactory- based on taste buds and smell and Non-olfactory

Question 12

What are the signalling events triggered by G proteins in the inhibitory G protein?

Answer 12

1. Activated GPCR
2. G subunit protein (inhibitory) blocks adenylate cyclase activity
3. Causes ATP to not turn into cAMP so deactivates Protein kinase A production
4. Example: 5-HT1 receptors

Question 13

What are the signalling events triggered by G proteins in calcium for this alpha subunit?

Answer 13

1. G alpha 11- promotes the release of intracellular calcium
2. Causes recruitment of a lipase called phospholipase C
3. Little lipids are involved in the membrane called PIP2
4. Substrate for the lipase that breaks down fats
5. Cleaves the lipid which stays in the plasma membrane called DAG- activates protein kinase C
6. And IP3 binds to receptors present on the endoplasmic reticulum which causes influx of Ca2+ into the cell
7. Transporters bring this back into the GPCR

Question 14

How does salbutamol work in terms of G proteins?

Answer 14

1. Works on the Beta 2 adrenoreceptors
2. That activate the G alpha s subunits
3. This increases cAMP and thus increases PKA

Question 15

How does Sumatriptan work in terms of G proteins?

Answer 15

1. Works on 5-HT 1b/d receptors
2. That activate the G alpha i/o subunits
3. This decreases cAMP and thus decreases PKA

Question 16

How does pilocarpine work in terms of G proteins?

Answer 16

1. Works on the M3 muscarinic ACh receptors
2. That activate the G alpha q subunits
3. This increases PKC and Ca2+

Question 17

What are the signalling events triggered by G proteins for this Beta and gamma subunit?

Answer 17

1. Have a 1 to 2 loop on the voltage gated calcium channels
2. Leads to a loss of function and inhibits the release of neurotransmitter release
3. Less calcium coming in
4. Example: GABAb receptors modulate the voltage gated calcium channels 2.2

Question 18

What is the desensitisation of GPCRs?

Answer 18

1. How to switch off the GPCR signal
2. Add an agonist that activates the receptors and you wash it away. Add the same agonist- that has a smaller or no effect. Won’t activate again.
3. Activates very quickly within seconds and occurs via phosphorylisation (done by kinases) or internally
4. Helps control uncontrolled signalling
   Two main types:
   -Homologous desensitisation- activate receptor A, all of A is desensitised
   - Heterologous desensitisation- activate receptor A, all of B is desensitised.
5. Down regulation: lysosomes of the cell gets rid of receptors that aren’t needed

Question 19

What are the desensitisation events triggered by G proteins?

Answer 19

1. Agonist attaches onto the G proteins which causes a conformational change
2. G protein receptor kinases get phosphorylated- changes their affinity- move to interact with the receptor at the cell surface
3. Beta- arrestins stop further signal transduction, facilitate internalisation
4. Attracted and bind to the Phosphorylated form of the GPCR
5. Scaffolding protein form cages and bind to the beta arrestins and intercellular vesicles we call endosomes
6. They are released and are dephosphorylated at phosphates
7. Can be recycled for resensitisation
8. or trafficked to the lysosome for degradation

Question 20

What are the mechanisms for tolerance at GPCRs?

Answer 20

1. Tolerance is the progressive reduction in the effectiveness of a drug, usually occurs over days or hours- slow
2. The cellular and molecular mechanisms are not directly specified
3. The underlying cellular and molecular mechanisms are often difficult to resolve
4. Tolerance is a problem in health care industry like morphine
5. Specifically targets and activates mew-opioid receptors

Question 21

How is tolerance developed in mew-opioid receptors for Enkephalin and Morphine?

Answer 21

1. Enkephalin bind to the GPCRs, promote phosphorylation of the kinases, Beta arrestin recruitment, internalisation, resensitisation and recycling. Don’t develop tolerance.
2. Morphine is only a partial agonist activate the Protein Kinase C Phosphorylation pathway. Remains desensitised for longer periods of time, can bind the morphine but cannot activate the signalling pathway to give you pain relief.

Both activate the same receptor but different signalling activities.