Pharmacology- Receptor classification

Question 1

Learning outcomes

Answer 1

• Using the example of the adrenoceptor family to illustrate general principles of receptor classification, provide examples of agonists and antagonists which display selectivity for each of the adrenoceptor subtypes
• Appreciate the need for an activated receptor protein to be linked to a signal transduction process to enable a cellular response to occur.
• Distinguish between the different structures and signalling mechanisms associated with each of the four receptor superfamilies, giving relevant examples and clinical applications of drugs targeting each superfamily.

Question 2

What is the conventional pharmacological approach to classifying receptors?

Answer 2

Division into receptor families based on
-Which mediator(s) receptor responds to

Division into family members (receptor subtypes) based on
•ranking of potency of endogenous mediator(s) at receptors for that mediator(s) in different tissues
•selective stimulation by agonist drugs
•selective blockade by competitive antagonist drugs

eg adrenoreceptors- family that respond to noradrenaline and adrenaline, contains several family members

a- adrenoreceptors, in blood vessels, have higher affinity for noradrenaline
b-adrenoreceptors in heart, higher affinity for adrenaline- e.g b2 selective agonist salbutamol and b1 antagoinst atenlol

Question 3

Selective agonist- e.g salbutamol

Answer 3

Drugs such as salbutamol (antagonist) can identify subgroups of b- adrenoreceptors
High occupancy in B2 receptors in airway and low in heart- too much salbutamol may stimulate some b1 receptors in the heart - tachycardia

Question 4

Selective agonist- e.g atenolol

Answer 4

Drugs such as atenlol (COMPETITVE REVERSIBLE antagonist) can identify subgroups of B- adrenoreceptors
atenolol (in B1) is a more potent antagonist in the heart than in the airways (b2)
SELECTIVE ANTAGONIST

Question 5

What is the Molecular Biological approach to receptor classification?

Answer 5

Molecular biological approach (more common today)- based on

• Molecular structure of receptor protein
• amino acid sequence
• 2ry and 3ry structure (a-helices etc.)
• location of (ant) agonist binding site(s)
• Signal transduction mechanism
• ion flux
• enzyme activity
• gene expression

Question 6

What are the 4 superfamilies?

Answer 6

1. Ligand- gated ion channels (ionotropic receptors)- time scale= milliseconds, nicotinic/ ACh receptor
2. G- protein coupled receptor (metabotropic)- seconds, muscarinic/ ACh receptor
3. Kinase linked receptors- Hours, cytokine receptor
4. Nuclear receptors- hours, oestrogen receptor

1-3 are cell surface receptors- for large/ water soluble mediators that cannot enter cell
4- small lipophilic mediators, intracellular receptors in cytoplasm/ nucleus- often associated w slow responses

See slide 9

Question 7

What similarities and differences exist between receptors in the same superfamily?

Answer 7

•possess basic molecular structure and functional properties of that superfamily

• Howevever they are encoded by different genes and differ in region of receptor that forms agonist binding site
• each family responds to a specific agonist mediator (hormone or neurotransmitter)

Question 8

What similarities and differences exist between receptors in the same class?

Answer 8

•possess basic molecular structure and functional properties of that (super) family

• but encoded by different genes and differ (subtly) in region of receptor that forms binding site
• individual family members (subtypes) differ in affinity for chemical mediator(s) associated with the family and in preferences for agonist and antagonist drugs

Question 9

Receptor classification

Answer 9

integration of molecular and pharmacological data
•sequencing of human genome reveals a finite number of genes encoding receptor proteins

• each protein can be assigned to one of four superfamilies (and to a family within a superfamily)
• distinct tissue distribution profile for each receptor (and distinct physiological affects associated with activation of that receptor)
• identification of new receptor proteins (family members) for existing chemical mediators –revision of pharmacological classification of adrenoceptors a1A ,a1B, a1D, a2A, a2B, a2C, b1, b2, b3

i.e. at least 3 proteins with conventional pharmacological characteristics of a1 adrenoceptors i.e. activated by phenylephrine and antagonised by prazosin

development of more selective agonists, antagonists e.g.tamsulosin novel selective antagonist of a1A receptors- example of an alpha blocker, antagonist of noradrenaline: relax smooth muscle in bladder neck and prostate, increased urine flow
•identification of receptor proteins (‘orphan’receptors) for which mediator currently unknown–novel therapeutic targets

Question 10

Superfamily 1- example of nicotinc acetylcholine receptor/ ion channel

Answer 10

•family of receptors for neurotransmitter acetylcholine also stimulated selectively by agonist nicotine

•competitive reversible antagonists (tubocurarine, vecuronium) -neuromuscular blocking agents prevent skeletal muscle contraction during surgery
Diagrams in lecture 51
•competitive irreversible antagonist abungarotoxin(from Bungarusmulticinctus),causes muscle paralysis, respiratory failure and death

Non-competitive antagonism -Ion channel blockers lidocaine and tetrodotoxin antagonise the action of acetylcholine at some point in the chain leading to response

Question 11

Subtypes (family members) of the nicotinic cholinoceptor

Answer 11

•several types of some (a1-10, b1-4)subunits occur–encoded by different genes
•various homo and heteropentameters formed
–NMJ receptors comprise 2a1, 1b1, 1d+ 1 other subunit (gor e)
–a4b2hetero-, a7homo-and a9a10heteropentamers common in CNS?
•subunit composition and ratio determine physiological and pharmacological properties of receptor
–influence affinity for e.g. acetylcholine, nicotine and cation selectivity
-tubocurarine (antagonist)-neuromuscular blocker discriminates NMJ receptors (a1,b1) from CNS/ganglia?
17 different building blocks

•novel therapeutic applications–partial agonists selective for a4b2 heteropentamers aids smoking cessation e.g. varenicline–↓ acute nicotine craving when quitting but also ↓ chronic nicotinic reinforcement (pleasure, reward)

Question 12

Example 2 for superfamily 1- GABA receptor/ chloride channel

Answer 12

•family of receptors for inhibitory neurotransmitter gaminobutyric acid (GABA) widely distributed in CNS
•target for facilitator modulator diazepam
multiple types of subunit give rise to various receptor subtypes

Question 13

Superfamily 2- GCPR

Answer 13

•largest superfamily >900 receptor proteins•targets for many currently prescribed drugs
Important Examples Autonomic (Para/sympathetic) nervous system

• adrenoceptor family–for noradrenaline and adrenaline
• muscarinic acetylcholine receptor family–for acetylcholine which are stimulated selectively by muscarine and antagonised by atropine

2 families of cholinoreceptor-
Nicotinic (NMJ)- LOW Ach affinity

Muscarinic (PNS)- HIGH Ach affinity

Question 14

What are receptors in superfamily 2 coupled to/ how are they related to signal transduction?

Answer 14

Heterotrimeric G-proteins:
•consist of 3 subunits (a b g)
•require GTP (when bound to G protein, it is on/active- encourages GDP to dissociate and GTP to bind) and GDP (off/inactive) for function
•molecular switches
•communicate between superfamily 2 receptor and an effector (enzyme or ion channel)

Alpha subunit of G-protein has GTP binding site, allowing activity of protein- GTPase can bind to effector protein interactive site, converting it to GDP and switching it off.
When binding occurs, the Ga subunit dissociates from the Gby subunits and altered activity by Ga of an effector such as adenylate cyclase

Look at basic structure slide 27

Question 15

Role of G-proteins and second messengers in Superfamily 2 receptor-effector coupling

Answer 15

First messenger binds to receptor, activating receptor and therefore the G protein, allowing GTP to bind and causing the A subunit to activate/regulate activity of effector (enzyme/ion channel). (M1 cannot cross cm, reliant on receptor binding on CSM to activate signal transduction and bring about response)

This leads to accumulation of accumulation of another chemical in the cell cytosol (enzyme and synthesis, or entering through I.Chan). The accumulation of this second messenger regulates an internal target, bringing about a cellular response.

Question 16

What is a second messenger?

Answer 16

‘intracellular chemical the concentration of which can be altered rapidly and reversibly on activation of a cell surface receptor by achemical mediator (first messenger) to regulate activity of an internal effector already present in cell to produce appropriate intracellular response’

Question 17

How are M2s generated within cells?

Answer 17

•synthesis inside cell catalysed by enzymes whose activity is regulated by cell surface receptors e.g. cAMP
OR
•influx of ions via channels whose activity is regulated (indirectly) by cell surface receptors e.g Ca2+

Question 18

G protein properties

Answer 18

•amplification
–receptor remains active long enough to active several G-proteins (or the same one several times)
–effector remains activated by Ga subunit long enough to generate many molecules of second messenger

•specificity-different Ga subunits (>28)
–each encoded by a different gene or splice variant
All Ga subunits have SIMILAR GTP/GDP site and GTPase activity- but different receptor interaction sites, and different effector interaction sites

Question 19

Concept of stimulatory/ inhibitory G-proteins

Answer 19

•stimulatory G-proteins induce conformational change in effector protein (channel or enzyme) to more active state (enhanced activity)
-switch enzymes on or open ion channels
•inhibitory G-proteins induce conformational change in effector protein (channel or enzyme) to less active state (reduced activity)
-turn enzymes off or close ion channels

Question 20

Some examples of effectors regulated by G-proteins

Answer 20

•adenylate cyclase (makes cyclic AMP)
–Gas activates, Gai inhibits
•phospholipase C-b(makes IP3and DAG)
–Gaq activates
•voltage-gated potassium channels
–Gai opens (K+ ion efflux, hyperpolarisation)
•voltage-gated calcium channels
–Gas opens (Ca2+ ion influx), Gao closes