1. INTRODUCTION 2. WHAT IS A RECEPTOR 3. HISTORY 4. CONCEPT OF CELL SIGNALLING 5. RECEPTOR SUPER FAMILIES 6. GPCRs- SIGNAL TRANSDUCTION & ITS SECOND MESSENGERS

1. Receptors as drug target
PC-520

2. Drugs produce their therapeutic effects by producing
biochemical/ physical changes:
o in the target tissues
o of the host
o of the organisms which invade the host.
These changes are due to;
o physical and chemical properties of drug
o action on the drug targets namely;
▪ Receptors
▪ Enzymes
▪ Carrier molecules
▪ Ion channels
Introduction

3. To get drug action, it is essential that-
1. Sufficient concentration of drug reaches the site of action
2. Remains there for a sufficient duration
3. The tissue is susceptible for drug action
o Magnitude of drug action is proportional to the concentration
of drug at the site of action.
o Receptor mechanism is very important to understand the
action and effect of a drug.
Introduction

4. Receptor:
o component of a cell or organism
o interacts with a drug
o initiates the chain of biochemical events
o leading to the drug’s observed effects
o They have specific binding sites that are definite in size
and shape
o Most are present on or near the membrane.
o Some lie in the enzymes or genes
o protein (polypeptide) in nature

5. HISTORY OF RECEPTORS
o Langley and Ehrlich introduced concept of receptor
o Langley (1852 – 1925);
o Studied the effects of atropine against pilocarpine
induced salivation in cats
o Postulated that there was a receptive substance in the
nerve ending or gland cell with which both atropine and
pilocarpine are capable of forming compounds

6. HISTORY OF RECEPTORS
Ehrlich (1854 – 1915) observed that;
o certain dyestuffs acted selectively, staining some cells
more deeply or in a different way from other cells
o suggested that drugs with selective actions on particular
cells could be developed
o Introduced the term “receptor”

7. Concept of Cell
Signaling
Process in which cells sense the
extracellular stimuli through
membranous or intracellular
receptors, transduce the signals
via intracellular molecules
-Regulate the biological
function of the cells.

12. Receptors that do not fall into these four receptor
families:
o Specific membrane ion pumps (e.g. Na+/K+ ATPase
o Specific enzymes (e.g. 5-phosphodiesterase)
o Structural proteins (e.g. colchicine to tubulin)
o Cytosolic proteins (e.g. ciclosporin to immunophilins)

15. 2. G-protein-coupled receptors (7-TM receptors)
2.1 Structure - Single protein with 7 transmembrane regions
Transmembrane
helix
C -Terminal chain
G-Protein
binding region
Variable
intracellular loop
Extracellular
loops
Intracellular loops
N -Terminal chain
HO2C
NH2
VII VI V IV III II I
Membrane

17. 2.2 Ligands
• Monoamines e.g. dopamine, histamine, noradrenaline, acetylcholine
(muscarinic)
• Nucleotides
• Lipids
• Hormones
• Glutamate
• Ca++
2. G-protein-coupled receptors (7-TM receptors)

18. 2.3 Ligand binding site - varies depending on receptor type
A) Monoamines - pocket in TM helices
B) Peptide hormones - top of TM helices + extracellular loops +
N-terminal chain
C) Hormones - extracellular loops + N-terminal chain
D) Glutamate - N-terminal chain
Ligand
B D
C
A
2. G-protein-coupled receptors (7-TM receptors)

19. 2.4 Bacteriorhodopsin & rhodopsin family
• Rhodopsin = visual receptor
• Many common receptors belong to this same family
• Implications for drug selectivity depending on similarity (evolution)
• Membrane bound receptors difficult to crystallise
• X-Ray structure of bacteriorhodopsin solved - bacterial protein similar
to rhodopsin
• Bacteriorhodopsin structure used as ‘template’ for other receptors
• Construct model receptors based on template and amino acid sequence
• Leads to model binding sites for drug design
• Crystal structure for rhodopsin now solved - better template
2. G-protein-coupled receptors (7-TM receptors)

20. Common ancestor
Endothelins
Opsins, Rhodopsins
Tachykinins
Monoamines
alpha beta
H2 1
muscarinic
H1
2 4 1
5 3 2A 2B 2C D1A D1B D5
D4 D3 D2 3 2 1
Bradykinin,
Angiotensin.
Interleukin-8
Muscarinic Histamine -Adrenergic Dopaminergic -Adrenergic
Receptor
types
Receptor
sub-types
2. G-protein-coupled receptors (7-TM receptors)
2.4 Bacteriorhodopsin & rhodopsin family

21. 2.5 Receptor types and subtypes
Reflects differences in receptors which recognise the same ligand
Receptor Types Subtypes
Alpha ()
Beta ()
1, 2A, 2B, 2C
1, 2, 3
Adrenergic
Muscarinic Nicotinic
Muscarinic M1-M5
2. G-protein-coupled receptors (7-TM receptors)

22. • Receptor types and subtypes not equally distributed amongst tissues.
• Target selectivity leads to tissue selectivity
Heart muscle - 1 adrenergic receptors
Fat cells - 3 adrenergic receptors
Bronchial muscle - 1& 2 adrenergic receptors
GI-tract - 1 2 & 2 adrenergic receptors
2. G-protein-coupled receptors (7-TM receptors)
2.5 Receptor types and subtypes

23. 2.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
GS-Protein - membrane bound protein of 3 subunits (, , g)
- S subunit has binding site for GDP
-GDP bound non covalently
 g

GDP
2. G-protein-coupled receptors (7-TM receptors)

24. ß

g
GDP GTP
Ligand
binding
Induced
fit
G-protein
binds
Induced
fit for
G-protein
G-Protein alters shape
GDP binding site distorted
GDP binding weakened
GDP departs
ß

g
Ligand
Receptor
G Protein
Cell membrane
ß

g
Binding site for G-protein opens
= GDP
a) Interaction of receptor with Gs-protein
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

25. ß

g
Binding site recognises GTP
GTP binds
Induced fit
G-protein alters shape
Complex destabilised
Fragmentation
and release
ß

g
• Process repeated for as long as ligand bound to receptor
• Signal amplification - several G-proteins activated by one ligand
• s Subunit carries message to next stage
ß

g
a) Interaction of receptor with Gs-protein
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

26. b) Interaction of s with adenylate cyclase
s Subunit recombines with ,g dimer
to reform Gs protein
Active site
(closed)
Binding site
for s subunit
cyclic AMP
ATP
Binding
Induced
fit
Active site
(open)
P
cyclic AMP
ATP
GTP hydrolysed
to GDP catalysed
by s subunit
s-subunit
Adenylate cyclase
GTP
GDP
s Subunit changes shape
Weaker binding to enzyme
Departure of subunit
Enzyme reverts to inactive
state
Active site
(closed)
Signal
transduction
(con)
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

27. N
N N
N
NH2
O
OH
OH
O
P
O
OH
O
P
O
OH
O
P
O
OH
HO
N
N N
N
NH2
O
OH
O
P O
O OH
ATP
Adenylate cyclase
H H
Cyclic AMP
H
H
H
H
• Several 100 ATP molecules converted before s-GTP deactivated
• Represents another signal amplification
• Cyclic AMP becomes next messenger (secondary messenger)
• Cyclic AMP enters cell cytoplasm with message
b) Interaction of s with adenylate cyclase
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

28. c) Interaction of cyclic AMP with protein kinase A (PKA)
• Protein kinase A = serine-threonine kinase
• Activated by cyclic AMP
• Catalyses phosphorylation of serine and threonine residues on protein substrates
• Phosphate unit provided by ATP
H
N C
O
OH
H
N C
O
O
P
HO O
OH
H
N C
O
HC OH
CH3
H
N C
O
HC O
CH3
P O
HO OH
Protein
kinase A
Serine
Protein
kinase A
Threonine
H H H H
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

29. cyclic AMP
ATP
Adenylate
cyclase
Enzyme
(active)
P
Enzyme
(inactive)
Chemical
reaction
Protein
kinase
Activation
c) Interaction of cyclic AMP with protein kinase A (PKA)
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

30. c) Interaction of cyclic AMP with protein kinase A (PKA)
Protein kinase A - 4 protein subunits
- 2 regulatory subunits (R) and 2 catalytic subunits (C)
Cyclic AMP binds to PKA
Induced fit destabilises complex
Catalytic units released and activated
Note
C
C
R
R
cAMP
cAMP
binding
sites
catalytic subunit
R
R
C
C
catalytic subunit
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

31. Phosphorylation of other proteins and enzymes
Signal continued by phosphorylated proteins
Further signal amplification
C
Protein
+ ATP
Protein
+ ADP
P
c) Interaction of cyclic AMP with protein kinase A (PKA)
2. G-protein-coupled receptors (7-TM receptors)
2.6 Signal transduction pathway

32. 2.7 Glycogen metabolism - triggered by adrenaline in liver cells
Catalytic
subunit of
PKA
cAMP
Protein kinase A
C
Inhibitor (inactive)
Inhibitor-P
(active)
Phosphatase
(inhibited)
Glycogen
synthase
(active)
Glycogen
synthase-P
(inactive)
Phosphorylase
kinase (inactive)
Phosphorylase
kinase-P (active)
Phosphorylase b
(inactive)
Phosphorylase a
(active)
Glycogen Glucose-1-phosphate
-Adrenoreceptor
Adrenaline
adenylate
cyclase
s s
2. G-protein-coupled receptors (7-TM receptors)

33. Coordinated effect - activation of glycogen metabolism
- inhibition of glycogen synthesis
Adrenaline has different effects on different cells - activates
fat metabolism in fat cells
2. G-protein-coupled receptors (7-TM receptors)
2.7 Glycogen metabolism - triggered by adrenaline in liver cells

35. 2.8 GI proteins
2. G-protein-coupled receptors (7-TM receptors)

36. 2.9 Phosphorylation
• Prevalent in activation and deactivation of enzymes
• Phosphorylation radically alters intramolecular binding
• Results in altered conformations
O
NH3
O
P
O
O
O
O
NH3
H
O
Active site
closed
Active site
open
NH3
O
O P
O
O
O
2. G-protein-coupled receptors (7-TM receptors)

37. 2.10 Drugs interacting with cyclic AMP signal transduction
Cholera toxin - constant activation of c.AMP - diahorrea
Theophylline and caffeine - inhibit phosphodiesterases
- phosphodiesterases responsible for metabolising cyclic AMP
- cyclic AMP activity prolonged
Theophylline
N
N
N
H
N
O
H3C
O
CH3
Caffeine
N
N
N
N
O
H3C
O
CH3
CH3
2. G-protein-coupled receptors (7-TM receptors)

38. 2.11 Signal transduction involving phospholipase C (PLC)
• Gq proteins - interact with different receptors from GS and GI
• Split by same mechanism to give q subunit
• q Subunit activates or deactivates PLC (membrane bound enzyme)
• Reaction catalysed for as long as q bound - signal amplification
• Brake and accelerator

Active site
(closed)
PLC
Active site
(open)

PLC

PLC
PIP2
Binding weakened
GTP hydrolysis q departs
Active site
(closed)
enzyme
deactivated

PLC
DG
IP3

PLC
PIP2
DG
IP3
Phosphate
2. G-protein-coupled receptors (7-TM receptors)

39. O
HO
O
O
OH
HO
CH2 CH CH2
O O
OH
C C
O
R R
O
C O
R
C
R
O
O
O
CH2
O
CH
CH2
P
O O
HO
OH
O
O
HO
O
+
IP3
PIP2
DG
PLC
H
H
H
H
H
H
P
P
P
P
P
2.11 Signal transduction involving phospholipase C (PLC)
Phosphatidylinositol diphosphate
(integral part of cell membrane)
Inositol triphosphate
(polar and moves
into cell cytoplasm)
Diacylglycerol
(remains in membrane)
R= long chain hydrocarbons = PO3
2-
P
2. G-protein-coupled receptors (7-TM receptors)

40. 2.12 Action of diacylglycerol
• Activates protein kinase C (PKC)
• PKC moves from cytoplasm to membrane
• Phosphorylates enzymes at Ser & Thr residues
• Activates enzymes to catalyse intracellular reactions
• Linked to inflammation, tumour propagation, smooth muscle activity etc
PKC
DG
Binding
site for DG
Cell membrane
Cytoplasm
PKC moves
to membrane
PKC
DG
Cytoplasm
DG binds to
DG binding site
Active site
closed
PKC
DG
Cytoplasm
Induced fit
opens active site
Enzyme
(inactive)
Enzyme
(active)
Chemical
reaction
2. G-protein-coupled receptors (7-TM receptors)

41. Drugs inhibiting PKC - potential anti cancer agents
2.12 Action of diacylglycerol
O
O
Me
Me
O
OH
H
O
O
C
H
Me
OH
Me
Me
C O
OH
H
CH
MeO2C
O
C
CH
O
CHCO2Me
H H
HO
H
Me
O
CH
CH
CH
CH3CH2CH2
Bryostatin (from sea moss)
2. G-protein-coupled receptors (7-TM receptors)

42. 2.13 Action of inositol triphosphate
• IP3 - hydrophilic and enters cell cytoplasm
• Mobilises Ca2+ release in cells by opening Ca2+ ion channels
• Ca2+ activates protein kinases
• Protein kinases activate intracellular enzymes
• Cell chemistry altered leading to biological effect
2. G-protein-coupled receptors (7-TM receptors)

43. IP3
Calcium
stores
Ca++
Calmodulin
Calmodulin Ca++
Activation
Protein
kinase
Activation
Protein
kinase
Enzyme
(inactive)
Enzyme
(active)
P
Cytoplasm
Cell membrane
Enzyme
(active)
Enzyme
(inactive)
P
Chemical
reaction
Chemical
reaction
2.13 Action of inositol triphosphate
2. G-protein-coupled receptors (7-TM receptors)

44. 2.14 Resynthesis of PIP2
IP3 + DG PIP2
several
steps
Li+ salts
Inhibition
Lithium salts used vs manic depression
2. G-protein-coupled receptors (7-TM receptors)

46. To be continue……….
Thank You