FM2003 | PT1445 | PT2201
Robert Callaghan
Lecture 2 Dept. Pharmacology & Therapeutics
[Link]@[Link]
Receptor mechanisms
Learning objectives
Describe the mechanisms by which the four receptor types act to
produce an effect.
Critically compare and contrast the four receptor types with one
another.
Provide examples of each receptor type.
Lecture 2 2
Signalling mechanisms as drug targets
Agonists bind to same site as ligand, producing same kind of signal,
or bind to another site on receptor, producing no signal themselves
but enhancing the signal of endogenous ligand (allosteric)
Antagonists bind to same site as ligand, diminishing or blocking the
signal competitively,
or bind to an allosteric site on the receptor, diminishing signal
produced by endogenous ligand,
or cross the membrane & intercept the signal downstream of the
receptor.
Lecture 2 3
Drug action at cell level R&D chapter 3
Endogenous ligand
Receptor Altered cell function
Drug (agonist) signal transduction
Receptor proteins
belong to one of 4 types that differ structurally & functionally
3 of these receptor groups are situated in the cell (plasma)
membrane; the fourth is intracellular (cytosol/nucleus)
all include a ligand-binding domain & an effector domain
Lecture 2 4
Receptor classes
R&D fig. 3.2
Lecture 2 5
1 Ion channels
4-5 subunits
ligand ligand-gated
conduct Na+, K+, Cl- or Ca2+
Cell
membrane alter cell membrane potential or
intracellular ionic composition
fast communication (s-ms)
Examples
Nicotinic acetylcholine receptor (nAChR)
GABAA receptor
Glycine receptor
Glutamate receptors
5-HT3 receptor
Lecture 2 6
P2x purine receptor
GABAA
nAChR
Na+
acetylcholine
Side view Top view
- - - -
0.65 nm pore
(cation selective)
Na+
Lecture 2 7
Ligand gated ion channel general
mechanism
❑ Agonist binds to ion channel at ligand binding site
❑ Agonist binding induces conformation change causing pore
opening in the ligand gated ion channel
❑ This pore is selective to specific ions based on the ion channel
type (e.g. nAChR specific to allow Na+ enter)
❑ Ion flows through pore based on concentration &
electrochemical gradients
❑ The influx/efflux of specific ions leads to cell
depolarisation/hyperpolarisation (depending on the receptor
type)
❑ Ligand disassociates and ion channel pore closes
2 G protein-coupled receptors
receptor G protein effector
N
cell membrane
C
GTP
GDP
second
messenger(s)
Receptors linked to effectors (adenylate cyclase/phospholipase C/ion channels)
that change intracellular level of second messenger(s), via GTP-binding proteins
(G proteins)
Lecture 2 9
GPCR general mechanism
ligand effector ion channel
membrane
(e.g. K+) hyperpolarization
receptor
effector
enzyme
GDP GDP
GTP
second messenger(s)
→ intracellular effects
Lecture 2 10
GPCR general mechanism
Agonist binds to GPCR ligand binding domain causing a
conformational change in the third intracellular loop
This allows G-protein to bind to GPCR allowing the
exchange of G-protein’s GDP for GTP -activating the G
protein by allowing the α subunit to dissociate from the
βγ dimer
G-protein subunits can then activate effector proteins
(e.g. adenylate cyclase) which generate second
messenger proteins (e.g. cAMP in the case of adenylate
cyclase) leading to intracellular effects. Subunits can also
activate effector ion channels leading to changes in
membrane polarisation
G-protein coupled receptors cont.
Single polypeptide chain (800-1,100 aa
N Ligand-binding residues), spanning membrane 7 times:
domain
-Helices most highly conserved
Third intracellular loop couples to G
protein & determines G-protein selectivity.
C
G-protein Smaller ligands bind within helix cluster,
coupling larger ligands at extracellular surface.
domain
e.g.
muscarinic acetylcholine receptor (mAChR) GABAB receptor
5-HT (serotonin) receptors (except 5-HT3) adrenoceptors
histamine H2, eicosanoid, many peptide & purine R dopamine receptors
(thrombin & other protease receptors) opioid receptors
Lecture 2 12
chemoreceptors
Variation on a theme – protease activated receptors
Thrombin receptor
Receptor activated by protease cleavage of N-terminus domain,
revealing a ‘tethered agonist’:
thrombin
(a serine protease)
tethered agonist
thrombin receptor
phosphorylated
active
inactive
desensitized
Lecture 2 13
G proteins
Subtypes either activate (Gs, Gq) or inhibit (Gi, G0) specific effectors
Signalling takes seconds to minutes
Scope for signal amplification
Second messengers generated in turn activate protein kinases that
phosphorylate target proteins
cAMP-dependent protein kinases (PKA)
cGMP-dependent protein kinases (PKG)
Ca2+- & Ca/calmodulin-dependent protein kinases (PKC)
Diacylglycerol-dependent PKC
etc.
Lecture 2 14
Effectors
(= ‘effectors’ on R&D fig. 3.14
previous slides)
(= end targets that
mediate cell response(s)) Lecture 2 15
Second messengers
cAMP synthesized from ATP by adenylate cyclase
activates PKA that phosphorylates target proteins
broken down by phosphodiesterases
cGMP formed from GTP by guanylate cyclase, (etc.)
diacylglycerol & IP3 (inositol tris phosphate)
formed from phosphoinositides by phospholipase C
IP3 triggers Ca2+ release from ER
DAG co-activates PKC
Ca2+ enters across cell membrane or released from
intracellular stores, via Ca channels
activates PKC that phosphorylates target proteins
Lecture 2 16
re-sequestered into ER or pumped out of cell
3 Receptors with direct kinase activity
Receptors for polypeptide hormones regulating cell proliferation &
differentiation
e.g. epidermal growth factor (EGF)
insulin-like growth factor (IGF-1)
nerve growth factor (NGF)
platelet-derived growth factor (PDGF)
These receptors are monomers - single polypeptide chain comprising a(n)
◼ extracellular domain containing the ligand-binding site
◼ single -helix crossing the membrane
◼ intracellular domain with catalytic activity tyrosine kinase
serine/threonine kinase
Lecture 2 17
Kinase-linked receptors continued
ligand
out
cell membrane
in
protein tyr-phosphorylated protein
ATP
ADP
Tyrosine kinase receptors
regulate growth, differentiation, development
e.g. receptors for insulin, epidermal growth factor (EGF), platelet-derived growth
factor (PDGF), etc.
Serine/threonine kinase receptors
e.g. tumour growth factor (TGF) receptor Lecture 2 18
Receptor tyrosine kinase (RTK)
ligand
cell membrane
P P P RAS
P
P activation
GDP
Raf
phosphorylation
MEK
GTP
altered gene various phosphorylation
transcription transcription MAP kinase
Grb2
phosphorylation
factors
Lecture 2 19
nucleus
RTK general mechanism
Binding of ligand to ligand binding domain of receptor leads to
conformational change causing dimerisation of kinase receptors
This dimerisation brings intracellular tyrosine kinase domains
into close proximity -allowing phosphorylation of tyrosine
residues on coupled receptor
This phosphorylation allows binding of Grb2 protein which itself
becomes phosphorylated -subsequently allowing the binding of
Ras which becomes activated through the exchange of GDP to
GTP
Following RAS activation subsequent proteins become
phosphorylated in a stepwise fashion Raf -> MEK -> MAPK ->
various transcription factors -leading to altered gene
transcription
Cell response to cytokines
Cytokines are soluble proteins that regulate cell growth, differentiation
& function.
They include
interferons formed in & secreted by virus-infected cells
bind to membrane receptors ➔
cell growth
expression of antiviral proteins
interleukins 2-7, 9-12, 15
Lecture 2 21
A variation on the RTK theme
Cytokine receptor
ligand
P P
P P
Stat
P P
Stat
altered gene Jak
transcription
Jak Lecture 2 22
4 Intracellular receptors
transcription-
N activating
Ligand-binding lipid-soluble domain
domain ligand
hsp90 DNA-binding
hsp90 domain
binds response alters transcription
Activated receptor
elements in DNA of specific genes
Process takes minutes to hours
Therapeutic consequences
Lag in response, e.g. slow onset in anti-inflammatory effect of glucocorticoids
23
Effects can persist long after agonist concentration is reduced to zero
Intracellular receptors continued
These mediate cell response to
Steroids (glucocorticoids, mineralocorticoids, sex steroids) & vitamin D
Thyroid hormone
Inducers of drug metabolism, acting via the Ah receptor
e.g. barbiturates, TCDD (tetrachlorodibenzodioxan)
Lecture 2 24
Glucocorticoid receptor
glucocorticoid-binding protein
GBP
G
glucocorticoid
G
hsp70 GR IPGR
hsp90
glucocorticoid receptor
altered gene transcription
glucocorticoid
response element GRE GRE
Lecture 2 25
cytosol nucleus
Nuclear receptors general mechanism
Lipophilic ligands must first cross cell membranes to reach
intracellular nuclear receptors
Ligand binds to nuclear receptor ligand binding domain leading
to a conformational change and expelling of chaperone
proteins such as hsp70, hsp90, and IP –allowing nuclear
receptors to subsequently dimerise
Nuclear receptor dimer then binds to response elements on
DNA leading to altered gene transcription
Intracellular receptors, continued
PPARs (peroxisome proliferator-activated receptors)
Bind fatty acids & FA metabolites (prostaglandins, polyunsaturated FAs)
→ regulate genes for lipid / carbohydrate metabolism/transport
PPAR role in dyslipidaemias, coronary heart disease, obesity
agonists include fibrates, plasticizers
PPAR1/2 roles in diabetes, atherosclerosis, cancer
agonists include thiazolidinediones (type 2 diabetes)
antagonists include the plasticizer bisphenol A
PPAR Lecture 2 27
Summary
Cell response to ligand or drug is via any of 4 general signalling pathways
that integrate extracellular chemical (& physical) information.
Receptors are located in the cell membrane or intracellularly.
Signalling rate ranges from rapid (ligand-gated ion channels) to slow
(intracellular receptors).
Specificity of signalling depends on target enzymes/ion channels
expressed in a particular cell type, resulting in appropriate response.
Receptor number & activity may vary over time.
Lecture 2 28
