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Chaps 4 & 5: Receptors as drug targets, structure, function & signal transduction
Cell signaling is part of a complex system of communication that governs basic cellular
activities and coordinates cell actions. Communication allows cells to perceive and
correctly respond to their microenvironment for proper development, tissue repair, and
immunity and normal tissue function. Errors in cellular information processing are
responsible for diseases such as cancer, autoimmunity, diabetes and more.
Understanding cell signaling is crucial for treating diseases effectively.
neighbor
g
talk
self
lf
talk
cell 1
signals
signals
((neurotransmitters))
receptors
cell 2
cell 1
cell 2
synapse
cell 2
receptor a
signals
(hormones)
signals
i l
(hormones)
cell 1
cell 5
receptor c
signals
(hormones)
signals
(hormones)
cell 3
synapse
receptor b
The same signal can
deliver different
messages to different
tissues (histamine).
(histamine)
etc.
synapse
cell 3
cell 4
receptor d
Important functions of receptors:
1. Globular proteins (receptors) acting as a cell’s ‘letter boxes’
2 Located mostly in the cell membrane
2.
3. Receive messages from chemical messengers coming from other cells
4. Transmit a message into the cell leading to a cellular effect
5. Different receptors specific for different chemical messengers
6. Each cell has a range of receptors in the cell membrane making it responsive to different
chemical messengers
© Oxford University Press, 2013
1
Signals can be divided into the 5 catagories below. Signal carriers (S) have to reach the proper
receptors (R) for the message to be recieved.
Intracrine signals are produced by the target cell that stay within the target cell.
cell a
S
R
S = signal
R = receptor
Autocrine signals are produced by the target cell, are secreted, and affect the target cell itself via
receptors. Sometimes
i
autocrine
i cells
ll can target cells
ll close
l
b if they
by
h are the
h same type off cell
ll as the
h
emitting cell. An example of this are immune cells.
cell a S R
Juxtacrine signals target adjacent (touching) cells. These signals are transmitted along cell membranes
via protein or lipid components integral to the membrane and are capable of affecting either the emitting
cell or cells immediately adjacent.
R
cell
ll a S R
cell
ll b
Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example.
cell
ll a S
R cell b S
synapse
R cell c S
synapse
etc.
synapse
Endocrine signals target distant cells. Endocrine cells produce hormones that travel through the blood to
reach all parts of the body (long-range allostery).
S
R2 cell c
S
S
S
S
S
S S
cell a S
S
R3 cell d
S
S
S
R1 cell b
© Oxford University Press, 2013
2
Central Nervous System (CNS)
Brain and spinal cord
Integrative and control centers,
process info out and info in
structure
function
Peripheral Nervous System (PNS)
Cranial nerves and spinal nerves
Communication lines between the
CNS and the rest of the body
Sensory (afferent) division
Motor (efferent) division (CNS)
Somatic and visceral sensory
nerve fibers
Conducts impulses from
receptors to the CNS
Motor nerve fibers
Sympathatic division
mobilizes body systems during
activity (fight or flight)
Parasympathetic division
Conducts impulses from the CNS
to effectors (muscles and glands)
Autonomic nervous system (ANS)
Somatic nervous system
Visceral motor (involuntary)
Somatic motor (voluntary)
Conducts impulses from the CNS to
cardiac muscles, smooth muscles,
and glands
Conducts impulses from the
CNS to skeletal muscles
Conserves energy
C
Promotes 'housekeeping'
functions during rest
© Oxford University Press, 2013
3
Central nervous system
The central nervous system consists of
the two major structures: the brain and
spinal cord. The brain is encased in the
skull,
k ll andd protected
t t d by
b the
th cranium.
i
The spinal cord is continuous with the
brain and lies on the backside to the
b i andd is
brain,
i protected
t t d by
b the
th vertebra.
t b
The spinal cord reaches from the base of
the skull, continues through or starting
below the foramen magnum, and
t
terminates
i t roughly
hl level
l l with
ith the
th first
fi t or
second lumbar vertebra, occupying the
upper sections of the vertebral canal.
The CNS iintegrates
Th
t
t iinformation
f
ti it
receives from, and coordinates and
influences the activity of, all parts of the
body and it contains the majority of the
nervous system.
t
Usually,
U ll the
th retina
ti andd
the optic nerve (2nd cranial nerve), as
well as the olfactory nerves (1st) and
olfactory epithelium are considered as
parts of the CNS
CNS, synapsing
s napsing directl
directly on
brain tissue without intermediate
ganglia..
© Oxford University Press, 2013
4
Peripheral nervous system
The peripheral nervous system
(PNS) is the part of the nervous
system that consists of the nerves
and ganglia on the outside of the
brain and spinal cord.
cord Its main
function is to connect the central
nervous system (CNS) to the
limbs and organs, essentially
serving as a communication relay
going back and forth between the
brain and spinal cord with the rest
of the body. Unlike the CNS, the
PNS is not protected by the bone
of spine and skull, or by the
blood–brain barrier, which leaves
it exposed to toxins and
mechanical injuries. The
peripheral nervous system is
divided into the somatic nervous
system and the autonomic nervous
system
5
© Oxford University Press, 2013
The autonomic nervous system is responsible for regulating the body's unconscious actions. The
parasympathetic system is responsible for stimulation of "rest-and-digest" or "feed and breed"
activities that occur when the body is at rest, especially after eating, including sexual arousal,
salivation,, lacrimation ((tears),
), urination,, digestion
g
and defecation. Its action is described as being
g
complementary to that of the sympathetic nervous system, which is responsible for stimulating
activities associated with the fight-or-flight response.
Parasympathetic nervous system
Sympathetic nervous system
© Oxford University Press, 2013
6
Somatic nervous system
y
The somatic nervous system is the part of the peripheral
nervous system associated with skeletal muscle voluntary
control of body movements. It consists of afferent nerves
(toward) and efferent nerves (out of).
of) Afferent nerves are
responsible for relaying sensation from the body to the
central nervous system (CNS); efferent nerves are
responsible for sending out commands from the CNS to
the body.
There are 43 segments of nerves in the human body. With
each segment, there is a pair of sensory and motor nerves.
In the body, 31 segments of nerves are in the spinal cord
and 12 are in the brain stem.
stem
The somatic nervous system consists of three parts:
Spinal nerves: They are peripheral nerves that carry
sensory information
i f
i into
i
andd motor commands
d out off the
h
spinal cord.
Cranial nerves: They are the nerve fibers that carry
information into and out of the brain stem. They
y include
smell, vision, eye, eye muscles, mouth, taste, ear, neck,
shoulders, and tongue.
Interneurons (association nerves): These nerves integrate
sensory input and motor output,
output numbering thousands
© Oxford University Press, 2013
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Nerve cells make contact to muscle cells, organs and other nerve cells
Can be 1000s of
axon inputs
p from
other nerve cells.
… or synaptic
button when
contact to neuron
8
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axon
Synaptic button
or neuromuscular
junction
Cell body
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9
Nerve cells and glial cells are incredibly complicated networks of connections. Perhaps,
100,000,000,000 neurons with 10,000 connections each  1,000,000,000,000,000 (quadrillion).
10
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Neurological disorders
Charcot–Marie–Tooth disease (CMT), also known as hereditary motor and sensory neuropathy
(HMSN) hhereditary
(HMSN),
dit
sensorimotor
i t neuropathy
th andd peroneall muscular
l atrophy,
t h is
i a heterogeneous
h t
inherited disorder of nerves (neuropathy) that is characterized by loss of muscle tissue and touch
sensation, predominantly in the feet and legs but also in the hands and arms in the advanced
stages of disease. Presently incurable, this disease is one of the most common inherited
neurological
l i l disorders,
di d
with
ith 37 in
i 100,000
100 000 affected
ff t d (1
( 1 in
i 2500).
2500)
Alzheimer's disease (AD), is a neurodegenerative disease characterized by progressive cognitive
deterioration together with declining activities of daily living and neuropsychiatric symptoms or
b h i l changes.
behavioral
h
Th
The di
disease process iis associated
i t d with
ith plaques
l
andd tangles
t l in
i the
th brain.
b i The
Th
most striking early symptom is loss of short-term memory (amnesia), which usually manifests as
minor forgetfulness that becomes steadily more pronounced with illness progression, with relative
preservation of older memories. As the disorder progresses, cognitive (intellectual) impairment
extends
t d to
t the
th domains
d
i off language,
l
skilled
kill d movements
t andd recognition,
iti andd functions
f ti
suchh as
decision-making and planning become impaired.
Parkinson's disease (PD), is a degenerative disorder of the central nervous system that often
i
impairs
i the
th sufferer's
ff
' motor
t skills
kill and
d speech.
h Parkinson's
P ki
' di
disease belongs
b l
to
t a group off
conditions called movement disorders. It is characterized by muscle rigidity, tremor, a slowing of
physical movement, and in extreme cases, a loss of physical movement). The primary symptoms
are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused
b the insufficient
by
ins fficient formation and action of dopamine,
dopamine which
hich is produced
prod ced in the dopaminergic
neurons of the brain. Secondary symptoms may include high level cognitive dysfunction and
subtle language problems. PD is both chronic and progressive.
© Oxford University Press, 2013
11
Myasthenia gravis is a neuromuscular disease leading to fluctuating muscle weakness and
fatigability during simple activities. Weakness is typically caused by circulating antibodies that
block acetylcholine receptors at the post-synaptic neuromuscular junction, inhibiting the stimulative
effect of the neurotransmitter acetylcholine.
Demyelination results in the loss of the myelin sheath insulating the nerves. When myelin
degrades, conduction of signals along the nerve can be impaired or lost, and the nerve eventually
withers. This leads to certain neurodegenerative disorders like multiple sclerosis, Guillain-barré
syndrome and chronic inflammatory demyelinating polyneuropathy.
Axonal degeneration
Although most injury responses include a calcium influx signaling to promote resealing of severed
parts, axonal injuries initially lead to acute axonal degeneration, which is rapid separation of the
proximal and distal ends within 30 minutes of injury. Early changes include accumulation of
mitochondria in the paranodal regions at the site of injury. Endoplasmic reticulum degrades and
mitochondria swell up and eventually disintegrate. The axon undergoes complete fragmentation.
The process takes about roughly 24 hrs in the PNS, and longer in the CNS.
Diabetic neuropathies are nerve damaging disorders associated with diabetes mellitus. These
conditions are thought to result from diabetic microvascular injury involving small blood vessels
that supply nerves (vasa nervorum) in addition to macrovascular conditions that can culminate in
diabetic neuropathy. Relatively common conditions which may be associated with diabetic
neuropathy include third nerve palsy; mononeuropathy; mononeuropathy multiplex; diabetic
amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy.
Diabetic neuropathy affects all peripheral nerves including pain fibers, motor neurons and the
12
autonomic nervous system. Symptoms usually develop gradually over years.
© Oxford University Press, 2013
cell potential = -50-89 mV because K+ can diffuse out, but negatively charged proteins cannot.
Sodium ions cannot move in because their ion channels are closed until triggered by a signal from the
presynaptic side of the neuron. Neurotransmitters are released from the prior cell and diffuse across
the synapse to bind to the ion-gate receptor and open an ion gate channel (Na+ by acetyl choline, Cl -by gama aminobutyric acid and others), Na+ influx depolarizes the cell down the axon and allows
Ca+2 in which causes vesicles to fuse with membrane and release the neurotransmitter
neurotransmitter, starting the
next impulse.
neurotransmitters
at synapse
y p
neurotransmitters bind
to receptors on the next
cell at the synapse
Some of these are
activated by the change
i membrane
in
b
potential.
t ti l
depolarization
milli seconds
proteins
Axon
neurotransmitters
leave cell by exocytosis
0
K
action potential
Na
Na
K
Na
Ca+2
action
potential
Cl
Cell Body
Ion gate
opens when
neurotransmitter
complexes
at receptor.
Ca+2
C
K
Na Cl
dendrites
Ca+2
H
O
O
O
O
acetyl choline
is an important
neurotransmitter,
Ca+2
B
H
B
H
N
O
O
O
O
choline retaken up by
presynaptic neuron
H
OH
N
diffuses across the synaptic
cleft and binds to a Na+
ion-gate channel on the
OH of a serine residue of
the receptor
H
O
O
B
B
receptor
H
B
H
N
H
B
O
B
H
receptor
receptor
hydrolyzed back to the serine "OH"
© Oxford University Press, 2013
13
Hormones are signaling molecules produced by glands in multicellular organisms that are
transported by the circulatory system to target distant organs to regulate physiology and behaviour.
Hormones have diverse chemical structures, mainly of 3 classes: icosanoids, steroids, and amino
acid derivatives (amines, peptides, and proteins). The glands that secrete hormones comprise the
endocrine signaling system. The term hormone is sometimes extended to include chemicals
produced by cells that affect the same cell (autocrine or intracrine signalling) or nearby cells
(paracrine signalling).
T3
T4
14
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15
© Oxford University Press, 2013
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© Oxford University Press, 2013
H
H
H
H
cholesterol
HO
many steps,
plus sunshine
H
H
H
7-Dehydrocholesterol
HO
H
H
pre-vitamin D3
H
HO
H
H
vitamin D3
HO
© Oxford University Press, 2013
17
Hormone binding to receptor - insulin
(Insulin protein requires post translational modification)
1. Insulin binds to tyrosine
kinase-linked receptor.
2. Phosphorylation of receptor,
starts sequence
q
leading
g to
synthesis of glycogen,
storage of glucose (and fat
too)
3. Allows influx of gglucose
4. Insulin-like molecules are
even found in the simplest
unicellular eukaryotes, over
1 billion yyears old
5. Our insulin is very similar to
pig and cow insulin, was
used before our genes were
spliced
p
into ggenetically
y
modified bacteria
(glucose transport)
http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.cfm
© Oxford University Press, 2013
18
Icosanoids are signaling molecules made by oxidation of either 20-carbon omega-3 (-3) or omega6 (-6) fatty acids. In general, the -6 eicosanoids are pro-inflammatory; -3s are much less so.
There are multiple subfamilies of eicosanoids,
eicosanoids including the prostaglandins,
prostaglandins thromboxanes,
thromboxanes and
leukotrienes, as well as the lipoxins and eoxins, and others.
They exert complex control over many bodily systems; mainly in growth during and after physical
activity inflammation or immunity after the intake of toxic compounds and pathogens,
activity,
pathogens and as
messengers in the central nervous system. Many are classified as hormones. The networks of controls
that depend upon icosanoids are among the most complex in the human body.
The amounts and balance of fats in a person
person'ss diet will affect the body
body'ss icosanoid
icosanoid-controlled
controlled
functions, with effects on cardiovascular disease, triglycerides, blood pressure, and arthritis.
prostaglandins
O
thromboxanes
O
OH
HO
OH
leukotrienes
O
O
OH
OH
OH
O
OH
alprostadil - inhibits
blood clotting , vasodilator
O
thromboxane A2 - vasoconstrictor,
hypertensive,
yp
blood clotting,
g
aspirin inhibits its formation
lipoxin B4 - eoxin, proimfalmmatory (white blood cells),
mast cells,
cells contributes to allergies,
allergies
various cancers, Hodgkin's lymphoma
© Oxford University Press, 2013
19
Steroids are organic compounds with four rings arranged in a specific configuration.
Examples include the dietary lipid cholesterol and the sex hormones estradiol and
testosterone. Dexamethasone is an anti-inflammatoryy drug.
g Steroids have two
principal biological functions: certain steroids (such as cholesterol) are important
components of cell membranes which alter cell membrane fluidity, and many
steroids are signaling molecules which activate steroid hormone receptors and can
lead to DNA activation and protein synthesis.
synthesis
C
A
D
B
glucocorticoids (adrenal cortex)
mineralocorticoids (adrenal cortex)
estrogens (gonads)
androgens (gonads)
progestins (ovaries and placenta)
vitamin D (diet and sun)
...and more
Letters are used to identify the 4 rings.
20
© Oxford University Press, 2013
CH3
OH
H
H
H
H
HO
Estradiol is the primary female sex hormone. It is important in the regulation of the estrous and menstrual
female reproductive cycles. Estradiol is essential for the development and maintenance of female
reproductive tissues but it also has important effects in many other tissues including bone. While estrogen
l l in
levels
i men are lower
l
compared
d to
t women, estrogens
t
h
have
essential
ti l functions
f ti
i men as well.
in
ll It is
i 2 steps
t
away from progesterone.
CH3
OH
H
H
CH3
H
H
O
Testosterone is secreted primarily by the testicles of males and, to a lesser extent, the ovaries of females.
Small amounts are also secreted by the adrenal glands. In men, testosterone plays a key role in the
d l
development
off male
l reproductive
d i tissues
i
such
h as the
h testis
i and
d prostate as well
ll as promoting
i secondary
d
sexual characteristics such as increased muscle, bone mass, and the growth of body hair. Levels of
testosterone in adult men are about 7–8 times as great as in adult females. As the metabolic consumption of
testosterone in males is greater, the daily production is about 20 times greater in men than women.
21
© Oxford University Press, 2013
H
Common steroid transformations
while similar in appearance,
everyone of these steroids has a
specific signal to transmit, such as
turning on a genetic switch. They
are extremely powerful chemicals,
some operating at the nanomolar
level.
H3C
V22, p. 703, p 1263
H3C
CH3
CH3
H
lanosterol - the compound
from which all steroids are
derived.
CH3
O
HO
3
sp oxidation
CH3
C
H
H
CH3
CH3
CH3
P
O
O
P
isopentenyl
phosphate
dimethylallyl
phosphate
HO
O
S
CoA
H
H
17-hydroxyprogesterone - sometimes
given to prevent premature birth
OH
sp oxidation
O
CH3
H
progesterone - involved
in menstrual cycle and
pregnacy
sp3 oxidation
OH
O
CH2
C
H
NADH
CH3
OH
H
CH3
H
H
H
H
11-deoxycorticosterone
intermediate to aldosterone
O
O
11-deoxycortisol (cortodoxone)
glucocorticoid (similar to cortosol)
sp3 oxidation
CH3
HO
sp3 oxidation
C
CH2
H
CH3
H
H
H reduced C=C is
dihydrotestosterone
H
H
(maybe with FADH2)
O
cortisol - glucocorticoid, increases
(more
potent
by
3x)
blood sugar, decreases immune
O
testosterone - produced
system (hydrocortisone)
in all vertebrates, 20x more
in males, male sex characteristics.
three well known members of the estrogen family
OH
CH3
OH
see
CH3
CH3
reactions
below
H
H
H
O
5 categories of steroid hormones
O
1. progestins - overies and placenta- mediate
menstrual cycle and maintains pregnacy
2. glococorticoids - adrenal cortex - affect
H
H
metabolism in diverse ways,
ways decrease
H
OH
inflamation,incease resistance to stress
3. mineralocorticoids - adrenal cortex - maintain
H
H
H
H
salt and water balance
H
H
4. androgens - gonads - maturation and function
NAD+
HO
of secondary sex organs, particularly in males,
sp3 oxidation HO estrone - known femaleoxidation
-estradiol - prominant estrogen HO
O
male sexual differentiation
in reproductive years, more potent
5. estrogens - gonads - maturation and function of
estriol - higher levels in pregnancy, carcinogen, causes breast tenderness,
than
estriol
(10x)
and
estrone
(80x)
in men causes anorexia
secondary sex organs, particularly in females
marker of fetal health
CH2
C
CH3
HO
OH
OH
O
OH
O
CH2
C
CH3
H
H
H
CH3
acetyl CoA
sp3 oxidation
CH3
dehydroisoandrosterone
d
h d i
d t
- most abundant
b d
circulating steroid, many uses
CH3
O
H
CH3
H
O
H
many steps
H
CH3
C
CH3
O
H
H
O
O
CH3
C
OH
Statins drugs
work here.
O
NAD+ and
isomerization O
3
? steps
H
pregnenolone neuroactive steroid
HO
O
17-hydroxypregnenolone - higher levels are
found at end of puberty and in pregnacy.
O
CH3
H
HO
O
H
OH
NAD+ and
isomerization
H
squalene
H
H
O
OH
CH3
CH3
cholesterol - essential component
of cell walls, precursor of steroid
hormones, bile acids and vit D
O
squalene oxide
H
? steps
H
about 19 steps
CH3
H
CH3
CH3
C
CH3
H
CH3
H3C
O
CH3
H
HO
H
sp3 oxidation
and
NAD+ oxidation
corticosterone - stress
hormone, immune system
O
H
O
C
HO
OH
C
CH2
OH
CH3
H
H
H
aldosterone - Na+,K+
balance, blood pressure reg.
© Oxford University Press, 2013
22
Amino acid derivatives (or from amino acids) – thyroxine, histamine, catacholamines (epinephrine, norepinephrine)
oxytocin, cytokines, glutamate, glycine, gama aminobutyric acid, acetylcholine (neurotransmitters and hormones)
O
HO
O
O
HO
H2
N
HO
epinephrine
adrenaline
HO
HO
NH3
HO
norepinephrine
O
NH3
glutamate
I
O
O
HO
O
O
-aminobutyric acid
glycine
I
O
H3N
H3N
NH3
O
I
th
thyroxine
i (T4)
HO
N
NH3
HO
histamine
Leu
Pro
O
N
N
nitrosyl
radical
di l
(nitric oxide)
Cys
oxytocin - 8 amino acid peptide
I
O
NH3
HN
dopamine
Gly
O
S
O
acetylcholine
Tyr
S
Cys
Asn
Ile
© Oxford University Press, 2013
23
Chemical Messengers (deliver messages), Receptors (receive messages)
Neurotransmitter: A chemical released from the end of a neuron which travels across a synapse to
bind with a receptor
p
on a target
g cell,, such as a muscle cell or another neuron. Usually
y short lived
and responsible for messages between individual neurons.
Hormone: A chemical released from a cell or a gland and which travels some distance to bind with
receptors
p
on target
g cells throughout
g
the body
y
Chemical messengers ‘switch on’ receptors without undergoing a reaction (different from enzymes)
Nerve 1
Synaptic button =,
connections to neurons,
acetylcholine
l h li
receptors
Blood supply,
supply ee.g.
g
insulin – absorb glucose
Glucagon – release glucose
Hormone
Neuromuscular end plates =
connections to muscles,
adrenoline
response
Nerve 2
Cell
secondary
messenger
Neurotransmitters
© Oxford University Press, 2013
24
Structure and function of receptors
Mechanism
Induced fit
Messenger
Messenger
Messenger
Cell
Membrane
Receptor
Receptor
Cell
Cell
Receptor
Cell
message
Message
M
Receptors contain a binding site (hydrophobic hollow or cleft on the receptor protein) that is recognised
by the chemical messenger
Binding of the messenger involves intermolecular bonds with amino acids
ion - ion
H bonds
dipole - dipole
dispersion forces
Binding results in an induced fit of the receptor protein and the messenger (alters shape)
Change in receptor shape results in a ‘domino’ effect (no reaction/catalysis, different from enzyme)
Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the
cell (amplification of signal is common)
Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently
bound (could find another receptor)
© Oxford University Press, 2013
25
Binding interactions
• Ionic
• H-bonding
• van der Waals
Induced fit - Binding site alters shape to maximise intermolecular
bonding
Phe
Phe
O
O
H
Ser
Ser
CO2
Asp
Intermolecular bonds not optimum
length for maximum binding strength
Induced
Fit
H
CO2
Asp
Intermolecular bond lengths optimised,
change of shape causes a change in
function (on or off)
© Oxford University Press, 2013
26
Overall Process of Receptor/Messenger Interaction
M
M
M = messenger
R = receptor
M
RE
R
RE
Signal transduction
Binding
Bi
di interactions
i
i
must be
b strong enoughh to hold
h ld the
h messenger sufficiently
ffi i l long
l
for signal transduction to take place
Interactions must be weak enough to allow the messenger to depart
Implies a fine balance
Designing molecules with stronger binding interactions can result in drugs that
block the binding site = antagonists (agonists turn on the effect)
© Oxford University Press, 2013
27
Receptor Superfamilies
RESPONSE
TIME
•ION CHANNEL RECEPTORS
•G PROTEIN COUPLED RECEPTORS
•G-PROTEIN
•KINASE LINKED RECEPTORS
msecs
MEMBRANE
BOUND
seconds
d
minutes
•INTRACELLULAR RECEPTORS
28
© Oxford University Press, 2013
Ion Channel Receptors
General principles
• Receptor protein is part of an ion channel protein complex
• Receptor binds a messenger leading to an induced fit
• Ion channel is opened or closed
• Ion channels are specific for specific ions (Na+, Ca2+, Cl-, K+)
• Ions flow across cell membrane down concentration gradient
• Polarises or depolarises nerve membranes
• Activates or deactivates enzyme-catalysed reactions within cell
© Oxford University Press, 2013
29
Ion Channel Receptors - General principles
hydrophilic
tunnel
Cell
membrane
hydrophobic
exterior
MESSENGER
Extra cellular
Extra cellular
ION
CHANNEL
(closed)
Cell
membrane
Cell
Ion
channel
ION
CHANNEL
(
(open)
)
RECEPTOR
BINDING
SITE
Induced fit
and opening
of ion channel
Lock
Gate
Ion
channel
MESSENGER
Cell
membrane
Cell
membrane
Ion
channel
Ion
channel
Cell
membrane
Cell
© Oxford University Press, 2013
30
Ion Channel Receptors - Gating
Receptor
Binding site
Cell
membrane
Messenger
Induced
fit
Cell
membrane
‘Gating’
(ion channel
opens)
Five glycoprotein subunits
traversing cell membrane
Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory
Anionic ion channels for Cl- (e.g. GABAA) = inhibitory
31
© Oxford University Press, 2013
Ion Channel Receptors
Structure of protein subunits (4-TM
(4 TM receptor subunits)
Neurotransmitter binding region

Cell
membrane




one of
these
Cell
membrane
Extracellular loop
H2N
CO2H
TM1
Intracellular
loop
p
TM2
TM3
TM4
Variable loop
4 Transmembrane (TM) regions
(hydrophobic on the outside of channel)
© Oxford University Press, 2013
32
Ion Channel Receptors
p
- Detailed Structure of Ion Channel
Protein
subunits
TM4
TM1
TM2
TM3
TM1
TM2
TM2
TM4
TM3
TM3
TM1
TM3
TM4
TM4
TM2
TM2
TM1
TM1
TM3
TM4
Transmembrane
regions
Note: TM2 of each protein subunit ‘lines’ the central pore
(TM2 is more polar, perhaps bonded to very polar carbohydrates, TM1,
TM33 and
d TM4 aree more
o e hydrophobic
yd op ob c and
d face
ce towards
ow ds ce
cell membrane.)
e b
e.)
© Oxford University Press, 2013
33
Ion Channel Receptors - Gating
Cell
membrane
TM2
Ion flow
TM2
TM2
TM2
TM2
TM2
TM2
TM2
TM2
Transverse view
TM2
TM2
Transverse view
TM2
Closed
Open
• Chemical messenger binds to receptor binding site
• Induced fit results in further conformational changes
• TM2 segments rotate to open central pore
• Fast response measured in msec
• Ideal for transmission between nerves
• Binding of messenger leads directly to ion flows across cell membrane
• Ion flow can lead to secondary effects (signal transduction)
• Ion concentration within cell alters
• Leads to variation in cell chemistry
© Oxford University Press, 2013
34
Ion Channel Receptors – Nicotinic receptor
Binding
Bi
di
sites
Ion channel


Cell
membranee







Two ligand binding sites
mainly on 
-subunits
subunits
x subunits
H3C
O
N
H3C

O
CH3
H3C
CH3
N
CH3
H
acetylcholine
neurotransmitter
H3C
N
O
CH3
CH3
nicotine
protonated at bodyy p
p
pH
Muscarine
OH
Muscarinic agonists activate muscarinic receptors while nicotinic agonists activate nicotine receptors.
Both are direct-acting cholinomimetics that mimic acetyl choline and act on the CNS.
© Oxford University Press, 2013
35
Ion Channel Receptors
p
– Glycine
y
receptor
p
Binding
sites
Ion channel




Cell
membrane






x
xx 
subunits
subunits
O
H3N
O
glycine
Three ligand binding sites
on -subunits
The glycine receptor, or GlyR, is the receptor for the amino acid neurotransmitter
glycine. GlyR is an ionotropic receptor that produces its effects through chloride
current. It is one of the most widely distributed inhibitory receptors in the central
nervous system and has important roles in a variety of physiological processes,
especially in mediating inhibitory neurotransmission in the spinal cord and
brainstem.
© Oxford University Press, 2013
36
G-Protein Coupled Receptors – General principles
• Receptor binds a messenger leading to an induced fit
• Opens a binding site for a signal protein (G-protein)
• G-Protein binds,, is destabilised then split
p
 

G protein
messenger
iinduced
d d
fit
closed
open
G-protein split into
α and β/ subunits
37
© Oxford University Press, 2013
G-Protein Coupled
p
Receptors
p
– General p
principles
p
• G-Protein subunit activates membrane bound enzyme
• Binds to allosteric binding site
• Induced fit results in opening of active site
• Intracellular reaction catalysed (makes cyclic AMP, PIP2, etc.)
Enzyme
Enzyme
α
α
active site
(closed)
active site
(
(open)
)
IIntracellular
t
ll l
reaction
© Oxford University Press, 2013
38
G-Protein
G
P t i Coupled
C
l d Receptors
R
t
- Structure
St t
Single protein with 7 transmembrane regions (7-TM receptor)
Extracellular
loops
NH3
extracellular
Membrane
N -Terminal chain
VII
VI
V
IV
III
II
Transmembrane
helix
I
G-Protein
binding region
O 2C
C -Terminal chain
cytoplasm
Variable
intracellular loop
Intracellular loops
G protein splits into an  subunit and an / subunit.
39
© Oxford University Press, 2013
G-Protein Coupled Receptors – Ligand binding site
varies depending on receptor type
Ligand
A
B
C
D
A) Monoamines: pocket in TM helices
B) Peptide hormones: top of TM helices + extracellular loops + N-terminal chain
C) H
Hormones: extracellular
t
ll l loops
l
+ N-terminal
Nt
i l chain
h i
D) Glutamate: N-terminal chain
Example receptors - ligands
• Monoamines - e.g. dopamine, histamine, noradrenaline, acetylcholine
• Nucleotides - e.g. Adenine, guanine, cytosine, uracil, thymine
• Lipids - e.g. Cholesterol, fatty acid, triglyceride, phospholipids
• Hormones - thyroxine,
y
, testosterone,, estrogen,
g , oxytocin,
y
, epinephrine
p p
++
• Glutamate, DOPA, glycine, acetylcholine, Ca
© Oxford University Press, 2013
40
SIGNAL TRANSDUCTION
neurotransmitter or hormone
signal binds at receptor site
of 7 TM receptor
Overview
There are many different types of G
proteins with about 20 different
types of α subunit proteins.

 
G protein
 ,
,
  subunits

 
 subunit splits off
and binds to another
protein to start signal
cascade inside the cell
G protein-coupled
receptors (7 TM receptors)
imbedded in membranes
continuing
reactions
inside cell


protein
protein


various
actions
inside cell
© Oxford University Press, 2013
41
DRUG TARGETS SIGNAL TRANSDUCTION
Signal Transduction involving Gs-Proteins provide one step in signal
transduction pathway.
GS-Protein
- membrane bound protein of 3 subunits ()
- S subunit
b it h
has bi
binding
di site
it ffor GDP
-GDP bound non covalently
 

Interaction of receptor with Gs-protein
GDP
ß


GTP binds
Binding site recognises GTP
GTP = guanine triphosphate
ß


Fragmentation
and release

ß

IInduced
d d fit
G-protein alters shape
Complex destabilised
• 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 to make cyclic AMP
© Oxford University Press, 2013
42
Signal Transduction involving Gs-Proteins
Interaction of s with adenylate cyclase (inside cell)
GTP
GDP
s-subunit
b it
Adenylate cyclase
Binding site
for s subunit
GTP hydrolysed
to GDP catalysed
by s subunit
Binding
αs
Induced
fit
ATP
Active site
(closed)
αs
P
cyclic AMP
Active site
(open)
ATP
Active site
(closed)
Activation
Protein
Kinase A
P
Enzyme
(i
(inactive)
ti )
cyclic AMP
Enzyme
( ti )
(active)
Enzyme-catalysed
reaction
s Subunit changes shape
Weaker binding to enzyme
Departure of subunit
Enzyme reverts to inactive
state
s Subunit recombines with 
dimer to reform Gs protein
43
© Oxford University Press, 2013
Signal Transduction involving Gs-Proteins
Interaction of cyclic AMP with protein kinase A (PKA)
P t i kinase
Protein
ki
A - 4 protein
t i subunits
b it
- 2 regulatory subunits (R) and 2 catalytic subunits (C)
cAMP
C
catalytic subunit released
C
R
R
R
cAMP
binding
sites
R
C
• Cyclic AMP binds to PKA
• Induced fit destabilises complex
• Catalytic units released and activated
C
catalytic subunit released
C
P
Protein
+ ATP
Protein
+ ADP
Phosphorylation of other proteins and enzymes
Signal continued by phosphorylated proteins
Further signal amplification
© Oxford University Press, 2013
44
Signal Transduction involving Gs-Proteins
Glycogen Metabolism - triggered by adrenaline in liver cells
Ad
Adrenaline
li
signal
received
s
s
adenylate
y
cyclase
 Adrenoreceptor
-Adrenoreceptor
cAMP
Glycogen Protein kinase A
synthase
(active)
Catalytic
C subunit of
PKA
Glycogen
synthase-P
(inactive)
Phosphorylase
kinase (inactive)
Inhibitor
(inactive)
Phosphatase
(inhibited)
Inhibitor-P
(active)
Phosphorylase
kinase P (active)
Phosphorylase b
(inactive)
Phosphorylase b
(active)
Glycogen
Glucose-1-phosphate
© Oxford University Press, 2013
45
Signal Transduction involving Gs-Proteins
Interaction of s with adenylate cyclase converts ATP to cAMP
NH2
ATP
adenosine triphosphate
B
P
O
N
H
O
O
O
NH2
N
O
P
O
N
O
O
P
O
O
H
O
H
O
H
H
H
B
N
N
cyclic AMP
N
O
enzyme
control
H
B
O
P
O
O
O
P
H
O
N
O
O
P
O
O
O
H
H
H
diphosphate
(pyrophosphate)
N
O
H
H
H
B
• 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 (possible further amplification)
© Oxford University Press, 2013
46
Signal Transduction involving Gs-Proteins
Glycogen Metabolism - triggered by adrenaline in liver cells
Coordinated effect: activation of glycogen metabolism ( glucose  glycolysis)  energy)
inhibition of glycogen synthesis (no gluconeogenesis)
Adrenaline has different effects on different cells
e.g. activates fat metabolism in fat cells ( acetyl CoA  glucose = slower)
Drugs interacting with cyclic AMP signal transduction
Cholera toxin causes constant activation of cyclic AMP leading to diahorrea
Theophylline and caffeine
-inhibit phosphodiesterases
-phosphodiesterases responsible for metabolizing cyclic AMP
-cyclic AMP activity is prolonged (keeps you wired)
O
H3C
O
N
N
O
H
N
H3C
N
O
CH3
N
N
N
CH3
CH3
Theophylline
Caffeine
N
© Oxford University Press, 2013
47
Signal Transduction involving Gs-Proteins
Interaction of cyclic AMP with protein kinase A (PKA)
Protein kinase A is a serine-threonine kinase
Activated by cyclic AMP
Catalyses phosphorylation of serine and threonine residues on protein
substrates
Phosphate unit provided by ATP
O
O
H
N
Protein
N
H
O
B
Protein
H
N
O
O
P
Protein
O
ATP
N
H
Protein
kinase A
ATP
ADP
O
O
O
H
Protein
O
P
ADP
O
O
serine or threonine
amino acid
phosphorylated serine
or threonine residue
Protein kinases modify other proteins by chemically adding phosphate groups to them
(phosphorylation). This usually results in a functional change of the target protein. The human genome
contains about 500 protein kinase genes and they constitute about 2% of all human genes.
genes Up to 30%
of all human proteins may be modified by kinase activity, and kinases are known to regulate the
majority of cellular pathways, especially those involved in signal transduction.
© Oxford University Press, 2013
48
G-Protein Coupled Receptors – bacteriorhodopsin / rhodopsin family
• Rhodopsin = visual receptor
• Many common receptors belong to this same family
(isozymes)
• Implications for drug selectivity depending on receptor
similarity – consequence of evolution
• Membrane bound receptors difficult to crystallise
• X-Ray
X Ray structure of bacteriorhodopsin solved - bacterial
protein similar to rhodopsin
• Bacteriorhodopsin structure used as ‘template’ for other
receptors
t
• Construct model receptors based on template and amino
acid sequence
• Leads
L d tto model
d l bi
binding
di sites
it for
f d
drug d
design
i
• Crystal structures for rhodopsin and 2-adrenergic
receptors now solved - better templates
© Oxford University Press, 2013
49
node
Ethene has a pi bond. Pi bonds are second
and third bonds and are generally weaker.
MO diagram of a pi bond.
higher,
higher
less stable
2pa
potential
energy
C
CC = 2pa - 2pb = antibonding MO
C
2 b
2p
LUMO
pi-star
antibond
tib d
C
C
lower,
more stable
HOMO
CC = 2pa + 2pb = bonding MO
C
pi bond
C
LUMO = lowest unoccupied molecular orbital
HOMO = highest occupied molecular orbital
four C empty, but available to accept electrons
Ethene MO diagram
H
CC = sp2a - sp2b = antibonding MO
H
C
LUMO
C
H
CC = p2a - p2b = antibonding MO
energy of starting
atomic orbitals
H
HOMO
CC = p2a + p2b = bonding MO
 63 kcal/mol
 146 kcal/mol
2C = 8 e's
4H = 4 e's
total bonding e's = 12 e's
CC = sp2a + sp2b = bonding MO  83 kcal/mol
four C full, with two electron each
bond order =
(12) - (0) = 6 bonds
2
© Oxford University Press, 2013
50
buta-1,3-diene - conjugated pi bonds
H
H
H
C
C
C
H
C
H
More nodes is less stable = no
electron density between adjacent atoms.
4 C
4 = 1 - 2
H
node
node node node
C
C
node
node
node
higher,
less stable
C
C
C
C
2
LUMO
1
LUMO
C
3 C
C
C
C
3 = 1 + 2 = LUMO
E is
larger
potential
energy
E is smaller
so  is longer
node
2 = 1 - 2 = HOMO
2 C
llower,
more stable
C
C
2
1
HOMO
C
C
C
C
C
C
HOMO
1
Energy gap between HOMO and
LUMO orbitals in pi bond versus diene.
E = h = hc(1/)
h = Plank's constant = 6.62x10-34 j-sec
c = 3.0x108 m/sec
C
C
C
1 = 1 + 2
These are
constants.
 = frequency (#/sec = Hz)
 = wave length (nm = 10-9m)
These are variables that indicate
the energy being measured. Only
one of them is needed to specify
© Oxford
the
energy.University Press, 2013
51
The HOMO/LUMO energy gap can be measured with light (frequency =  or wave length = )
 systems
H2C
max (wave length measure of HOMO/LUMO energy gap
CH2
174 nm
217 nm
*
250 nm
larger E (HOMO/LUMO energy gap)
smaller , indicates
a larger E
*
280 nm
E = (hc) 1

*
310 nm
*
340 nm
*
370 nm
smaller E (HOMO/LUMO energy gap)
*
400 nm
* = estimated value
1 2 3 4 5 6 7 8
100 nm
200 nm
300 nm
ultraviolet light
400 nm
V
I
larger , indicates
a smaller E
500 nm
B
G
600 nm
Y
700 nm
O
R
800 nm
900 nm
infrared light
visible light
IR is lower energy electromagnetic
UV is higher energy electromagnetic
Increasing
Energy
radiation that we sense as heat
radiation that can damage chemical bonds
-carrotene (this is the most common structure given, but there are hundreds of variations known in nature)
color (?) =
lycopene (found in tomatoes and other vegetables, also has hundreds of variations known in nature)
color (?) =
© Oxford University Press, 2013
52
higher energy

LUMO
E
transition
in UV
E

100 nm
200 nm
300 nm
Ultra Violet
lower energy
Increasing Energy
400 nm
I
V
500 nm 600 nm
B
G
Y
700 nm
O
R
800 nm
900 nm
Infrared
eyes detect electromagnetic
radiation (light) in this region
transitions in visible
-carrotene
HOMO
oxidation
O
hydration
OH
oxidation
id ti
OH
rhodopsin
OH
recycles through vitamin A via
1. hydrolysis
2. reduction
R HOD
OP
oxidation
vitamin A
H
O
SI
N
isomerization 11-trans-retinal
OP
Binding is weak when
trans configuration.
SI
N
protein
11-cis-retinal
H
N
H
Absorption of photon (h) causes
isomerization of  system to the
more stable trans configuration.
The release of retinal causes polarization of the cell
membrane, ion channels open up and Na+ rushes in and
K+ rushes out and an impulse travels to the synapse
where neurotransmitters are released and complex at
receptors of the next cell (neuron) which causes that
cell to polarize and continue the signal to the visual part
of your brain where it constructs an image that you see.
binds to
opsin protein
R HO D
OP
SI
N
Binding is strong
H
when cis configuration.
H
N
H
O
Aldehyde and 1o amine
bind together in an imine
linkage with loss of water.
H 3N
Rhodopsin
p absorbs visible light
g ((depends
p
on the protein charge and twist in
conformation), and uses the energy to break
the cis pi bond, isomerizing cis to trans,
which causes a release of 11-trans-retinal
from the protein and starts a cascade of
reactions (cell polarization) leading to
"vision".
© Oxford University Press, 2013
53
How does vision work?
Slightly different protein environments, changes HOMO/LUMO gap and what part of visible region gets absorbed. Your genetics determines your protein
structure and the colors that you see. Different variations of protein complexes (twisting and charge distribution) absorb different wavelengths of light.
E = h  = h c (1 / )
What would happen if one
of these was defective?
E = h
protein
A
V
I
 = 400 nm
protein
C
protein
B
B
G
R = red
O = orange
Y = yellow
G = green
B = blue
I = indigo
V = violet
C
B
A
Y
O
R
 = 700 nm
complementary
color wheel
violet
red
dk blue
orange
lt blue
yellow
green yel-grn
Rhodopsin complex imbedded in cell membrane has retinal bound
perpendicular to 7 transmembrane protein helicies. Isomerization
triggers cell action potential (polarization) and Na
Na+ flows in and K
K+
flows out (1 millisec). In some cells this is followed by Ca+2 ions
efflux (100 millisec).
y p , neurotransmitters are released from vesicles,,
At a synapse,
triggering the next neuron to fire and tranmit the signal to
the appropriate parts of the brain
cell membrane
cell membrane (lipid bilayer)
O
AC = acetylcholine used
AC
in peripheral and
AC AC
centrall nervous systems
ion channels
N
Na+
O
vesicles with neurotransmitters
fuse with cell wall and release
1000s of molecules = exocytosis
Rhodopsin
complex
O
GLU = glutamate / glutamic acid
GLU
important neurotranmitter
GLU
in the brain and spinal cord
GLU
O
NH3
C
O
O
HO2C
NH3
DPA = dopamine used in several HO
parts of the brain, cocaine and
methamphetamine act on dopamine
receptors, Parkinson's is caused
DPA by loss of dopamine secretion HO
DPA
DPA
cell membrane
NH3
K+
Ca+2
ion channels
© Oxford University Press, 2013
54
G-Protein Coupled Receptors – bacteriorhodopsin / rhodopsin family
Common ancestor
divergent evolution
Monoamines
muscarinic
Endothelins
Bradykinin
Angiotensin
Interleukin-8
2
Opsins,
Rhodopsins
beta
alpha
Tachkinins
Receptor
types
4
5
3
Muscarinic
1
H1
H2
Histamine
1 2A 2B 2C
D4 D3 D2
-Adrenergic
convergent evolution
D1A
D1B D5
Dopaminergic
3
2
1
Receptor
subtypes
-Adrenergic
convergent evolution
55
© Oxford University Press, 2013
G-Protein Coupled Receptors – Receptor types and subtypes
Reflects differences in receptors which recognise the same ligand
Receptor
OH
Types
H
HO
N
CH3
Adrenergic
adrenoline
HO
O
Subtypes
1, 2A, 2B, 2C
Alpha
Al
h (())
Beta ()
1, 2, 3
CH3
O
acetylcholine
N
CH3
CH3
Cholenergic
Nicotinic
Muscarinic

•Receptor types and subtypes are not equally distributed
amongst tissues
• Target selectivity leads to tissue selectivity
Heart muscle
Fat cells
Bronchial muscle
GI-tract
1 adrenergic receptors
3 adrenergic receptors
1& 2 adrenergic receptors
1 2 & 2 adrenergic receptors
© Oxford University Press, 2013
56
Signal Transduction involving Gi-Proteins (i = inhibition)
• Bind to different receptors from those used by Gs proteins
• Mechanism of activation is identical
• I subunit binds to adenylate cyclase and inhibits it
• Adenylate cyclase is under dual control (brake/accelerator)
• Background activity due to constant levels of s and i
• Overall effect depends on dominant alpha subunit (s or i)
• Dominant alpha subunit depends on receptors activated
© Oxford University Press, 2013
57
Phosphorylation Reactions
• Prevalent in activation and deactivation of enzymes
• Phosphorylation radically alters intramolecular binding
• Results
R lt in
i altered
lt d conformations
f
ti
NH3
NH3
NH3
might turn on,
might turn off
O
O
H
O
O
Active site
closed
P
O
O
O
O
O
P
O
O
O
Active site
open
© Oxford University Press, 2013
58
Phosphorylation Reactions
• Prevalent in activation and deactivation of enzymes
• Phosphorylation radically alters intramolecular binding
• Results
R lt in
i altered
lt d conformations
f
ti
CO2
OH
CO2
CO2
-2
PO3
might turn on,
might turn off
O
O
P
O
O
Active site
open
Active site
open
Active site
closed
© Oxford University Press, 2013
59
Signal Transduction involving Gq Proteins
Interaction with phospholipase C (PLC)
• Gq proteins - interact with different receptors from those recognised by GS and GI
• Split by the same mechanism to give an q subunit
• The q subunit activates or deactivates PLC (membrane bound enzyme)
• Reaction catalysed for as long as q bound - signal amplification
• Brake and accelerator effect
Active site
(open)
Active site
(closed)


PLC

PLC
DG
PLC
PIP2
IP3
GTP hydrolysis

DG
PLC
Phosphate
Binding
weakened
PIP2
IP3
Active site
(closed)
q departs

PLC
Enzyme
deactivated
© Oxford University Press, 2013
60
Signal Transduction involving Gq Proteins
Interaction with phospholipase C (PLC)
O
R
R
C
C
O
H
O
H2 C
B
O
C
H
O
P
O
O
P
O
OH
PLC
OH
phospholipase
p
p
p
C
O
PIP2
phosphatidylinositol diphosphate
O
OH
O
OH
B
H
P
O
P
O
O
O
HO
H
H2
C
P
Phosphatidylinositol diphosphate
(integral part of cell membrane)
R= long chain hydrocarbons
P
O
O
R
R
C
C
O
O
H2C
C
H
O
OH
CH2
P
IP3
inositol triphosphate
DG
diacylglycerol
Inositol triphosphate
(polar and moves
into cell cytoplasm)
Diacylglycerol
(remains in
membrane)
= PO32© Oxford University Press, 2013
61
Signal Transduction involving Gq Proteins
Action of diacylglycerol
• Activates protein kinase C (PKC)
• PKC moves from cytoplasm to membrane
• Phosphorylates
Ph h l t S
Ser & Th
Thr residues
id
off protein
t i substrates
bt t
• Activates enzymes to catalyse intracellular reactions
• Linked to inflammation, tumour propagation, smooth muscle activity etc
Cell membrane
DG
Binding
site for DG
DG
DG
PKC
Active site
closed
PKC
Cytoplasm
PKC moves
to membrane
Cytoplasm
DG binds to
DG bindingg site
PKC
Enzyme
(inactive)
Enzyme
(active)
Enzyme-catalysed
Enzyme
catalysed
reaction
Cytoplasm
Induced fit
p
active site
opens
© Oxford University Press, 2013
62
Signal Transduction involving Gq Proteins
Resynthesis of PIP2 – phosphatidylinositol diphosphate
IP3
phosphatase
IP2 phosphatase IP phosphatase inositol PI synthase PI PI-4-kinase
PI-4-P-5-kinase PIP
2
PIP3
inhibition
Li salts for
manic depressive illness
O
O
P
O
HO
O
O
H
O
OH
H
H
HO
O
O
O
O
O
R
O
P
O
OH
O
O
O
O
H
PIP2
O
P
HO
H
R
O
O
P
HO
R
O H
HO H HO
IP3
O
O
H
H HO
H
O
OH
O
HO
H
H
O
O
P
OH
DG = diacylglycerol
O
P
O
H
O
H
HO
inositol
R
O
O
O
63
© Oxford University Press, 2013
Signal Transduction involving Gq Proteins
Action of diacylglycerol
Drugs inhibiting Protein Kinase C (PKC) - potential anti cancer agents
O
Me
Me
H
O
O
O
H
Me
HO
OH
Me
O
O
HO
O
Me
H
O
H
O
O
H
O
O
Me
OH
H
Me
B
Bryostatin
t ti (from
(f
sea moss))
Text, Fig 21.70 p. 566
64
© Oxford University Press, 2013
Signal Transduction involving Gq Proteins
Action of inositol triphosphate
•IP3 is hydrophilic and enters the cell cytoplasm
• Mobilizes Ca2+ release in cells by opening Ca2+ ion channels
• Ca2+ activates protein kinases
Calmodulin = calcium modulated protein,
• Protein kinases activate intracellular enzymes
interacts with kinases and phosphatases
• Cell chemistry is altered leading to a biological effect
Cell membrane
IP3
Cytoplasm
Calmodulin
Calcium
stores
Ca++
Calmodulin
Activation
Protein
kinase
Enzyme
(inactive)
Ca++
Activation
P
Enzyme
(active)
Enzyme-catalysed
Enzymereaction
Protein
kinase
Enzyme
(inactive)
inactive)
P
Enzyme
(active)
Enzyme-catalysed
Enzymereaction
© Oxford
University Press, 2013
Tyrosine Kinase Linked Receptors
•Bifunctional receptor / enzyme
• Activated
A i
db
by h
hormones
•Protein serves dual role - receptor plus enzyme
• Receptor binds messenger leading to an induced fit
• Protein changes shape and opens intracellular active site
• Reaction catalysed within cell
• Overexpression related to several cancers
messenger
messenger
iinduced
d d
fit
closed
active site
open
intracellular reaction
closed
66
© Oxford University Press, 2013
Tyrosine Kinase Linked Receptors - Structure
Extracellular
N-terminal
chain
Ligand binding region
NH3
H d hili
Hydrophilic
transmembrane
region (
(-helix)
Cell membrane
Catalytic binding region
(closed in resting state)
Intracellular
C-terminal
chain in cytosol
C O2
Tyrosine kinase
67
© Oxford University Press, 2013
Tyrosine Kinase Linked Receptors – reaction catalyzed by tyrosine kinase
O
O
H
N
Protein
N
H
Protein
O
O
P
O
ATP
Tyrosine
kinase
Mg+2
H
N
Protein
N
H
Protein
O
O
O
Tyrosine
amino acid
H
B
ATP
ADP
O
Phosphorylated
tyrosine residue
O
P
O
O
68
© Oxford University Press, 2013
ADP
Tyrosine Kinase Linked Receptors – Epidermal growth factor receptor (EGF- R)
EGF
Ligand binding
& dimerisation
Cell
membrane
Phosphorylation
OH
HO
OH
Inactive EGF-R
monomers
OH
OP
PO
ATP
ADP
OP
OP
Induced fit
opens tyrosine kinase
active sites
Binding site for EGF
EGF - protein hormone - bivalent ligand
Active site of tyrosine kinase
Active site on one half of dimer catalyses phosphorylation of Tyr residues on other half
• Dimerisation of receptor is crucial
• Phosphorylated regions act as binding sites for further proteins and enzymes
• Results
l in
i activation
i i off signalling
i lli proteins
i andd enzymes
• Message carried into cell
© Oxford University Press, 2013
69
Tyrosine Kinase Linked Receptors Insulin receptor (tetrameric complex)
Insulin
Phosphorylation
p y
Cell
membrane
OH ATP
HO
OH
Insulin binding site
Kinase active site
OH
ADP
PO
OP
OP
OP
Kinase active site
opened by induced fit
70
© Oxford University Press, 2013
Tyrosine Kinase Linked Receptors - Growth hormone receptor
Tetrameric complex constructed in presence of growth hormone
GH
GH binding
&
dimerisation
Binding
of kinases
Activation and
phosphorylation
ATP
GH receptors
(no kinase activity)
HO
Kinases
OH
OH
HO
OH
OH
OH
OH
ADP
PO
OP
OP
Kinase active site
opened by induced fit
Growth hormone binding site
Kinase active site
71
© Oxford University Press, 2013
OP
Signal Transduction - Tyrosine Kinase Linked Receptors
Si
Signalling
lli pathways
th
1-TM Receptors
Tyrosine kinase
inherent or associated
Guanylate cyclase
cGMP
Signalling proteins
PLC
IP3
DG
IP3
kinase
PIP3
GAP
Grb2
O
Others
N
cyclic GMP
N
O
O
P
O
NH2
H
H
O
N
O
H
Ca++ PKC
H
N
H
H
© Oxford University Press, 2013
72
Signal Transduction - Tyrosine Kinase Linked Receptors
Example
p of a signalling
g
gp
pathway
y
Growth
factor
1) Binding of
growth factor
Dimerisation
Phosphorylation
2) Conformational
change
HO
HO
HO
HO
OH
OH
HO
OH
OH
HO
PO
OP
PO PO PO
HO
OH
OH
OH OH OH
PO
OP
OP
OH
Binding
Ras and
Grb2
Binding and
phosphorylation
of Grb2
OP
PO
OP
PO PO PO
PO
Grb2
OP
OP
GTP/GDP
exchange
OP
PO
PO
PO
OP
PO PO
OP
OP
Ras
GDP
GTP
involved in cell growth,
diff
differentiation
ti ti andd survival
i l
© Oxford University Press, 2013
73
Signal Transduction - Tyrosine Kinase Linked Receptors
Example
p of a signalling
g
gp
pathway
y
Growth
factor
1) Binding of
growth factor
Dimerisation
Phosphorylation
2) Conformational
change
HO
HO
HO
HO
OH
OH
HO
OH
OH
HO
PO
OP
PO PO PO
HO
OH
OH
OH OH OH
PO
OP
OP
OH
Binding
Ras and
Grb2
Binding and
phosphorylation
of Grb2
OP
PO
OP
PO PO PO
PO
Grb2
OP
OP
GTP/GDP
exchange
OP
PO
PO
PO
OP
PO PO
OP
OP
Ras
GDP
GTP
involved in cell growth,
diff
differentiation
ti ti andd survival
i l
© Oxford University Press, 2013
74
Signal Transduction - Tyrosine Kinase Linked Receptors
Example of a signalling pathway
Ras
OP
PO
OP
PO PO PO
PO
OP
Gene transcription
OP
Raf (inactive)
Raf (active)
Mek (inactive)
Mek (active)
Map kinase (inactive)
Map kinase (active)
Transcription
factor (inactive)
Transcription
factor (active)
© Oxford University Press, 2013
75
Intracellular Receptors - Structure
• Chemical messengers must cross cell membrane
• Chemical messengers must be hydrophobic
• Example - steroids and steroid receptors
CO2
Steroid
binding region
Zinc
DNA binding region
(‘zinc fingers’)
H3N
Zinc fi
Zi
fingers contain
t i C
Cys residues
id
(SH)
Allow S-Zn interactions with proteins that bind to DNA and RNA
© Oxford University Press, 2013
76
Intracellular Receptors – Mechanism
Co-activator
protein
Receptor
DNA
Messenger
Receptor-ligand
complex
Cell
membrane
1.
2.
3.
4.
5.
6.
7
7.
Dimerisation
Messenger crosses membrane
Binds to receptor
Receptor
p dimerisation
Binds co-activator protein
Complex binds to DNA
Transcription switched on or off
P t i synthesis
Protein
th i activated
ti t d or inhibited
i hibit d
© Oxford University Press, 2013
77
Intracellular Receptors – Estrogen receptor
Binding
site
AF-2
g
regions
H12
Coactivator
Coactivator
Estradiol
DNA
Estrogen
receptor
Dimerisation &
exposure of
AF-2 regions
CH3
Nuclear
transcription
factor
Transcription
OH
CH3
H
H
CH3
H
H
H
OH
How?
H
H
H
HO
O
Testosterone
Estradiol / Estrogen
© Oxford University Press, 2013
78
The oxidation of testosterone to make estradiol shows yet another way of
losing a methyl group through a conjugated C=C bond.
Methyl is removed
and an aromatic ring
is formed.
formed
OH
CH3
CH3
CH3
H
H
H
H
H
testosterone
a male sex hormone
O
H2C
O
H
C
N
+3
Fe
+4
O
O
B
H
H
H
O
O
R
B
H
H
H
O
H
N
O
NADH
H
R
R
N
R
NAD+
oxidation
O
O
B
O
H
H
O
C
O
N
B
FADH2
H
H
H
O
B
H
O
O
H
B
N
R
H
dehydrogenation
B
H
d
decarboxylation
b l ti
R
tautomers
O
R
C
FAD
H
B
R
H
N
N
H
R
H
C
C
NAD+
oxidation
O
R
Fe
B
H
R
CH2
O
R
sp3 C-H oxidation
O
H
CH2
Fe +4
H
-estradiol
a female sex hormone
HO
R
B
H
HO
H
O
OH
H
O
© Oxford University Press, 2013
79
Overview of signal transduction pathways
80
© Oxford University Press, 2013
Signal transduction occurs when an extracellular signaling molecule activates a specific receptor
located on the cell surface or inside the cell. That receptor triggers a biochemical chain of events
inside the cell. Depending on the cell, the response alters the cell's metabolism, shape, gene
expression ability to divide and more.
expression,
more The signal can be amplified at any step.
step
G protein-coupled receptors integral transmembrane proteins that possess seven transmembrane domains
and are linked to a heterotrimeric G protein. (includes adrenergic receptors and chemokine receptors.)
Tyrosine kinase receptors are transmembrane proteins with an intracellular kinase domain and an
extracellular domain that binds ligands; (includes growth factor receptors such as the insulin receptor)
Integrins play a role in cell attachment to other cells and the extracellular matrix and in the transduction
of signals from extracellular matrix components such as fibronectin and collagen.
Toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a
signal. Thousands of genes are activated by TLR signaling, implying that this method constitutes an
important gateway for gene modulation.
A ligand-gated ion channel, upon binding with a ligand, changes conformation to open a channel in the
cell membrane through which ions relaying signals can pass. An example of this mechanism is found in
the receiving cell of a neural synapse.
Intracellular receptors,
receptors such as nuclear receptors and cytoplasmic receptors,
receptors are soluble proteins
localized within their respective areas. The typical ligands for nuclear receptors are non-polar hormones
like the steroid hormones testosterone and progesterone and derivatives of vitamins A and D. To initiate
signal transduction, the ligand must pass through the plasma membrane by passive diffusion. On binding
with the receptor, the ligands pass through the nuclear membrane into the nucleus, altering gene
expression.
i
N l i receptors
Nucleic
t have
h
DNA bi di domains
DNA-binding
d
i containing
t i i zinc
i fingers
fi
and
d a ligand-binding
li d bi di
domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone.
Second messengers: Ca+2, lipophilics (diacylglycerol), nitric oxide and other redox signaling molecules 81
© Oxford University Press, 2013