Cell signalling

CELL SIGNALLINGCELL SIGNALLING
BASIC MECHANISM
REGULATION OF SIGNAL
PATHWAYS
Jan 18, 2017 1

• All cells receive and respond to signals from their
environment
• The behaviour of cell signalling is accomplished by a
variety of signalling molecules that are either
secreted or expressed on the cell surface and bind
to receptors expressed by other cells.
• The bonding between signalling molecules and
receptors initiates a series of intracellular reactions
that regulate virtually all aspects of cellular
behaviour (metabolism, movement, proliferation,
survival and differentiation).
Jan 18, 2017 2

SIGNAL MOLECULES AND THEIRSIGNAL MOLECULES AND THEIR
RECEPTORSRECEPTORS
Jan 18, 2017 3

• Many different kinds of molecules transmit
information between the cells.
• Although all these molecules act as ligands that
bind to the receptors expressed by their target
cells, there is a considerable variation in the
structure and function of the different types of
molecules that serve signal transmitters.
• Some of these molecules carry signals over long
distances whereas others act locally.
• Some signalling molecules are able to cross the
plasma membrane and bind to intracellular
receptors in the cytoplasm or nucleus.
Jan 18, 2017 4

MODES OF CELL-CELL SIGNALLING
Jan 18, 2017 5

• Cell signalling can result either from the direct
interaction of a cell with its neighbour or from the
action of secreted signalling molecules.
• Signalling by direct Cell-Cell interactions play a
critical role in regulating the behaviour of cells in
animal tissues- in the embryonic development as
well as in the maintenance of adult tissues.
• The varieties of signalling are frequently divided
into three general categories based on the distance
over which the signals are transmitted.
– A) Endocrine Signalling
– B) Paracrine Signalling
– C) Autocrine SignallingJan 18, 2017 6

• In endocrine signalling, the signalling molecules
(hormones) are secreted by specialised endocrine
cells and carried to the target cells through
circulation. A classical example is provided by the
steroid hormone estrogen, which is produced by
the ovary and stimulates development and
maintenance of the female reproductive system,
secondary sexual characters. In animals more than
50 different hormones are produced by endocrine
glands.
• In contrast, some signalling molecules act locally to
affect the behaviour of nearby cells. In paracrine
signalling, molecules released by one cell……………..Jan 18, 2017 8

• ……..acts on neighbouring target cells. An example
is provided by the action of neurotransmitters in
carrying signal between the nerve cells at a
synapse.
• Finally, some cells respond to signalling molecules
that are produced by themselves. One important
example of such autocrine signalling is the response
of cells of vertebrate immune system to foreign Ag.
Certain types of T cells respond to antigenic
stimulation by synthesising a growth factor that
drives their own proliferation, thereby increasing
the number of responsive T cells and amplifying
immune response. It is also noteworthy that………..Jan 18, 2017 9

• …….abnormal autocrine signalling frequently
contributes to the uncontrolled growth of
cancerous cells. In this situation a cancer produces
a growth factor to which it also responds, thereby
continuous driving its own unregulated
proliferation.
Jan 18, 2017 10

STEROID HORMONES AND NUCLEARSTEROID HORMONES AND NUCLEAR
RECEPTOR SUPERFAMILYRECEPTOR SUPERFAMILY
Jan 18, 2017 11

• In many cases, receptors are expressed on the
target cell surface but some receptors are
intracellular proteins located in the cytosol or in the
nucleus.
• These intracellular receptors respond to small
hydrophobic signalling molecules that are able to
diffuse across the plasma membrane.
• The steroid hormones are the classical examples of
this group of signalling molecules which also include
thyroid hormones, Vitamin D3 and retinoic acid.
Jan 18, 2017 12

• The steroid hormones (testosterone, estrogen,
progesterone, corticosteroids and ecdysone) are all
synthesised from cholesterol.
• The testosterone, progesterone and estrogen are
sex steroids, which are produced by gonads. The
corticosteroids are produced by adrenal cortex-they
include glucocorticoids, mineralocorticoids etc. The
ecdysone is an insect hormone that play a key role
in development by triggering the metamorphosis of
larvae to adults.
• Although thyroid hormone, vitamin D3 and retinoic
acid are structurally distinct from steroids, they
share a common mechanism of action in their
target cells.
Jan 18, 2017 13

• Thyroid hormone (thyroxin) is synthesised in the
thyroid gland; it plays important role in
development and regulation of metabolism.
• Vitamin D3 regulates calcium metabolism and bone
growth .
• Retinoic acid and related compounds synthesised
from vitamin A plays an important role in
vertebrate development.
Jan 18, 2017 14

• Because of their hydrophobic character, the steroid
hormones, thyroid hormone, vitamin D3 and
retinoic acid are all able to enter cells by diffusing
across the plasma membrane. Once inside, they
bind to intracellular receptors. These receptors
which are members of a family of proteins known
as the Nuclear Receptor Superfamily, are
transcription factors that contain related domains
for (a)ligand binding, (b)DNA binding and
(c)transcriptional activation.
• Ligand binding regulates functions as Activators or
Repressors of their target genes, so the steroid
hormones and related compounds directly regulate
gene expression.
Jan 18, 2017 15

• Ligand binding has distinct effect on different
receptors.
• Some members of the steroid receptor superfamily
are unable to bind DNA in the absence of hormone.
Estrogen receptors, for example, bind to the Hsp 90
chaperones in the absence of hormone. The binding
of estrogen induces a conformational change in the
receptor, displacing Hsp 90 and leading to the
formation of receptor dimers that bind to
regulatory DNA sequences and activate
transcription of target genes
Jan 18, 2017 16

• In other cases, the thyroid hormone receptor for
example, is associated with a Corepressor Complex
and represses transcription of its target genes.
Hormone binding induces a conformationational
change that results in the interaction of the
receptor with Coactivators rather than
Corepressors, leading to transcriptional activation
of thyroid hormone-inducible genes.
Jan 18, 2017 18

NITRIC OXIDE AND CARBONNITRIC OXIDE AND CARBON
MONOXIDEMONOXIDE
Jan 18, 2017 19

• The simple gas nitric oxide (NO) is a major paracrine
signalling molecule in the nervous, immune and
circulatory systems.
• Like steroid hormones, NO is able to diffuse directly
across the plasma membrane of its target cells. The
molecular action of NO is however, distinct from
that of steroid hormones- rather than binding to
the receptors that regulate transcription, NO alters
the activity of intracellular target enzymes.
• NO is synthesised from amino acid arginine by the
enzyme nitric oxide synthase. Once synthesised, NO
diffuses out of the cell and can act locally to affect
nearby cell.Jan 18, 2017 20

• Its action is restricted to such local action because
NO is extremely unstable with a half life of only few
seconds.
• The major intracellular target of NO is guanylyl
cyclase. NO binds to a haeme group of active site of
this enzyme stimulating synthesis of second
messenger cyclic GMP. In addition, NO may directly
modify some target proteins by nitrosylation of
cysteine residue.
Jan 18, 2017 21

• A well-characterised example of NO action is signalling
the dilation of blood vessels. The first step in this
process is the release of neurotransmitters, such as
acetylcholine, from the termini of nerve cells in the
blood vessel wall. These neurotransmitters act on
endothelial cells to stimulate NO synthesis. NO then
diffuses to neighbouring smooth muscle cells where it
activates guanylyl cyclase resulting in the synthesis of
cGMP, which induces muscle cell relaxation and blood
vessel dialatation.
• It is also interesting to note that the medical use of
nitroglycerine in treatment of heart disease is based on
its conversion to NO, which dilates coronary blood
vessels and increases blood flow to the heart.Jan 18, 2017 23

• Another simple gas carbon monoxide (CO) also
functions as signalling molecule in the nervous
system.
• CO is closely related to and appears to act similarly
as a neurotransmitter and mediator of blood vessel
dilation. The synthesis of CO in brain cells, like that
of NO is stimulated by neurotransmitters. In
addition, CO can stimulate guanylyl cyclase, which
may also represent the major physiological target of
CO signal.
Jan 18, 2017 24

Nitric oxide and Carbon Monoxide
NO, a simple gas, is able to diffuse across the membrane, and alters the
activity of intracellular target enzymes. It’s extremely unstable, so its effects are
local.
Mechanism.
Acetylcholine is released from the terminus of nerve cell in the blood
vessel wall. The endothelial cells are stimulated to produce NO (from arginine),
which causes an increased synthesis of GMP, a second messenger responsible for
blood vessel dilation.
Ach
Nerve cell endothelial cell
NO GMP Vessel dilationAchR
Jan 18, 2017 25

NEUROTRANSMITTERSNEUROTRANSMITTERS
Jan 18, 2017 26

Neurotransmitters
They signal from neuron to neuron or from neuron to other target cell
(ex. muscle cell ).
Acetylcholine
Glycine
Glutamate
Dopamine
Epinephrine
Serotonin
Histamine
GABA.
Common features: hydrophilic molecules that bind to cell surface receptors.
The binding induces conformational changes that open ion channels
ion fluxes in the cell.
Jan 18, 2017 27

• The neurotransmitters (NTs) carry signals between
neurons or from neurons to other type of target cells
(muscle cells).
• They are diverse group of small hydrophlic molecules
including acetylcholine, dopamine, epinephrine,
serotonin, histamine, glutamate, glycine and GABA etc.
• The release of NTs is signalled by the arrival of an
action potential at the terminus of a neuron.
• The NTs then diffuse across the synaptic cleft and bind
to receptors on the target cell surface.
• Note that some NTs can also act as hormones- for
example- epinephrine functions both as NT and as a
hormone produced by adrenal to signal glycogen break
down in muscle cells.Jan 18, 2017 28

• Because the NTs are hydrophilic molecules they are
unable to cross the PM of their target cells.
Therefore, in contrast to the steroid hormones, NO
and CO, the NTs act by binding the cell surface
receptors.
• Many NT receptors are ‘ligand-gated ion channels’ ,
such as the acetylcholine receptor. NT binding to
these receptors induces a conformational change
that opens ion channels, directly resulting in
changes ion flux in the target cell.
• Other NT receptors are coupled to G proteins – a
major group of signalling molecules that that link
cell surface receptors to a variety of intracellular
responses.
Jan 18, 2017 29

PEPTIDE HORMONES AND GROWTHPEPTIDE HORMONES AND GROWTH
FACTORSFACTORS
Jan 18, 2017 31

• The widest variety of signalling molecules in animals
are peptides. This group includes peptide
hormones, neuropeptides and a diverse array of
growth factors.
• Well known example of peptide hormones include
insulin, glucagon and the hormones produced
pituitary gland.
• The neuropeptides, such as enkaphalins and
endorphins, function not only as NTs at synapse but
also as neurohormones that act on distant cells. The
enkaphalins and endorphins have been widely
studied because of their activities as natural
analgesics that decrease pain responses.Jan 18, 2017 32

• The pp growth factors include a wide variety of
signalling molecules.
• The first of this factor is nerve growth factor (NGF).
• NGF is a member of family of pp growth factor called
neurotrophins that regulate development and survival
of neurons.
• Epidermal growth factor (EGF) a 53 AAs pp, regulates
cell proliferation.
• A good example of growth factor activity is provided by
the activity of platelet-derived growth factor (PDGF) in
wound healing.
• PDGF is stored in blood platelets and released during
blood clotting at the site of wound. It then stimulates
the proliferation of fibroblasts, thereby contributing to
regrowth of damaged tissue.Jan 18, 2017 33

• Members of another large group of pp growth
factors, called cytokines, regulate development and
differentiation of blood cells and control the
activities of lymphocytes during immune response.
• Other pp growth factors, membrane-anchored
growth factors (MAGF) remains associated with the
plasma membrane and function specifically as
signalling molecule during direct cell-cell
interaction.
Jan 18, 2017 34

EICOSANOIDSEICOSANOIDS
Jan 18, 2017 35

• Several types of lipids serve as signalling molecules
and these are member of a class of lipid family,
called ecosanoids which include prostaglandins,
prostacyclin, thromboboxanes and leukotrienes.
• They act autocrine and paracrine signalling
pathways.
• They stimulate a variety of responses including
blood platelets aggregation, inflammation and
smooth muscle contraction.
Jan 18, 2017 36

FUNCTIONS OF CELL SURFACEFUNCTIONS OF CELL SURFACE
RECEPTORSRECEPTORS
Jan 18, 2017 37

• Most ligands are responsible for cell-cell signalling
bind to the receptors on the surface of their target
cells.
• Some neurotransmitter receptors are ion gated ion
channel that directly control ion flux across the
plasma membrane.
• Other cell surface receptors (peptide hormones,
growth factors) act by regulating the activities of
intracellular proteins. These proteins then transmit
signals from the receptors to a series of additional
intracellular targets.
• Ligand binding to the receptor on the surface thus
initiates a chain of intracellular reactions, ultimately
reaching the target cell nucleus.Jan 18, 2017 38

G Protein CoupledG Protein Coupled
ReactionReaction
Jan 18, 2017 39

• The largest family of cell surface receptors transmit
signals to intracellular targets via the intermediary
action guanine nucleotide binding proteins called G
Proteins. More than a thousand such G protein
receptors have been identified.
• The G protein coupled receptors are structurally
and functionally related to the proteins
characterised by seven membrane spacing α
helices.
• The binding of ligands to the extracellular domain
of these receptors induces a conformational change
that allows the cytosolic domain of the receptor to
bind to a G protein associated with the inner face of
plasma membrane.
Jan 18, 2017 40

• This interaction activates G protein, which then
dissociates from the receptor and carries the signal
to an intracellular target, which may be an enzyme.
• The discovery of G proteins came from studies of
hormones that regulate the synthesis of cAMP in
their target cells. cAMP is an important second
messenger that mediates cellular responses to a
variety of hormones.
Jan 18, 2017 41

• G proteins consist of three subunits designated α, β
and γ. They are frequently called heterotrimeric G
proteins to distinguish them from other guanine
nucleotide binding proteins.
• The α subunit binds guanine nucleotides, which
regulate G protein activity. In the resting state, α is
bound to GDP in a complex with β and γ. Hormone
binding induces a conformational change in the
receptor, such that the cytosolic domain of receptor
interacts with G protein and stimulates the release
of bound GDP and its exchange for GTP. The
activated GTP-bound α subunit then dissociates
from β and γ, which remain together and functions
as a βγ complex.
Jan 18, 2017 42

• Both the active GTP-bound α subunit and the βγ
complex then interact with their targets to elicit an
intracellular response.
• In addition to regulating target enzymes, both the α
and βγ subunits of some G proteins directly
regulate ion channels. A good example is provided
by the action of neurotransmitter acetylcholine on
heart muscle, which is distinct from its effect on
nerve and skeletal muscle.
Jan 18, 2017 43

G-Protein – Coupled Receptors
(Largest family of cell surface receptors)
cAMP is a second messenger that mediates cellular responses to a variety of
hormones.
Jan 18, 2017 44

Pathways of Intracellular SignalPathways of Intracellular Signal
TransductionTransduction
Jan 18, 2017 45

• Many cell surface receptors stimulate intracellular
target enzymes, which may be either directly linked
or indirectly coupled to receptors by G proteins.
• These intracellular enzymes serve as downstream
signalling elements that propagate and amplify the
signal initiated by ligand binding.
• In most cases, a chain of receptors transmits signals
from the cell surface to a variety of intracellular
targets- a process called intracellular signal
transduction.
Jan 18, 2017 46

The cAMP Pathway : SecondThe cAMP Pathway : Second
Messenger and ProteinMessenger and Protein
PhosphorylationPhosphorylation
Jan 18, 2017 47

• The intracellular signalling was first elucidated by
the studies the action hormone (epinephrine),
which signals the breakdown of glycogen to
glucose.
• In 1958, Sutherland discovered that the action of
epinephrine was mediated by an increase in
intracellular concentration of cAMP, leading to the
concept that cAMP is second messenger in
hormonal signalling.
• cAMP is formed from ATP by the action of adenylyl
cyclase and degraded to AMP by cAMP
phosphodiesterase.
Jan 18, 2017 48

ATP → cAMP → AMP
Jan 18, 2017 49

The cAMP pathway : Second messengers and
Protein phosphorylation.
Cyclic AMP is synthesized from ATP by adenylyl cyclase and
degraded to AMP by cAMP phosphodiesterase.
Jan 18, 2017 50

The epinephrine receptor is
coupled to adenylyl cyclase via a G
protein that stimulates enzymatic
activity, thereby increasing the
intracellular concentration of cAMP.
Jan 18, 2017 51

How does cAMP then signal the breakdown of
glycogen?
•This and most other effects of cAMP in animal cells
are mediated by the action “cAMP dependent Protein
Kinase”or Protein Kinase A (PKA).
•The inactive form of PKA is a tetramer, consisting of
two catalytic and two regulatory subunits.
•cAMP binds to the regulatory subunits leading to
their dissociation from catalytic subunits.
•The free catalytic subunits are then enzymatically
active and able to phosphorylate the target proteins.
Jan 18, 2017 53

• In the regulation of glycogen metabolism, PKA
phosphorylates two target enzymes.
• The first is another protein kinase, phosphorylase
kinase, which is phosphorylated and activated by PKA.
The phosphorylase kinase, in turn, phosphorylates and
activates glycogen phosphorylase, which catalyses the
breakdown of glycogen to glucose-1-phosphate.
• In addition, PKA phosphorylates the enzyme glycogen
synthase , which catalyses glycogen synthesis. In this
case, however, phosphorylation inhibits enzymatic
activity.
• The elevation of cAMP and activation of PKA thus
blocks further glycogen synthesis at the same time as it
stimulates glycogen breakdown.Jan 18, 2017 54

This is an example of a cAMP mediated pathway: PKA is
activated by 4 molecules of cAMP that bind to the regulatory
subunits releasing the catalytic subunits of the PKA.
The active protein kinase phosphorylates two key enzymes that
control degradation of glycogen.
cAMP
Jan 18, 2017 56

• The chain of reactions leading from the epinephrine
receptor to glycogen phosphorylase provides a good
illustration of signal amplification (SA) during
intracellular signal transduction.
• Each molecule of epinephrine activates only a single
receptor. However, each receptor may activate up to a
hundred molecules of G protein signal transduction
(Gs).
• Each molecule of Gs then stimulates the enzymatic
activity of adenylyl cyclase , which can catalyse the
synthesis of many molecules of cAMP.
• SA continues as each molecule of PKA phosphorylates
many molecules of phosphorylase kinase, which in
turn, phosphorylates many molecules of glycogen
phosphorylase.Jan 18, 2017 57

• In many animal cells, increase in cAMP activates the
transcription of specific target genes that contain a
regulatory sequence called “cAMP response
element” (CRE). In this case, the signal is carried out
from the cytoplasm to the nucleus following its
release from regulator subunit.
• Within the nucleus PKA phosphorylates a
transcription factor called CREB (CRE binding
protein), leading to the recruitment of co-activators
and transcription of cAMP-inducible genes.
• Such regulation of gene expression by cAMP plays
important role in controlling proliferation, survival
and differentiation of a wide variety of animal cells,
as well as being implicated in learning and memory.
Jan 18, 2017 58

• Although most effects of cAMP are mediated by
PKA, cAMP can also directly regulate ion channels,
independent of protein phosphorylation. cAMP
functions in this way as a second messenger
involved in sensing smells.
• Many of odorant receptors in the sensory neurons
in the nose are G protein coupled receptors that
stimulate adenylyl cyclase, leading to an increase
intracellular cAMP.
• Rather than stimulating PKA, cAMP in the system
directly opens sodium channels in the plasma
membrane, leading to membrane depolarisation
and initiation of nerve impulse.Jan 18, 2017 59

Another example of a signaling pathway mediated by cAMP
Jan 18, 2017 61

Phospholipids and Ca++
(another group of second messengers)
PIP2: PHOSPHATIDYLINOSITOL 4,5-BIPHOSPHATE
(a component of the plasma membrane)
Hydrolysis of PIP2 is activated by PLC (Phospholipase
C), and yields diacylglycerol and inositol phosphate
(IP3).
DAG: Diacylglycerol activates the protein kinase C
family, that play a crucial role in cell growth and
differentiation.
Jan 18, 2017 62

Ca++
Calcium cellular concentration is maintained low by pumps that
transport calcium across the plasma membrane and from the
cytosol inside the endoplasmic reticulum (ER).
High concentrations of calcium activate the functions of
proteins including protein kinase and phosphatases.
Many of the effects of calcium are mediated by the Ca++-
binding protein calmodulin, which is activated by calcium
binding when the concentration of cytosolic calcium increases
from 0.1 to 0.5 micromolar.
Calmodulin, in turn, binds to a variety of target proteins
including protein kinases (CaM).
Jan 18, 2017 64

One of the proteins activated by Ca/calmodulin is a kinase
called CaM
Jan 18, 2017 65

Summary
Signaling molecules and their receptors;
-Modes of cell-cell signaling (endocrine, paracrine, and autocrine)
-Steroid hormones and steroid receptor superfamily
-Nitric oxide and carbon oxide (paracrine signaling molecules
important in the nervous system.)
-Neurotransmitters (hydrophilic, carry signals between neurons or neuron
and other cell type, often bind to ion channels)
-Peptide hormones and growth factors (widest variety of signaling
molecules)
-Eicosanoids (paracrine and autocrine; aspirin inhibits their function)
Jan 18, 2017 66

The endThe end
Jan 18, 2017 68

Cell signalling

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