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Signal Transduction
Signal Transduction
(Old terms:Signal transmission or sensory transduction)
In this phenomenon a mechanical or
chemical stimulus is converted into a
specific cellular response. It starts with a
signal to a receptor, and ends with a
change in cell function or response.
Signal Transduction
• Cells respond to their environment by re-organizing their
structure, regulating the activity of proteins and altering
patterns of gene expression. The stimulus for such responses
is known as signal. It may be a small molecule, a
macromolecule or a physical agent such as light, temperature,
water etc. Signals interact with the responding cell through
specific molecules called receptors.
• Small molecules often act as diffusible signals. In unicellular
organizations diffusible signals may be environmental or may
be released from other cells e.g. yeast mating-type
pheromones or cAMP in Dictyostelium (a species of soil-living
amoebae). In metazoans (syn: the Animal Kingdom), signals
may be released from nearby cells and diffuse over short
distances (Paracrine signaling), or they may be released from
distant cells and reach their target through vas. system
(endocrine signaling). Macromolecular signals are associated
with the extracellular matrix or displayed on the surface of
neighboring cells (Juxtacrine signaling). A molecular signal
that binds to a receptor is called ligand.
Signal Transduction in Prokaryotes
• Animal (mammals) possess a well developed nervous system by
which they are able to sense and respond to environment.
• Many processes such as (1) stimulus detection (2) signal
amplification (3) appropriate output responses are present in all cell
sensory systems including bacteria.
• Many bacterial signaling pathways consist of molecular units called
transmitters and receivers. This is called two-component regulatory
system.
• Bacteria use two component regulatory systems to sense
extracellular signals. They sense chemicals in the environment by
means of a small familyof cell surface receptors, each involved in
the response to a specific group of chemicals called ligands. A
protein in the plasma membrane of bacteria binds directly to a ligand
or binds to a soluble protein that has already attached to the ligand,
in the periplasmic space between the PM and the cell wall.
• Upon
binding,
the
membrane
protein
undergoes
a
conformationational change that passes across the membrane to
the cytosolic domain of the receptor protein. This conformational
change initiates the signaling pathway that leads to the response.
Signaling via bacterial two-component-systems
• The sensor protein present on cell wall detects
the stimulus via the input domain and transfers
the signal to the transmitter domain by means of
conformational change (first dashed arrows).
The transmitter domain of the sensor then
communicates with the response regulator by
protein phosphorylation of the receiver domain.
Phosphorylation of the receiver domain induces
a conformational change (second dashed
arrows) that activates the output domain and
brings about the cellular response.
• For instance, osmoregulation, chemotaxis and
sporulation are regulated by two component
systems.
• The signal is passed from transmitter domain to receiver
domain due to protein phosphorylation. Transmitter
domains have the ability to phosphorylate themselves
using ATP on a histidine near the amino terminus.
Because of this autophosphorylation, the sensor proteins
containing
transmitter
domains
are
called
autophosphorylating histidine kinases. Immediately after
the transmitter domain becomes autophosphorylated on
a histidine residue the (P) is transferred to a specific
aspartate residue near the middle of the receiver domain
of the response regulator protein. As a result, the
aspartate
residue
becomes
phosphorylated.
Phosphorylation of the aspartate residue causes the
response regulator to undergo a conformational change
that results in activation.
Parkinson (1993)
Signal Transduction in Eukaryotes
• In complex multi-cellular organisms two main systems
have been evolved
– (i) nervous system
– (ii) endocrine system
• In contrast, plants lack nervous system but they have
evolved hormones as chemical messengers. However,
generally plant signal transduction pathways differ to a
great extent from those of animals. But nevertheless
there are some common signal mechanisms in both
animals and plants that we are discussing here.
Hormones
• There are two classes of hormones depending
on their ability to move across the plasma
membrane.
– Lipophilic hormones: which diffuse readily across
the hydrophobic bilayer of plasma membrane e.g.
Androgens,
glucocorticoids,
estrogens
and
brassinosteroids.
– Water soluble hormones which are unable to enter
the cell by their own e.g. antidiuretic hormone (ADH),
glucagons, Thyroid Stimulating Hormone (TSH)
• Lipophilic hormones bind mainly to receptors in
the cytoplasm or nucleus, whereas water soluble
hormones bind to receptors located on the cell
surface.
• In both cases ligand binding changes the
receptor by causing a conformational change.
• Some receptors such as steroid hormone
receptors can regulate gene expression directly.
• But in most cases the receptor initiates one or
most sequences of biochemical reactions that
connect the stimulus to a cellular response.
Such a sequence of reactions is called a signal
transduction pathway.
• The final or end result of signal transduction
pathways is to regulate transcription factors
which in turn regulate gene expression.
Signal transduction pathways
• Signal transduction pathways often involve generation of second
messengers. They actually transmit secondary signals inside the
cell that greatly amplify the original signal.
• Molecules inside the cells that act to transmit signals from a
receptor to a target. The term second messenger is used to
distinguish them from hormones and other molecules that
function outside the cell as “first messengers” in the transmission
of biological information.
• Most common second messengers are cAMP, cGMP, 1,2
diacylglycerol (DAG), or inositol 1,4,5 triphosphate or inositol
4,5,6 triphosphate (IP3) and Ca2+.
• Hormones binding normally causes enhanced levels of one or
more of these second messengers resulting in the activation or
inactivation of enzymes or regulatory proteins.
Steroid Receptors can act as transcription factors
• Steroid Receptors can act as transcription factors. Almost all the
steroid hormones can freely pass through the PM because of their
lipophilic/hydrophobic nature and they bind to the intra-cellular
receptor proteins. On binding, these proteins function as transcription
factors. All such steroid receptor proteins have similar DNA binding
domains. Steroid response elements are typically located in enhancer
regions of steroid stimulated genes.
• Most steroid receptors are localized in the nucleus, where they get
anchored to nuclear proteins in an inactive form. When the receptor
binds to the steroid, it is released from the inactive anchor protein and
becomes activated as a transcription factor. The activated
transcription factor then binds to the enhancer and stimulates
transcription.
• Not all intracellular steroid receptors are localized in the nucleus. The
receptor for cortisol (glucocorticoid hormone) differs from the others in
that it is located in the cytosol, anchored in an inactive cytosolic
protein.
Transport of water soluble hormones into the cell
• The cell surface receptors of water soluble hormones can interact
with G-proteins (GPCRs) known as G-protein-coupled receptors
• All water soluble (hydrophilic) hormones of mammals bind to cell
surface receptors. The cell receptors interact with signal –
transducting GTP binding proteins known as heterotrimeric G
proteins. The activated G proteins, in turn, activate an effector
enzyme. The activated effector enzyme generates an intracellular
second messenger, which stimulates numerous cellular processes.
• Receptors using G-proteins are structurally similar but functionally
different. The receptor proteins consist of seven trans-membrane a
helices which are known as seven arch spanning, seven-pas or
serpentine receptors.
• Heterotrimeric G proteins cycle b/w active and inactive forms
The G proteins that transmit/transduce the signals from the sevenspanning receptors are called heterotrimeric G-Proteins because
they consist of three different subunits, a, b and g. The
heterotrimeric G-proteins act as molecular switches b/w active and
inactive forms. The b & g subunits form a tight complex that anchors
the trimeric G protein to the membrane on the cytoplasmic side.
• The G-protein becomes activated after binding
to the activated seven-spanning receptor. In an
inactive form, G exists as a trimer with GDP
bound to the a. Binding to the receptor – ligand
complex induces the a to exchange GDP for
GTP. The exchange causes the a – submit to
associate with an effector enzyme.
• The a submit has a GTPase activity that is
activated when it binds to the effector enzyme
e.g. adenylyl cyclase. GTP is hydrolyzed to GDP
thereby inactivating the a subunit, which in turn
inactivates adenylyl cyclase. The a subunit
bound to GDP re-associates with the b and g
subunits, and it starts another cycle to bind to
the hormone-receptor complex.
Nobel Prize in Chemistry 2012
• Robert J. Lefkowitz and Brian K. Kobilka
won the 2012 Nobel Prize in Chemistry for
their research on G-protein-coupled
receptors (GPCRs). These receptors allow
cells in the body to sense and respond to
outside signals and are the target for
several commonly prescribed drugs
including β-blockers (protection for heart
attack), antihistamines and antidepressants.
Schematic diagram of the general structure of G protein-coupled receptors
All receptors of this type have the same orientation in the membrane
Contain seven transmembrane α-helical regions (H1-H7)
Four extracellular segments (E1-E4)
Four cytosolic segments (C1-C4)
The carboxylic-terminal segment (C4), the C3 loop, and , in some receptors, also the
C2 loop are involved in interactions with a coupled trimeric G protein
Activation of adenylyl cyclase increases the
level of cyclic AMP
• cAMP is an important signaling molecule in
prokaryotes, animal cells, and plant cells. In
higher animals, adenylate cyclase is an integral
membrane
protein
that
contains
two
clusters/groups of six-membrane spanning
domains, two of which extending into the
cytoplasm as catalytic domains. Activation of
adenylyl cyclase by heterotrimeric G proteins
increases the conc. of cAMP in the cell, which is
normally maintained at a low level by the action
of cAMP phosphodiesterase which hydrolyzes
cAMP to 5 AMP. Now the evidence is growing to
affirm the presence of adenylate cyclase in
plants (Gehring 2010).
• Plant-activated bacterial receptor
adenylate cyclases modulate epidermal
infection in the Sinorhizobium melilotiMedicago symbiosis (Tian et al. 2012,
from France) published in PNAS.
IP3- second messenger of water soluble hormones
Activation of Phospholipase C initiates the IP3 pathway
Calcium serves as a second messenger for many signaling events
in both animals and plants. The concentration of free Ca2+ in the
cytosol is very low (0.1µM). Ca-ATPases on PM and ER pump
calcium out of cell and into the ER lumen, respectively. In plant cells
most of the Ca of the cell accumulates in the vacuole. The transport
of Ca at tonoplast is carried out by proton pumps via Ca2+-H+
antiporter.
• In animal cells, certain hormones can induce a transient rise in the
cytosolic Ca up to 5 uM. The rise in cytosolic Ca occurs due to
opening of intracellular compartments. The coupling of hormone
binding to the opening of intra-cellular Ca channels is mediated by
another second messenger IP3.
• Phosphatidylinositol (PI) is a minor phospholipid component
of cell membrane which can be converted to PI phosphate
(PIP) and PI bisphosphate (PIP2) by kinases. PIP2 though not
abundant as PI, it plays a crucial role in signal transduction.
The activated phospholipase C (PLC) readily hydrolyzes PIP2
to give rise IP3 and diacylglycerol (DAG). They both play
important role in cell signaling.
• The IP3 generated by activated phospholipase – C diffuses
through the cytosol and binds to IP3 binding sites on the ER
and tonoplast. These binding sites are IP3 – gated Ca2+
channels that open when they bind to IP3. Ca diffuses into
cytosol and causes a cellular response. This response is
terminated when IP3 is broken down by specific
phosphatases or when the released Ca is pumped out of the
cytoplasm by Ca2+ - ATPases.
• The Ca signal often originates in a localized region of the cell
and propagates as a wave throughout the cytosol. Repeated
waves called Ca oscillations can follow the original signal,
each lasting from a few sec to several minutes. The oscillation
may play a role in avoiding the toxicity that results from high
conc. of Ca in the cytosol.
Diacylglycerol activates protein kinase C
• Breakdown of PIP2 by phospholipase C produces IP3
and DAG. IP3 being hydrophilic diffuses rapidly into the
cytoplasm but DAG being a lipid remains in the
membrane. In animal cells DAG can come close to
protein kinase C (PKC) and activates it. The PKC is
located in the cytosol in an inactive form, but upon
binding to the Ca2+ it undergoes conformational change
and associates with a PKC receptor protein that
transports it to the inner surface of the PM where it
encounters DAG. PKC activity has also been detected in
plants. G proteins, phospholipase C and various protein
kinases have been discovered in plant membranes.
Role of Ca - Calmodulin (Ca-modulated protein (CaM)) in
activation of Protein kinases -- act as on-off switches
• In addition to directly binding of Ca with some proteins, such as
channels, most of the effects of Ca are due to its binding to
regulatory proteins such as calmodulin (17 kDa 148 amino acids).
Calmodulin widely occurs in eukaryotes both animals and plants as
well as now found in prokaryotes (Shemarova and Nesterov, 2005).
• There are some other Ca-binding proteins in addition to calmodulin
that possess a similar Ca-binding site e.g. in protein parvalbumin
(EF hand = E & F are two ά helices)
• Each calmodulin molecule binds 4 Ca2+ ions and changes
conformation thereby enabling it to bind and activate other proteins.
Ca2+ - calmodulin can stimulate some enzymes directly such as PM
Ca2+ -ATPase which pumps Ca2+ out of the cell. In addition, most of
the effects of Ca are brought about by activation of Ca2+ calmodulin dependent protein kinases (CaM kinases). The CaM
kinases phosphorylate serine or threonine residues of their target
enzymes, causing enzyme activation.
Enzymes regulated by Ca or Calmodulin
•
•
•
•
•
•
•
•
Adenylyl cyclase
Ca – dependent protein kinase
Ca2+ Mg2+ - ATPase
NAD Kinase
Phospholipase kinase
PA carboxylase
PA dehydrogenase
PA kinase
Some water soluble hormones bind to
Receptor Tyrosine Kinases (RTK)
•
•
•
•
Over 50 RTKs have been characterized. They are divided into 14 families based on
structure. 58 RTKs have been characterized in man.
In animal cells, RTKs are the important class of cell surface receptors. The RTK
possess a hormone ligand-binding domain, a trans-membrane domain, and
catalytic domain (in cytosol). Since the trans-membrane domain consists of a single
ά helix, the hormone cannot transmit a signal directly to the cytosolic side.
However, binding of the hormone to the receptor induces dimerization of adjacent
receptors which allows the two catalytic domains to come into contact and
autophosphorylate the tyrosine residues.
Similar to RTKs, in plants Leucine-rich repeat receptor kinases (LRR-RKs)
comprise the largest subfamily of transmembrane receptor-like kinases in plants,
with over 200 members in Arabidopsis. LRR-RKs regulate a wide variety of
developmental and defense-related processes including cell proliferation, stem cell
maintenance, hormone perception, host-specific as well as non-host-specific
defense response, wounding response, and symbiosis. However, true RTKs have
been recently discovered in some fungi.
On auto-phosphorylation, the catalytic site of the RTKs binds to different cytosolic
signaling proteins. After binding to the RTK, the inactive signaling protein is itself
phosphorylated on specific tyrosine residues. Some transcription factors are
activated like this. However, the signaling initiated by RTKs begins with the small
monomeric (only ά unit) G proteins called as Ras.
• There are three common monomeric G proteins: Ras,
Rab and Rho/Rac. All belong to Ras super-family of
monomeric GTPases. Rho and Rac relay signals from
surface receptor to the actin cytoskeleton, whereas
members of the Rab family of GTPases are involved in
regulating intracellular membrane vesicle traffic. Ras
proteins transmit signals from RTKs to the nucleus. Ras
is a G protein that cycles b/w an inactive GDP – binding
form and an active GTP – binding form. Ras can also act
as GTPase to hydrolyze bound GTP to GDP thereby
terminating the response.
• The crystal structure of Arabidopsis thaliana
RAC7/ROP9 has been determined: The first RAS
superfamily GTPase from the plant kingdom (Sormo et
al., 2006; Sormo et al. 2011 Trends Plant Sci)
•
• Binding of hormone to the RTK induces dimerization followed
by auto-phosphorylation of the catalytic domain. Autophosphorylation of the receptor causes binding to the Grb2
protein which is tightly bound to another protein known as
Sos. The Grb–Sos complex then attaches to the RTK. Upon
binding to Sos, Ras exchanges GDP with GTP and Ras then
becomes active.
• The activated Ras is then able to bind to another soluble
serine/threonine kinase known as Raf. Upon binding to Ras,
Raf becomes active and initiates a chain of phosphorylation
reactions called the MAPK cascade. In plants the ethylene
receptors pass signals to a protein kinase of Raf family.
• MAPK (mitrogen – activated protein kinase) cascade refers to
a series of protein kinases that phosphorylate each other in a
specific sequence. Ultimately, the phosphorylated MAPK
enters the nucleus and activates transcription factors. The
activated transcription factors then stimulate gene expression.
Ras Signaling Pathway