Signal transduction pathways

DR AAMIR
(presenter)
1ST YEAR RESIDENT
DEPT OF
PATHOLOGY
19-01-2016.
DR MAHESH KUMAR
(moderator)

1. INTRODUCTION.
2. SIGNAL TRANSDUCTION.
3. RECEPTORS.
4. EXTRACELLULAR RECEPTORS.
4a. G-PROTEIN COUPLED RECEPTORS.
4b. RECEPTORS WITH KINASE ACTIVITY.
1/19/2016SIGNAL TRANSDUCTION MECHANISMS
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4b1. RECEPTOR TYROSINE KINASE.
4b2. NON RECEPTOR TYROSINE KINASE.
JAK-STAT PATHWAYS.
4c. INTEGRINS.
4d. TOLL GATE RECEPTORS.
4e. LIGAND-GATED ION CHANNELS.
5. INTRACELLULAR RECEPTORS.
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1.INTRODUCTION
 In human body, numerous processes are required for
coordinating individual cells to support the body as a whole.
 At the cellular level, Sensing of environments and cell
communication for coordination relies on signal transduction;
modeling signal transduction systems as self-organizing allows
one to explain how equilibria are maintained.
 Many disease processes, such as diabetes and heart
disease arise from defects or dysregulations in these pathways,
highlighting the importance of these processes in human
biology and medicine
4

 Cell communication occurs through chemical signals and cellular
receptors by either the
 1) direct contact of molecules on two cells surfaces or the
 2) release of a "chemical signal" recognized by another cell (near
or far).
 Hormones are carried by the circulatory systems to many sites.
 Growth factors are released to act on nearby tissues.
 Ligands are signals that bind cell surface receptors (as observed
with insulin (a ligand) and the insulin receptor) or that can pass
into the cell and bind an internal receptor (such as the steroid
hormones).
5

2. SIGNAL TRANSDUCTION
 Any process occurring within cells that convert one kind of
signal/stimulus into another type.
 It also known as cell signaling in which the transmission of
molecular signals from a cell's exterior to its interior.
 Signals received by cells must be transmitted effectively into the
cell to ensure an appropriate response. This step is initiated by
cell-surface receptors which triggers a biochemical chain of
events inside the cell, creating a response.
6

 It is also defined as the ability of a cell to change behavior in
response to a receptor-ligand interaction.(signal)
 The ligand is the primary messenger.
 As the result of binding the receptor, other molecules or second
messengers are produced within the target cell.
 Second messengers relay the signal from one location to another
(such as from plasma membrane to nucleus) leading to cascade
of events/changes within a cell
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 Messenger molecules may be amino acids, peptides, proteins,
fatty acids, lipids, nucleosides or nucleotides.
 Hydrophilic messengers bind to cell membrane receptors.
 Hydrophobic messengers bind to intracellular receptors which
regulate expression of specific genes.
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 A ligand binds its receptor through a number of specific weak
non-covalent bonds by fitting into a specific binding site or
"pocket".
 In situations where even low concentrations of a ligand will result
in binding of most of the cognate receptors, the receptor affinity
is considered to be high.
 Low receptor affinity occurs when a high concentration of the
ligand is required for most receptors to be occupied.
 The dissociation constant (Kd) is the concentration of ligand
required to occupy one half of the total available receptors.
10

 With prolonged exposure to a ligand (and occupation of the
receptor) cells often become desensitized.
 Desensitization of the cell to a ligand depends upon receptor
down-regulation.
 Desensitization may lead to tolerance, a phenomenon that results
in the loss of medicinal effectiveness of some medicines that are
over prescribed.
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3.RECEPTORS
 Receptors can be roughly divided into two major
classes: intracellular receptors and extracellular receptors.
4.EXTRACELLULAR RECEPTORS
 Extracellular receptors are integral transmembrane proteins and
make up most receptors.
 They span the plasma membrane of the cell, with one part of the
receptor on the outside of the cell and the other on the inside.
 Signal transduction occurs as a result of a ligand binding to the
outside region of the receptor (the ligand does not pass through
the membrane).
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SIGNAL TRANSDUCTION MECHANISMS
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Various Extracellular Receptors
 G protein-coupled receptors.
 Receptors with Kinase activity.
 Integrin receptors.
 Toll gate receptors.
 Ligand-gated ion channel receptors.
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4a. G PROTEIN–COUPLED RECEPTORS (GPCRs)
 Also known as seven-transmembrane domain receptors, 7TM
receptors, heptahelical receptors, and G protein–linked
receptors (GPLR).
 These constitute a large protein family of receptors that sense
molecules outside the cell and activate inside signal
transduction pathways and, ultimately, cellular responses.
 Coupling with G proteins, they are called seven-transmembrane
receptors because they pass through the cell membrane seven
times.
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 The ligands that bind and activate these receptors include
 light-sensitive compounds,
 odors, pheromones, hormones,
 and neurotransmitters, and vary in size from small molecules
to peptides to large proteins.
 G protein–coupled receptors are involved in many diseases, and
are also the target of approximately 40% of all modern medicinal
drugs.
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 G proteins, also known as guanine nucleotide-binding
proteins, are a family of proteins that act as molecular
switches inside cells, and are involved in transmitting signals
from a variety of stimuli outside a cell to its interior.
 When they are bound to GTP, they are 'on', and, when they
are bound to GDP, they are 'off'.
 G proteins belong to the larger group of enzymes
called GTPases
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 There are two classes of G proteins.
 The first function as
monomeric small GTPases,
 The second form and function
as heterotrimeric G protein
complexes.
 Heterotrimeric class of complexes is made up
of alpha (α), beta (β) and gamma (γ) subunits. The beta and
gamma subunits can form a stable dimeric complex referred to as
the beta-gamma complex while alpha subunit dissociates on
activation.
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MECHANISM
 It is known that in the inactive state, the GPCR is bound to
a heterotrimeric G protein complex.
 Binding of an agonist to the GPCR results in
a conformation change in the receptor that is transmitted to the
bound Gαsubunit of the heterotrimeric G protein.
 The activated Gα subunit exchanges GTP in place of GDP which in
turn triggers the dissociation of Gα subunit from the Gβγ dimer
and from the receptor.
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 CONTD..
 The dissociated Gα and Gβγ subunits interact with other
intracellular proteins to continue the signal transduction cascade.
 While the freed GPCR is able to rebind to another heterotrimeric
G protein to form a new complex that is ready to initiate another
round of signal transduction.
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 There are two principal signal transduction pathways involving
the G protein–coupled receptors:
 A. the cAMP signal pathway and
 B. the phosphatidylinositol signal pathway.
 A. cAMP-DEPENDENT PATHWAY,
 It is also known as the adenylyl cyclase pathway.
 In a cAMP-dependent pathway, the activated Gs alpha subunit
binds to and activates an enzyme called adenylyl cyclase, which,
in turn, catalyzes the conversion of ATP into cyclic adenosine
monophosphate (cAMP).
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 Increases in concentration of
the second messenger cAMP may
lead to the activation of:
 cyclic nucleotide-gated ion channels.
 exchange proteins activated by
cAMP (EPAC) such as RAPGEF3
 popeye domain containing proteins
(Popdc)
 an enzyme called protein kinase
A (PKA).

 The PKA enzyme is also known as cAMP-dependent enzyme
because it gets activated only if cAMP is present. Once PKA is
activated, it phosphorylates a number of other proteins including:
 enzymes that convert glycogen into glucose
 enzymes that promote muscle contraction in the heart leading to
an increase in heart rate
 transcription factors, which regulate gene expression
24

Molecules that activate cAMP pathway include:
 cholera toxin - increase cAMP levels
 caffeine and theophylline inhibit cAMP phosphodiesterase, which
degrades cAMP - thus enabling higher levels of cAMP than would
otherwise be had.
 pertussis toxin, which increase cAMP levels by inhibiting Gi to its
GDP (inactive) form. This leads to an increase in adenylyl cyclase
activity, thereby increasing cAMP levels, which can lead to an
increase in insulin and therefore hypoglycemia
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 DEACTIVATION
 The Gs alpha subunit slowly catalyzes the hydrolysis of GTP to
GDP, which in turn deactivates the Gs protein, shutting off the
cAMP pathway.
 The pathway may also be deactivated downstream by directly
inhibiting adenylyl cyclase or dephosphorylating the proteins
phosphorylated by PKA.
 Molecules that inhibit the cAMP pathway include:
 cAMP phosphodiesterase dephosphorylates cAMP into AMP,
reducing the cAMP levels
 Gi protein, which is a G protein that inhibits adenylyl cyclase,
reducing cAMP levels.
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 B. PHOSPHATIDYLINOSITOL SIGNAL PATHWAY;
 In the phosphatidylinositol signal pathway, the extracellular signal molecule
binds with the G-protein receptor (Gq) on the cell surface and
activates phospholipase C, which is located on the plasma membrane.
27
phospholipase C

28
 IP3 binds with the IP3 receptor in the membrane of the
smooth endoplasmic reticulum and mitochondria to open
Ca2+ channels.
 DAG helps activate protein kinase C (PKC), which
phosphorylates many other proteins, changing their catalytic
activities, leading to cellular responses.

29

 The effects of Ca2+ are also remarkable:
 It cooperates with DAG in activating PKC and can activate
the CaM kinase pathway, in which calcium-modulated
protein calmodulin (CaM) binds Ca2+, undergoes a change in
conformation, and activates CaM kinase.
 The kinase then phosphorylates target enzymes, regulating their
activities. The two signal pathways are connected together by
Ca2+-CaM, which is also a regulatory subunit of adenylyl cyclase
and phosphodiesterase in the cAMP signal pathway.
30

WHEN G PROTEIN SIGNALING IS DISRUPTED
 G protein-related diseases are characterized by either deficient or
excessive G protein signal transmission, which arises through
abnormal signal initiation, defective termination, or reduced
levels of G proteins
 Deficient G protein signaling can arise through either reduced
levels of G proteins, or through decrease signal initiation.
31

 Diseases involving a decrease in the production of G proteins
include;
 Night Blindness, where mutations in G(t) protein a subunits affect
the response of rod cells to light.
 Pseudo hypoparathyroidism, where the genetic loss of G(s)
protein a subunits results in non-responsiveness to parathyroid
hormone.
 Other abnormalities involve decreased signal initiation through
the inability of G proteins to switch to active states, e.g. the
symptoms of Whooping Cough (Pertussis) like hypoglycemia
32

 Excessive G protein signaling can arise through either increased
signal initiation, or defective signal termination.
 Increased signal initiation occurs in Testotoxicosis, where a
mutation in the receptor for luteinizing hormone can over-
stimulate G(s) proteins, resulting in the excessive production of
testosterone.
 Diseases arising from defective signal termination result from the
persistent elevated activity of downstream effectors, such as in
Cholera, the symptoms of which results from the action of a
bacterial toxin that lead to stimulation of adenylyl cyclase and the
subsequent secretion of salt and water leading to fatal diarrhea.
33

 Two other diseases involve defective termination through
mutations in G(s) protein a subunits, including;
 Adenomas, in which G proteins lose their ability to hydrolyze GTP
through mutation, resulting in the excessive secretion of growth
hormone and the increased proliferation of somatotrophs.
 McCune-Albright Syndrome, where scattered regions of skin
hyper-pigmentation arise from the hyper-functioning of one or
more endocrine glands.
34

4b. RECEPTORS WITH KINASE ACTIVITY;
 A kinase is a type of enzyme that transfers phosphate groups
from high-energy donor molecules, such as ATP (see below) to
specific target molecules (substrates); the process is
termed phosphorylation.
 For every phosphorylation event, there is a phosphatase, an
enzyme that can remove phosphate residue and thus modulate
signaling
 Kinase enzymes that specifically phosphorylate tyrosine amino
acids are termed tyrosine kinases.
35

1. RECEPTOR TYROSINE KINASE(RTKs)
 It is a cell surface receptor that also has a tyrosine kinase activity.
 The signal binding
domain of the receptor
tyrosine kinase is on the
cell surface, while the
tyrosine kinase enzymatic
activity resides in the
cytoplasmic part
of the protein.
36

 A transmembrane alpha helix connects these two regions of the
receptor.
 As is the case with GPCRs, proteins that bind GTP play a major
role in signal transduction from the activated RTK into the cell.
 In this case, the G proteins are members of the
Ras, Rho, and Raf families, referred to collectively as
small G proteins
37

 The most important groups of signals that bind to receptor
tyrosine kinases are:
 peptide growth factors like nerve growth factor (NGF) and
epidermal growth factor (EGF)
 peptide hormones, like insulin.
 Binding of signal molecules to the extracellular domains of
receptor tyrosine kinase molecules causes two receptor
molecules to dimerize.
 This brings the cytoplasmic tails of the receptors close to each
other and causes the tyrosine kinase activity of these tails to be
turned on.
38

 The activated tails then phosphorylate each other on several
tyrosine residues. This is called autophosphorylation.
 The phosphorylation of tyrosines on the receptor tails triggers
the assembly of an intracellular signaling complex on the tails.
 The newly phosphorylated tyrosines serve as binding sites for a
variety of signaling proteins that then pass the message on to yet
other proteins.
 An important protein that is subsequently activated by the
signaling complexes on the receptor tyrosine kinases is
called RAS.
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6
SIGNAL TRANSDUCTION MECHANISMS
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 The RAS protein has the following important features:
 1. It is associated with the cytosolic face of the plasma membrane
2. It is a monomeric GTP-binding protein (in fact, it is a lot like
the alpha subunit of trimeric G-proteins).
3. Just like the alpha subunit of a G-protein, Ras is active when
GTP is bound to it and inactive when GDP is bound to it. Like the
a subunit, Ras can hydrolyze the GTP to GDP
 This excited signal-emitting state is short-lived, however, because
the intrinsic (GTPase) activity of RAS hydrolyzes GTP to GDP,
returning the protein to its quiescent GDP-bound state
42

 Activated RAS
triggers a
phosphorylation
cascade of
protein kinases,
which relay and
distribute the signal.
43

 These protein kinases are members of a group called the MAP
kinases (Mitogen Activated Protein Kinases).
 The final kinase in this cascade phosphorylates various target
proteins, including transcriptional activators (e.g.myc) that
regulate gene expression.
 RAS is the most commonly mutated proto-oncogene in human
tumors. Indeed, approximately 30% of all human tumors
contain mutated versions of the RAS gene, and the frequency is
even higher in some specific cancers (e.g., colon and pancreatic
adenocarcinomas).
44

 2. NON RECPETOR TYROSINE KINASE (nRTKs)
 Non-receptor tyrosine kinases are a subgroup of protein
family tyrosine kinases, enzymes that can transfer the phosphate
group from ATP to a tyrosine residue of a protein
(phosphorylation).
 Unlike the receptor tyrosine kinases (RTKs), the second subgroup
of tyrosine kinases, the non-receptor tyrosine kinases are
cytoplasmic enzymes.
 nRTKs regulate cell's growth, proliferation, differentiation,
adhesion, migration and apoptosis and they are critical
components in the regulation of the immune system.
45

 The main function of nRTKs is their involvement in signal
transduction in activated T- and B-cells in the immune system.
 CD4 and CD8 receptors on T lymphocytes require for their
signaling the Src family member Lck.
 Src, cellular homolog of transforming protein of Rous Sarcoma
Virus, is prototype for an important family of such nRTKs
46

 Src contains unique functional regions such as Src-homology 2
(SH2) and Src- homology 3 (SH3).
 SH2 domains typically bind to receptors phosphorylated by
another kinase, allowing the aggregation of multiple enzymes.
 SH3 domains mediate other protein-protein interactions.
47

JAK-STAT Signaling pathway
 The JAK-STAT signaling pathway transmits information from
chemical signals outside the cell, through the cell membrane, and
into gene promoters on the DNA in the cell nucleus, which causes
DNA transcription and activity in the cell.
 The JAK-STAT system is a major signaling alternative to
the second messenger system and is an example of nRTK.
 The JAK-STAT system consists of three main components:
 1. a receptor
 2. Janus kinase (JAK) and
 3. Signal Transducer and Activator of Transcription (STAT).
48

 Many JAK-STAT pathways are expressed in white blood cells, and
are therefore involved in regulation of the immune system.
 MECHANISM.
 The binding of the ligand to the receptor triggers activation of
JAKs.
 With increased kinase activity, they phosphorylate tyrosine
residues on the receptor and create sites for interaction with
proteins that contain phosphotyrosine-binding SH2 domains.
49

 STATs possessing SH2 domains capable of binding these
phosphotyrosine residues are recruited to the receptors, and
are themselves tyrosine-phosphorylated by JAKs
 These phosphotyrosines then act as binding sites for SH2
domains of other STATs, mediating their dimerization.
Different STATs form hetero- or homodimers.
 Activated STAT dimers accumulate in the cell nucleus and
activate transcription of their target genes
50

51

 JAK-STAT pathway mutations are associated with many
hematological malignancies caused by gaining constitutive
functions e.g.
 MYELOPROLIFERATIVE NEOPLASMS; Ph negative MPNs
 MDS
 MULTIPLE MYELOMA.
52

4c. INTEGRINS
 Integrins are produced by a wide variety of cells; they play a role
in:
 1. Cell attachment to other cells and the extracellular matrix and
 2. In the transduction of signals from extracellular matrix
components such as fibronectin and collagen.
 Ligand binding to the extracellular domain of integrins changes
the protein's conformation, clustering it at the cell membrane to
initiate signal transduction.
53

 Integrins lack kinase activity; hence, integrin-mediated signal
transduction is achieved through a variety of intracellular protein
kinases and adaptor molecules, the main coordinator being
integrin-linked kinase.
 Integrin signaling exist in two places mainly;
 Integrin-signaling
in circulating blood cells
and non-circulating cells
such as epithelial cells.
54

 Important differences exist between two is that integrins of circulating cells
are normally inactive.
 For example, cell membrane integrins on circulating leukocytes are
maintained in an inactive state to avoid epithelial cell attachment; they are
activated only in response to stimuli such as those received at the site of an
inflammatory response.
 In a similar manner, integrins at the cell membrane of
circulating platelets are normally kept inactive to avoid thrombosis.
 Epithelial cells (which are non-circulating) normally have active integrins at
their cell membrane, helping maintain their stable adhesion to underlying
stromal cells that provide signals to maintain normal functioning
55

4c. TOLL GATE RECEPTORS
 Toll-like receptors (TLRs) are a class of proteins that play a key
role in the innate immune system.
 TLRs are a type of pattern recognition receptor (PRR) and
recognize molecules that are broadly shared by pathogens but
distinguishable from host molecules, collectively referred to
as pathogen-associated molecular patterns (PAMPs).
56

57

4e. LIGAND-GATED ION CHANNEL
 Ligand-gated ion channels (LGICs) are a group
of transmembrane ion channel proteins which open to allow ions
such as Na+, K+,Ca2+, or Cl− to pass through the membrane in
response to the binding of a chemical messenger (i.e.
a ligand), such as a neurotransmitter.
 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.
58

 An example of this mechanism is found in the receiving cell of
a neural synapse.
 The influx of ions that occurs in response to the opening of
these channels induces action potentials, such as those that
travel along nerves, by depolarizing the membrane of post-
synaptic cells, resulting in the opening of voltage-gated ion
channels.
 Ligand-gated ion channels are likely to be the major site at
which anaesthetic agents and ethanol have their effects, in
particular, the GABA and NMDA receptors are affected
by anaesthetic agents.
59

60

5. INTRACELLULAR RECEPTORS
 Intracellular receptors are receptors located inside the cell rather
than on its cell membrane.
 Classic hormones that use intracellular receptors include thyroid
and steroid hormones.
 Examples are:
 Class of nuclear receptors located in the cell
nucleus and cytoplasm
 IP3 receptor located on the endoplasmic reticulum.
61

 The ligands that bind to them are usually:
 Intracellular second messengers like inositol trisphosphate (IP3)
 Extracellular lipophilic hormones like steroid hormones.
 Activated nuclear receptors attach to the DNA at receptor-
specific hormone-responsive element (HRE) sequences, located in
the promoter region of the genes activated by the hormone-
receptor complex.
 Due to their enabling gene transcription, they are alternatively
called inductors of gene expression.
62

 All hormones that act by regulation of gene expression have
two consequences in their mechanism of action;
 1. their effects are produced after a characteristically long
period of time
 2. their effects persist for another long period of time, even
after their concentration has been reduced to zero, due to a
relatively slow turnover of most enzymes and proteins that
would either deactivate or terminate ligand binding onto the
receptor. E.g. Thyroid Hormone
63

64

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CONCLUSION
 The entire signal transduction system normally works
astonishingly well, but serious problems can occur.
 Cancer is unregulated cell growth and occurs when the
machinery tightly regulating cell growth breaks down.
 Mutations in growth factor receptors, G proteins, MAP kinases,
and other molecules frequently contribute to cancer, and
generally result in these molecules losing their normal switching
function, staying in the activated form and therefore
inappropriately stimulating these important enzyme cascades.
66

 The complexity of the signaling system makes for challenging
research, but once understood it holds the promise for better
treatments for cancer and other diseases.
 This is because each step in each pathway provides one or more
targets for drugs.
 Designing a drug that could quiet the excess signaling caused by
defective MAP kinase, for example, might provide a promising
cancer treatment.
67

 The examples given thus far provide only an outline of how
signal transduction cascades work and an overview of a few of
the most important enzymes.
 The actual process is much more complex, and there is much
about the process that remains mysterious.
 Perhaps the biggest mystery is how the cell makes sense of all of
the input from different growth factors, hormones, extracellular
substrates, and so on to produce an appropriate response.
68

 The solution to this problem will result from a complete
understanding and computer modeling of the biochemical and
kinetic properties of the components of all these signaling
cascades.
_______________________.____________________________
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70

 REFRENCES
1. KUMAR, ABBAS AND COTRAN, ROBBINS BASIC PATHOLOGY OF DISEASES,
NINTH EDITION
2. GUYTON AND HALL, TEXT BOOK OF MEDICAL PHYSIOLOGY, ELEVENTH
EDITION.
3. KIM E BARRET, SUSAN M BARMAN, SCOT BIOTANO,GANONG’S MDEICAL
PHYSIOLOGY, TWENTY THIRD EDITION.
4. INTERNET SOURCES.
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