Cell signaling

dr. Dwi Ngestiningsih, M.Kes, Sp.PD-KGer | dr. Lusiana Batubara, M.Si.Med

Introduction

• Cell communications occurred by cell signaling

• There are many signaling pathways

• Signaling pathways may categorized and termed by:

 The receptor types used
 The second messenger employed
 The transduction protein used

• Signaling pathways may yield:

 Activate proteins present
 Activate some genes
 Signal transduction:
Process of converting
extracellular signals
into intra-cellular
responses.

Occurs by several
mechanisms

A simple intracellular signaling pathway

activated by an extracellular signal molecule
Receptors for extracellular signaling molecules that regulating the activity of transcription factors

13-1
G-Protein-Coupled Receptors (GPCR)
G-Protein-Coupled Receptors (GPCR)
Ligands:
Epinephrine, glucagon, serotonin, vasopressin, ACTH,
adenosine, and many others (mammals); odorant molecules,
light; mating factors (yeast)

Receptors:
Seven transmembrane helices;
Sytosolic domain associated with a membrane-tethered
trimeric G protein

Signal transduction:
(1) Second-messenger pathways involving cAMP (cAMP
pathway) or IP3/DAG (IP3/DAG pathway);
(2) linked ion channels
(3) MAP kinase pathway
G-Protein-Coupled Receptors (GPCR)

Mechanism of Activation of GPCR:

1. Binding of ligand to extracellular domain of GPCR induces
conformational change that allows cytosolic domain of the
receptor to bind to inactive G protein.
2. This interaction activates the G protein
3. Activated G protein  subunit can now bind GTP instead of
GDP, causing dissociation into activated  vs.  subunits.
Each of these can go on to activate target proteins.
cAMP Pathway

• Ligand binding
• Activated GPCR
• Activated α subunit of G
protein
• Activated adenylyl cyclase
• Rise in cAMP
• Activated Protein kinase A
(PKA)
• PKA translocating to the
nucleus
• Activated CREB
transcription factor
 Cytokine Receptors
Receptor Tyrosine Kinases

Two receptor classes, cytokine receptors and receptor tyrosine

kinases, transduce signals via their associated or intrinsic
protein tyrosine kinases.
Ligand binding triggers formation of functional dimeric receptors
and phosphorylation of the activation lip in the kinases,
enhancing their catalytic activity
General structure and ligand-induced activation of receptor tyrosine
kinases (RTKs) and cytokine receptors

The mechanisms by which cytokine receptors and receptor tyrosine kinases

become activated by ligands are very similar

Receptor Tyrosine cytokine receptors

Kinases (RTKs)
General structure and ligand-induced activation of receptor tyrosine
kinases (RTKs) and cytokine receptors

(step 1) The cytosolic domain of RTKs contains a protein tyrosine kinase catalytic
site, whereas the cytosolic domain of cytokine receptors associates with a
separate JAK kinase.

(step 2 ) In both types of receptor, ligand binding causes a conformational change

that promotes formation of a functional dimeric receptor, bringing together two
intrinsic or associated kinases, which then phosphorylate each other on a
tyrosine residue in the activation lip

(step 3 ) Phosphorylation causes the lip to move out of the kinase catalytic site,
thus allowing ATP or a protein substrate to bind. The activated kinase then
phosphorylates other tyrosine residues in the receptor’s cytosolic domain

The resulting phosphotyrosines function as docking (binding) sites for various

signal-transduction proteins (SH2 domains or PTB domains).
General structure and ligand-induced activation of receptor tyrosine
kinases (RTKs) and cytokine receptors

Receptor Tyrosine cytokine receptors

Kinases (RTKs)
General structure and ligand-induced activation of receptor tyrosine
kinases (RTKs) and cytokine receptors

STAT

Recruitment of signal-transduction proteins to the cell membrane

by binding to phosphotyrosine residues in activated receptors.
Cytokine receptors
Cytokine receptors
Ligands:
Interferons, erythropoietin, growth hormone, some interleukins (IL-2, IL-
4), other cytokines

Receptors:
• Single transmembrane helix
• cytosolic domains are closely associated with a member of a family of
cytosolic protein tyrosine kinases, the JAK kinases.

Signal transduction:
(1) Direct activation of cytosolic STAT transcription factors;
(2) PI-3 kinase pathway;
(3) IP3/DAG pathway;
(4) Ras-MAP kinase pathway
Cytokine receptors

Cytokines control many aspects of

growth and differentiation of specific
types of cells.

Erythropoietin, a cytokine secreted

by kidney cells, prevents apoptosis
and promotes proliferation and
differentiation of erythroid
progenitor cells in the bone marrow.

An excess of erythropoietin or
mutations in its receptor that
prevent down-regulation result in
production of elevated numbers of
red blood cells.
Cytokine receptors

JAK-STAT signaling pathway

• The JAK-STAT pathway operates downstream of all cytokine receptors.

• STAT monomers bound to receptors are phosphorylated by receptor-

associated JAKs, then dimerize and move to the nucleus, where they
activate transcription.

• Short peptide sequences containing phosphotyrosine residues are bound by

SH2 and PTB domains, which are found in many signal-transducing proteins.

• Signaling from cytokine receptors is terminated by the phosphotyrosine

phosphatase SHP1 and several SOCS proteins.

SH2: Src homology 2

Cytokine receptors

STAT = signal transducer and activator of transcription

• A family of eukaryotic transcription factors that mediate the

response to many cytokines and growth factors.

• Upon receptor activation, STAT proteins dimerize, translocate

to the nucleus, and bind to specific promoter sequences on
target genes.

• Seven different genes have been so far identified in mammals.

• Example: STAT1_HUMAN, 750 amino acids (87.33 kDa)

Cytokine receptors

JAK-STAT signaling pathway

Because the STAT homodimer has two phosphotyrosine–SH2 domain

interactions, whereas the receptor-STAT complex is stabilized by only one such
interaction, phosphorylated STATs tend not to rebind to the receptor.

The STAT dimer, which has two exposed nuclear-localization signals (NLS),
moves into the nucleus, where it can bind to promoter sequences and activate
transcription of target genes.
Cytokine receptors

Associated with
cytosolic JAK
kinases

Activate cytosolic
STAT transcription
factors by
phosphorylation
Cytokine receptors

Overview of signal-transduction pathways triggered by ligand

binding to the erythropoietin receptor (EpoR), a typical cytokine
receptor.
Cytokine receptors
JAK-STAT signaling pathway

• Ligand binding
• Functional dimeric receptor
• Activation of an associated JAK kinase
• JAK phosphorylates several tyrosine
residues on the receptor’s cytosolic
domain.
• STAT binds to a phosphotyrosine in the
receptor (by SH2 domain)
• STAT is phosphorylated by active JAK
• Phosphorylated STATs spontaneously
dissociate from the receptor and
spontaneously dimerize.
• STAT (transcription factor) translocate to
the nucleus
Receptor tyrosine kinases
Receptor tyrosine kinases

Ligands:
Insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF),
neurotrophins, other growth factors

Receptor:
Single transmembrane helix;
intrinsic protein tyrosine kinase activity in cytosolic domain

Signal transduction:
(1) Ras–MAP kinase pathway;
(2) IP3/DAG pathway;
(3) PI-3 kinase pathway  insulin

RTK signaling pathways have a wide spectrum of functions including regulation

of cell proliferation and differentiation, promotion of cell survival, an
modulation of cellular metabolism
Receptor tyrosine kinases

Ras–MAP kinase pathway (Step 1)

• RTKs are linked indirectly to Ras via two proteins:

1. GRB2 (growth factor receptor-bound protein 2)  an adapter protein
2. Sos (Son of Sevenless)  which has GEF (guanine nucleotide–exchange
factor) activity

• The SH2 domain in GRB2 binds to a phosphotyrosine in activated RTKs, two

SH3 domains bind Sos  bringing Sos close to membrane-bound Ras-GDP
 activating its nucleotide exchange activity.

• Binding of Sos to inactive Ras causes a large conformational change that

permits release of GDP and binding of GTP, forming active Ras.
Receptor tyrosine kinases

• Ras is an intracellular GTPase switch protein that acts

downstream from most RTKs.
• Like trimeric G protein, Ras cycles between an inactive GDP-
bound form and an active GTP-bound form.
• Ras cycling requires the assistance of two proteins, a guanine
nucleotide–exchange factor (GEF) and a GTPase-activating
protein (GAP).
• GEF  Sos
• GAP  accelerates GTP hydrolysis
Receptor tyrosine kinases

Activation of RTK
Receptor tyrosine kinases

• Ligand Binding
• Functional dimeric receptor
• Transphosphorylation of
Receptor Tyrosine Kinases
• Activated Receptor Tyrosine
Kinases
• RTK bind to SH2 domain in
GRB2
• SH3 domain in GRB2 bind to
SOS

Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs)

Receptor tyrosine kinases

• Sos bind to Ras

• Sos promotes
dissociation of GDP from
Ras
• GTP bind to Ras
• Activated Ras dissociates
from Sos

Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs)

Receptor tyrosine kinases

Ras–MAP kinase pathway (Step 2)

• Activated Ras triggers a kinase cascade in which Raf, MEK, and MAP kinase
are sequentially phosphorylated and thus activated.

• Activated MAP kinase dimerizes and translocates to the nucleus.

• Activation of MAP kinase following stimulation of a growth factor receptor

leads to phosphorylation and activation of two transcription factors, TCF
and SRF.

• These associate into a trimeric complex that promotes transcription of

various early-response genes.
Receptor tyrosine kinases

Ras–MAP kinase pathway

• Active Ras phosphorylated Raf
• Activated Raf
Receptor tyrosine kinases

• Actives Raf phosphorylated

MEK
• Activated MEK
• Actives MEK phosphorylated
MAP Kinase
• Activated MAP kinase
• Translocates to nucleus
• Activates many transcription
factors (example: TCF and SRF)
Transforming growth factor-β (TGF-β ) receptors
 TGF-β receptors

Ligands:
Transforming growth factor β superfamily (TGF β, BMPs),
activin, inhibins (mammals); Dpp ( Drosophila )

Receptors:
Type I and type II (RI and RII): Dimeric transmembrane proteins
with serine/threonine kinases as part of their cytosolic
domains.
Type III (RIII): The most abundant TGF receptor, binds and
concentrates TGF near the cell surface.

Signal transduction:
Direct activation of cytosolic Smad transcription factors

BMP: Bone morphogenetic protein

TGF-β receptors

Smad

• Act as transcription factors.

• The name is derived from a fusion of the gene names

sma (in Caenorhabditis elegans) and
mad (in Drosophila).

• Smads fall into one of three classes:

(1) Receptor-regulated Smads (R-Smads: 1, 2, 3, 5, and 8)
(2) Common-partner Smads (Co-Smads: 4 and 4b)
(3) Inhibitory Smads (I-Smads: 6 and 7).
TGF-β receptors

• Stimulation by TGF β leads to activation of the intrinsic serine /

threonine kinase activity in the cytosolic domain of the type I (RI)
receptor, which then phosphorylates an R-Smad, exposing a nuclear-
localization signal (NLS).

• After phosphorylated, R-Smad binds a co-Smad  the resulting

complex translocates into the nucleus, where it interacts with various
transcription factors to induce expression of target genes.

• Oncoproteins (e.g., Ski and SnoN) and I-Smads (e.g., Smad7) act as
negative regulators of TGF β signaling.

• TGF β signaling generally inhibits cell proliferation. Loss of various

components of the signaling pathway contributes to abnormal cell
proliferation and malignancy.
TGF-β receptors

TGF β-Smad signaling pathway

Step1a :
In some cells, TGF β binds to the type III TGF β receptor (RIII),
which presents it to the type II receptor (RII).
Step1b :
In other cells, TGF β binds directly to RII (a constitutively phosphorylated and
active kinase)
Step2 :
Ligand-bound RII recruits and phosphorylates the type I receptor (RI), which
does not directly bind TGF β.
Step3 :
Activated R1 then phosphorylates Smad3 (or another R-Smad) causing a
conformational change that unmasks its nuclear-localization signal (NLS).
TGF-β receptors

TGF β-Smad signaling pathway

Step 4:
Two phosphorylated molecules of Smad3 interact with one co-Smad (Smad4)
and with importin b (Imp- b), forming a large cytosolic complex.

Steps 5 and 6:
After the entire complex translocates into the nucleus, Ran-GTP causes
dissociation of Imp- b.

Step 7:
A nuclear transcription factor (e.g., TFE3) then associates with the
Smad3/Smad4 complex, forming an activation complex that cooperatively binds
in a precise geometry to regulatory sequences of a target gene.
TGF β-Smad signaling pathway

• Ligand binding
• RII recruits and phosphorylates RI
• Activated RI then phosphorylates
Smad3
• 2 activated Smad3 interact with a co-
Smad (Smad4) and Imp- b
• translocates into the nucleus
• Dissociation of Imp- b by Ran-GTP
• Smad3/Smad4 complex associates
with a nuclear transcription factor
(e.g., TFE3)

Shown at the bottom is the activation complex

for the gene encoding plasminogen activator
inhibitor (PAI-1).
 Ligands:
Atrial natriuretic factor and
related peptide hormones
Receptor:
• Single transmembrane
helix;
• Intrinsic guanylate
cyclase activity in
cytosolic domain
Signal transduction:
Generation of cGMP

Receptor guanylyl cyclases

 T-cell receptors
Ligands:
Small peptides associated with
major histocompatability (MHC)
proteins in the plasma membrane
of macrophages and other
antigen-presenting cells
Receptors:
• Single transmembrane helix;
• Several protein kinases associated
with cytosolic domain;
• Found only on T lymphocytes

Signal transduction:
(1) Activation of cytosolic protein tyrosine kinases;
(2) PI-3 kinase pathway;
(3) IP3/DAG pathway;
(4) Ras–MAP kinase pathway
Intracellular Receptors
Intracellular Receptors

Nuclear Receptor Pathways

Ligands:
Lipophilic molecules including steroid hormones, thyroxine, retinoids,
and fatty acids in mammals and ecdysone in Drosophila

Receptors:
• Highly conserved DNA-binding domain
• hormone-binding domain
• located within nucleus or cytosol

Signal transduction:
Activation of receptor’s transcription factor activity by ligand binding
Intracellular Receptors

Nitric oxide pathway

Ligands: Nitric oxide (NO)

Receptor: Cytosolic guanylyl cyclase
Signal transduction: Generation of cGMP
 Pathways That Involve
Signal-Induced Protein Cleavage
(Irreversible pathway)

• Wnt pathway
• Hedgehog (Hh) pathway
• Notch/Delta pathway
• NF-kB pathways
NF-kB pathways

Ligands:
Tumor necrosis factor (TNF-α), interleukin-1 (mammals)

Receptors:
Various in mammals; Toll and Toll-like receptors in Drosophila

Signal transduction:
Phosphorylation-dependent degradation of inhibitor protein with
release of active NF-β transcription factor in the cytosol
NF-kB pathways
• Stimulation by TNF- or IL-1
induces activation of TAK1
kinase
• activation of the trimeric
I-B kinase
• phosphorylation of I-B by
I-B kinase and binding of
E3 ubiquitin ligase 
polyubiquitination of I-B
 triggering its
immediate degradation by
a proteasome

The removal of
I-B unmasks the nuclear-
localization signals (NLS) in
both subunits of NF-B,
allowing their translocation
to the nucleus
Notch/Delta pathway

Ligands:
Membrane-bound Delta or Serrate protein

Receptors:
Extracellular subunit of Notch receptor noncovalently associated
with transmembrane-cytosolic subunit

Signal transduction:
Intramembrane proteolytic cleavage of receptor transmembrane
domain with release of cytosolic segment that functions as co-
activator for nuclear trascription factors
Notch/Delta pathway

TACE (tumor necrosis

factor alpha
converting enzyme),
releasing the
extracellular segment.

Presenilin 1, an
integral membrane
protein, catalyzes an
intramembrane
cleavage that releases
the cytosolic segment
of Notch.
 Wnt signaling pathway

• The name Wnt is a portmanteau of int and Wg

and stands for "Wingless-related integration
site“.
• Wnt protein in Human:
– WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4,
WNT5A, WNT5B, WNT6, WNT7A, WNT7B,
WNT8A, WNT8B, WNT9A, WNT9B, WNT10A,
WNT10B, WNT11, WNT16
• Wnt comprises a diverse family of secreted lipid-modified
signaling glycoproteins that are 350–400 amino acids in
length.
• The type of lipid modification that occurs on these proteins is
palmitoylation of cysteines in a conserved pattern of 23–24
cysteine residues.
• Palmitoylation is necessary because it initiates targeting of the
Wnt protein to the plasma membrane for secretion and it
allows the Wnt protein to bind its receptor due to the
covalent attachment of fatty acids.
• Wnt proteins also undergo glycosylation, which attaches a
carbohydrate in order to ensure proper secretion.
• In Wnt signaling, these proteins act as ligands to activate the
different Wnt pathways via paracrine and autocrine routes.
Figure 1. Wnt doesn't bind to the receptor. Axin, GSK and APC form a
"destruction complex," and β-Cat is destroyed.
Figure 2. Wnt binds to (activates) the
receptor. Axin is removed from the
"destruction complex." β-Cat moves into
the nucleus, binds to a transcription factor
on DNA, and activates transcription of a
protein. "P" represents phosphate.
Wnt signaling pathways are
• the canonical Wnt pathway,
• the noncanonical planar cell polarity pathway,
• the noncanonical Wnt/calcium pathway.

•  canonical or noncanonical.

• The functions of these categories have a very impact purpose

to the glycerol shape and intestinal homeostasis.
• a canonical pathway involves the protein β-catenin while a
noncanonical pathway operates independently of it.
(Rao TP, Kühl M (Jun 2010). "An updated overview on Wnt signaling pathways: a prelude for
more". Circulation Research. 106 (12): 1798–806.)
Canonical Wnt pathway
Noncanonical Wnt/calcium pathway
Noncanonical PCP pathway
 Clinical implications

• Cancer
• Type II diabetes
Diagram illustrating the interaction between the Wnt and insulin signaling
pathways.
Thank you