Signal Transduction

Prof. Dr. Volker Haucke

Institut fr Chemie-Biochemie
Takustrasse 6
Tel. 030-8385-6920 (secretary)
030-8385-6922 (direct)
e-mail: vhaucke@chemie.fu-berlin.de
http://userpage.chemie.fu-berlin.de/biochemie/aghaucke/teaching.html
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors)
& 13,14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13,
14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Signals in animal cells
a. autocrine (acting on itself)
b. paracrine (acting on near neighbour)
c. endocrine (carried via blood
to distant target cell)
Signals in animal cells
a. autocrine (acting on itself)
b. paracrine (acting on near neighbour)
c. endocrine (carried via blood
to distant target cell)
Characteristics of signal transduction events
Characteristics of signal transduction events
Characteristics of signal transduction events
example: walking from bright sunlight
into a dark room
Characteristics of signal transduction events
example: walking from bright sunlight
into a dark room
-high affinity of receptors for their ligand (K
d
very low)
-cooperativity in ligand-receptor interaction
-amplification of the signal by enzyme cascades
Sensitivity in signal transduction
General features of signal transduction
Step 1: Signal/ ligand interacts with receptor
Step 2: Activated receptor interacts with cellular machinery
(second signal or change in activity of cellular protein)
Step 3: Change in (metabolic) activity of the target cell
Step 4: Signal termination (cell returns to prestimulus state)
General types of signal transducers
General types of signal transducers
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) &
13, 14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Lipid-soluble hormones that bind to nuclear receptors
Steroid hormones
example derived from
Cholesterol
(carbohydrate metabolism)
(synthesized in:
several endocrine tissues incl.
adrenal cortex (cortisol), ovaries
(estrogens) & testes (testosterone)
Lipid-soluble hormones that bind to nuclear receptors
Steroid hormones
example
Retinoids
derived from
Cholesterol
Vitamin A
(retinol)
(carbohydrate metabolism)
(cell differentiation,
growth, survival)
(synthesized in:
several endocrine tissues incl.
adrenal cortex (cortisol), ovaries
(estrogens) & testes (testosterone)
(retinol synthesized in liver from
!-carotene; RA synthesized in
many tissues)
Lipid-soluble hormones that bind to nuclear receptors
Steroid hormones
example
Retinoids
Thyroid hormones
derived from
Cholesterol
Vitamin A
(retinol)
Tyrosine (via iodination
& proteolysis of
thyroglobulin)
(carbohydrate metabolism)
(cell differentiation,
growth, survival)
(stimulates energy yielding
metabolism in liver & muscle)
(synthesized in:
several endocrine tissues incl.
adrenal cortex (cortisol), ovaries
(estrogens) & testes (testosterone)
(synthesized in the thyroid gland)
(T
4
)
(retinol synthesized in liver from
!-carotene; RA synthesized in
many tissues)
Nuclear hormone receptors form a superfamily that
displays a common design
Characteristics:
-variable region-C
4
zinc finger DNA binding domain; ligand binding domain
-ligand binding domain: contains hormone-dependent activation domain (transcr.
activation); may function as repression domain in absence of ligand
Nuclear hormone receptors contain a repeat of the C
4
zinc
finger DNA binding motif
Characteristics:
-zinc ions serve to stabilize loop structure and orientation of helices
-interaction between DNA & individual zinc finger is weak; usually requires several Zn
2+
fingers
-nuclear hormone receptors bind to DNA usually as homo- or heterodimers
Nuclear hormone receptors contain a repeat of the C
4
zinc
finger DNA binding motif
Characteristics:
-zinc ions serve to stabilize loop structure and orientation of helices
-interaction between DNA & individual zinc finger is weak; usually requires several Zn
2+
fingers
-nuclear hormone receptors bind to DNA usually as homo- or heterodimers
Hormone response elements (HRE) contain inverted or direct repeats
glucocorticoid receptor (GRE)
estrogen receptor (ERE)
vitamin D
3
receptor (VDRE)
thyroid hormone receptor (TRE)
retinoic acid receptor (RARE)
inverted repeat recognition:
by symmetrical homodimeric hormone receptors
direct repeat recognition:
by heterodimers with common RXR receptor monomer
Hormone-dependent gene activation involves translocation of homodimeric
nuclear receptors into the cell nucleus
1. hormone diffusion through the
plasma membrane
2. binding to receptor (conformational
change) & dissociation of inhibitor
proteins in the cytoplasm
3. translocation of hormone-receptor
complex into the cell nucleus
4. DNA binding & transcriptional
activation
Hormone-dependent gene activation involves translocation of homodimeric
nuclear receptors into the cell nucleus
1. hormone diffusion through the
plasma membrane
2. binding to receptor (conformational
change) & dissociation of inhibitor
proteins in the cytoplasm
3. translocation of hormone-receptor
complex into the cell nucleus
4. DNA binding & transcriptional
activation
Hormone-dependent gene activation involves translocation of homodimeric
nuclear receptors into the cell nucleus
1. hormone diffusion through the
plasma membrane
2. binding to receptor (conformational
change) & dissociation of inhibitor
proteins in the cytoplasm
3. translocation of hormone-receptor
complex into the cell nucleus
4. DNA binding & transcriptional
activation
Hormone-dependent gene activation involves translocation of homodimeric
nuclear receptors into the cell nucleus
+ hormone
- hormone
1. hormone diffusion through the
plasma membrane
2. binding to receptor (conformational
change) & dissociation of inhibitor
proteins in the cytoplasm
3. translocation of hormone-receptor
complex into the cell nucleus
4. DNA binding & transcriptional
activation
Hormone-dependent gene activation involves translocation of homodimeric
nuclear receptors into the cell nucleus
1. hormone diffusion through the
plasma membrane
2. binding to receptor (conformational
change) & dissociation of inhibitor
proteins in the cytoplasm
3. translocation of hormone-receptor
complex into the cell nucleus
4. DNA binding & transcriptional
activation
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors) & 13,
14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Overview of the major classes of cell surface signaling receptors
Receptor(s)
Ligand(s)
Quantitative determination of ligand binding to receptors
Quantitative determination of ligand binding to receptors
Quantitative determination of ligand binding to receptors
bound
free
[RL]
[L]
=
=
1
K
d
([R
T
] - [RL])
bound
total
[RL]
[R
T
]
= =
1
1 + K
d
/ [L]
(=R
T
)
Scatchard Plot
Overview of the major classes of cell surface signaling receptors
Receptor
Second
messenger
Response
(i.e.
transcription)
Common second messenger as intracellular signaling molecules
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors)
& 13, 14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Overview of the major classes of cell surface signaling receptors
Receptor
Second
messenger
Response
(i.e.
transcription)
General structure of G protein-coupled receptors
features:
7 transmembrane "-helices (H1,..,H7)
N-term. exoplasmic; C-term. cytosolic
C3/ C4 loop as interacting sites for heterotrimeric G proteins
G protein-coupled receptors (GPCRs) may signal via
different effector pathways including adenylyl cyclase
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
(i.e. adenylyl cyclase
[AC])
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
G protein-coupled receptors (GPCRs) may signal via
different effector pathways including adenylyl cyclase
Signal cascade involving G protein-coupled receptors (GPCRs)
& adenylyl cyclase
Epinephrine signals via different types of G protein coupled
adrenergic receptors
Adrenaline= Epinephrine
-released from adrenal gland
-regulates energy metabolism
in muscle, liver and adipose tissue
(i.e. glycogen and fat breakdown)
- antagonizes insulin action
-four types of receptors:
"1, "2, !1, !2; !-receptors found in
muscle, liver, and adipose tissue
(also used as neurotransmitter in
adrenergic neurons)
Common second messenger as intracellular signaling molecules
Transduction of the epinephrine signal: the !-adrenergic pathway
Transduction of the epinephrine signal: the !-adrenergic pathway
Adenylyl cyclase (AC):
-integral plasma membrane
protein
-active site on cytoplasmic side
Interaction of (active) G
s"
with adenylyl cyclase
AC catalyses the synthesis of cAMP from ATP
enzymatic activity stimulated by active G
sa
Transduction of the epinephrine signal: the !-adrenergic pathway
cAMP activates protein kinase A (PKA)
Epinephrine signals breakdown of glycogen via
!-adrenergic receptor activation
Signal amplification in the epinephrine cascade
Termination of the cAMP signal
Signal termination (1): cAMP hydrolysis via cyclic
nucleotide phosphodiesterase (PDE)
Caffeine & theophylline: inhibit PDE, increase cAMP half-life, and potentiate
AC-mediated effects
Signal termination (2): phosphorylation-dependent
receptor desensitization
(!ARK is a member of
the G protein-coupled receptor
kinases (GRKs))
G protein-coupled receptors (GPCRs) may signal via
different effector pathways including adenylyl cyclase
"
1
# and !
2
-adrenergic receptors are coupled to different G proteins
liver, adipose tissue
i.e. cardiomyocytes regulation of
contraction
lipolysis,
glycogenolysis
Expression Effect
1
G protein-coupled receptors (GPCRs) may signal via
different effector pathways including phospholipase C
Common second messenger as intracellular signaling molecules
Ligand induced activation of signal-transducing G proteins
associated with GPCRs
(i.e. phospholipase C-!
[PLC !])
G
"o
or G
"q
Common second messenger as intracellular signaling molecules
DAG and IP
3
are second messengers derived from phosphatidyl-
inositol 4,5-bisphosphate (PIP
2
) by PLC-mediated hydrolysis
Signaling via DAG and IP
3
involves opening of calcium channels in
the plasma membrane & the ER as well as PKC activation
Calcium and IP
3
mediated signaling events
SUMMARY I - Signaling
1. Signaling in animal cells can be autocrine, paracrine or endocrine and are
characterized by specificity, amplification, desensitization, and integration. Scatchard
analysis can be used to quantitatively describe receptor-ligand interactions.
2. Steroid & thyroid hormones as well as retinoids bind to nuclear hormone receptors
that contain related C
4
-zinc finger DNA binding domains. Upon ligand association
dimerized receptors activate gene transcription in the cell nucleus by binding to hormone
response elements (HREs) consisting of direct or inverted repeats.
3. G protein-coupled receptors activate heterotrimeric (",!,$) GTP- binding (G)
proteins. In the case of the !#adrenergic receptor G
sa
then activates adenylyl
cyclase (AC) which generates cAMP as a second messenger, thus amplifying the signal.
4. Calcium and IP
3
are also second messengers acting downstream of G protein coupled
and other receptors that are generated by phospholipase C (PLC)-mediated cleavage of
the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP
2
).
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors)
& 13, 14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Receptor enzymes - receptor tyrosine kinases
The Ras-MAPK pathway regulates development of R7
photoreceptors in flies via a receptor tyrosine kinase
sevenless mutation:
loss of R7 photoreceptors
molecular reason:
mutated EGF receptor encoded
by sev gene (sev
-
)
The Ras-MAPK pathway is conserved from mammals to flies
where it regulates cell proliferation and development
Activation of Ras following ligand binding to receptor tyrosine kinases
(RTKs)
Activation of Ras following ligand binding to receptor tyrosine kinases
(RTKs)
Activation of Ras following ligand binding to receptor tyrosine kinases
(RTKs)
Sos (GEF) binding to Ras facilitates GTP binding by inducing release
of GDP via a conformational change that results in Ras activation
Ras defines a superfamily of related small GTPases involved in
signaling and membrane traffic
A kinase cascade transmits signals downstream from activated
Ras protein to MAP kinase
Cytokine receptors & the JAK-STAT pathway
Cytokine receptors & the JAK-STAT pathway
Cytokines:
family of small (about 160 amino acids), secreted
proteins that control cell growth & differentiation
examples:
IL-2: proliferation of T cells in the immune system
interferons: secreted in response to viral infections
Epo: produced by kidney cells in response to low
oxygen; induces red blood cell differentiation from
hematopoietic stem cells
Erythropoietin regulates hematopoiesis via
a cytokine receptor & the JAK-STAT pathway
Erythropoietin binds to a dimeric
receptor (EpoR) on erythroid
progenitor cells (CFU-E)
Erythropoietin binds to a dimeric
receptor (EpoR) on erythroid
progenitor cells (CFU-E)
The Epo receptor (EpoR) and JAK are essential for
development of erythrocytes
fetal liver
(day 13 embryo)
Epo binding to its receptor (EpoR) triggers the JAK-STAT
signaling pathway
JAK (=janus kinase):
N-term. receptor binding domain
C-term. kinase domain (Tyr kinase)
Epo binding:
(1) induces conformational change
in the EpoR
(2) autophosphorylation of JA at
Tyr of activation lip (approximation)
(3) activated JAK Tyr-phosphorylates
EpoR on cytoplasmic tail
(4) binding of STAT via SH2 domain
to P-Tyr; phosphorylation of C-term.
Tyr residues in STAT by JAK
Epo binding to its receptor (EpoR) triggers the JAK-STAT
signaling pathway
JAK (=janus kinase):
N-term. receptor binding domain
C-term. kinase domain (Tyr kinase)
Epo binding:
(1) induces conformational change
in the EpoR
(2) autophosphorylation of JA at
Tyr of activation lip (approximation)
(3) activated JAK Tyr-phosphorylates
EpoR on cytoplasmic tail
(4) binding of STAT via SH2 domain
to P-Tyr; phosphorylation of C-term.
Tyr residues in STAT by JAK
Epo binding to its receptor (EpoR) triggers the JAK-STAT
signaling pathway
JAK (=janus kinase):
N-term. receptor binding domain
C-term. kinase domain (Tyr kinase)
Epo binding:
(1) induces conformational change
in the EpoR
(2) autophosphorylation of JA at
Tyr of activation lip (approximation)
(3) activated JAK Tyr-phosphorylates
EpoR on cytoplasmic tail
(4) binding of STAT via SH2 domain
to P-Tyr; phosphorylation of C-term.
Tyr residues in STAT by JAK
Epo binding to its receptor (EpoR) triggers the JAK-STAT
signaling pathway
(5) STAT dimer goes into the nucleus
& activates transcription
JAK (=janus kinase):
N-term. receptor binding domain
C-term. kinase domain (Tyr kinase)
Epo binding:
(1) induces conformational change
in the EpoR
(2) autophosphorylation of JA at
Tyr of activation lip (approximation)
(3) activated JAK Tyr-phosphorylates
EpoR on cytoplasmic tail
(4) binding of STAT via SH2 domain
to P-Tyr; phosphorylation of C-term.
Tyr residues in STAT by JAK
Termination of the JAK-STAT signaling pathway
Dephosphorylation of JAK
Blocking and JAK degradation
Erythropoietin binds to a dimeric
receptor (EpoR) on erythroid
progenitor cells (CFU-E)
Generation of phosphatidylinositol 3-phosphates
downstream of RTKs and cytokine receptors
PI 3-kinase:
-several isoforms; i.e. p85-p110
dimer
-recruited to membrane via SH2
domain downstream of
phosphotyrosine formation
-PI-3 phosphates produced which
transduce signals, i.e. via
protein kinase B (PKB; a Ser/ Thr
protein kinase)
Phosphatidylinositol 3-phosphates activate protein kinase B via
binding to its PH domain
Protein kinase B (PKB; a Ser/ Thr protein kinase)
-PH (=pleckstrin homology) domain of PKB binds to inositol 3-phosphates leading to
partial activation at the PM
-co-activated by PDK1 which also harbors a PH domain
Introduction: Lehninger, Principles of Biochemistry, 3rd ed., chapter 13;
In depth: Lodish, Molecular Cell Biology, 5th ed., chapters 11 (nuclear hormone receptors)
& 13, 14 (cell surface signaling)
1. Overview over the molecular mechanisms of signal transduction
2. Nuclear hormone receptors (i.e. steroid hormone receptors etc.)
3. Cell surface receptors & intracellular signaling molecules
4. G protein-coupled receptors
5. Tyrosine kinase & kinase associated receptors (Ras-MAPK
pathway, JAK-STAT pathway)
6. Communication by gap junctions and ion channels
Signal Transduction
Gap junctions mediate communication by coupling cells
Gap junctions are intercytoplasmic junctions that allow
diffusion of small molecules and ions
Gap junctions
-intercytoplasmic junction
-transport of ions & small molecules
(<1-2 kDa)
(i.e second messengers) between
adjacent cells; metabolic coupling
(i.e. in muscle to create a syncytium)
-opening regulated by calcium, pH
etc.
-composed of connexons built from
12 connexin proteins (transmembrane
protein with
4 transmembrane segments); 2
connexon hemi-
channels
Neuronal signaling via gated ion channels
-transmembrane potential of -60mV
(membrane is polarized) is generated
by active transport
-passive ion fluxes include:
a. depolarization by: efflux of anions or
influx of cations
b. hyperpolarization: influx of anions or
efflux of cations
The transmembrane electrical potential forms the basis for signaling
via voltage- and ligand-gated ion channels
The transmembrane electrical potential forms the basis for signaling
via voltage- and ligand-gated ion channels
-ion fluxes depend on electrochemical
gradient: %G
0
= RT lnK + nF V
m
-ions move passively until equilibrium
potential E is reached (%G
0
=0)
- specific channel opening leads to change
of membrane potential towards E for that ion
-action potential first opens Na
+
, and then
K
+
channels
-electrical signal moves along the axon until
it reaches nerve terminal
-Ca
2+
channels open leading to neurotrans-
mitter release
-neurotransmitter binds to postsynaptic receptor
(ligand-gated ion channels)
-ion flux on postsynaptic side may elicit an
action potential
Ion channels are involved in neuronal signaling
Ion channels are involved in neuronal signaling
-action potential first opens Na
+
, and then
K
+
channels
-electrical signal moves along the axon until
it reaches nerve terminal
-Ca
2+
channels open leading to neurotrans-
mitter release
-neurotransmitter binds to postsynaptic receptor
(ligand-gated ion channels)
-ion flux on postsynaptic side may elicit an
action potential
Action potentials involve consecutive opening of voltage-sensitive
sodium (Na
+
) and potassium (K
+
) channels
-action potential first opens Na
+
, and then
K
+
channels
-electrical signal moves along the axon until
it reaches nerve terminal
Ligand-gated ion channels in neuronal signaling
-action potential opens Na
+
and
K
+
channels
-electrical signal moves along the axon until
it reaches nerve terminal
-Ca
2+
channels open leading to neurotrans-
mitter release
-neurotransmitter binds to postsynaptic receptor
(ligand-gated ion channels)
-ion flux on postsynaptic side may elicit an
action potential
Membrane depolarization involves opening of ligand-gated ion
channels such as the acetylcholine receptor
Gating of the nACh receptor, a ligand-gated ion channel
Many postsynaptic neurotransmitter receptors are
ligand-gated ion channels
Glutamate
Glycine
nicotinic acetylcholine receptor
(AChR)
Ligand/ Neurotransmitter
Neurotransmitter receptor
(ligand-gated ion channel)
serotonin receptor
glutamate receptors
(AMPAR, NMDAR)
glycine receptor (GlyR)
SUMMARY II - Signaling
1. Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR)
trigger Sos-induced activation of Ras which in turn activates the MAP kinase cascade to
regulate transcription of target genes.
2. Cytokine receptors which are not enzymes themselves among others activate the receptor-
associated kinase JAK which becomes autophosphorylated, and further activated by STAT.
STAT phosphorylated by JAK dimerizes via its SH2 domain, translocates to the nucleus and
activates transcription. The Epo-EpoR system regulates hematopoiesis via the JAK-STAT
pathway.
3. Many RTKs or cytokine receptors may also signal via recruitment of phosphatidylinositol
3-kinase and subsequent PH-domain-dependent activation of protein kinase B (PKB).
4. Connexins form connexon hemi-channels that assemble into intercytoplasmic gap junctions
that allow electrical coupling between muscle cells or exchange of metabolites and signaling
molecules.
5. The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel at the
postsynaptic membrane. Opening allows cation flux and may elicit an action potential in the
postsynaptic cell.