[VII]. Regulation of Gene Expression Via
Signal Transduction
Reading List VII:


Signal transduction
Signal transduction in biological systems
External Signal Regulating the Expression of Genes
Signals
Signal transduction cascades
or
Gene Expression
Cytoplasmic
mechanism/muscle
contraction/etc.
New Proteins
mRNAs
Proteins
Communication between
Matting Yeast Cells
•
•
•
•
Yeast cells use chemical
signaling to communicate
with the opposite mating
types and initiate mating
process
Two mating type factors are
a and b
The mating factors are
peptides of about 11 amino
acid residues
Receptors on the surface of
the yeast cells recognize
the specific mating type
factor
Communication among Bacteria
Individual cells
Aggregation in progress Spore forming
Myxobacteria (Myxococcus xanthus, slime bacteria) use chemical
signaling to share information about nutrient availability. When food
is limited, starving cells secrete a molecule that enters neighboring
cells and stimulate them to aggregate. The cells form a structure that
produces thick-walled spores capable of surviving until the
environment improves.
Iron-Dependent Regulation of Translation Degradation
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•
•
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Iron binds to transferrin in the
circulation and transported into the
cell by binding to transferrin-receptor
In the cytoplasm, Iron is bound to
ferritin
IRE-BP: iron-response element binding
protein. Active form of IRE-BP can
bind to IREs (iron response element).
IRE-BP can regulate the translation of
ferritin mRNA and the stability of
transferrin receptor
Under low concentration of intracellular concentration, IRE-BP is active
and can bind to the IREs in the 5’-end
of ferritin mRNA and inhibit the
translation of ferritin mRNA
The 3’end of transferrin receptor
mRNA has IREs sequence. Binding of
IRE-BP to these IREs will result in
degradation of transferrin receptor
mRNA
Characteristics of Signals
•
•
•
Have specificity:
unique, can only be
detected by the
molecular machinery
designed for the
detection
Small and easy
traveling to the site of
action
Easily made, mobilized,
altered relatively
quickly, and easily
destroyed
Signaling Via Cell-Surface Receptors (I)

Synthesis and release of
signaling molecules by
signaling cells (step 1&2)

Transport of signaling
molecules to the target
cells (step 3)

Binding of the signaling
molecule with a specific
receptor protein on the
membrane leading to
activation (step 4)
Signaling Via Cell-Surface Receptors (II)

Initiating one or more
intracellular signaltransduction pathways
initiated by the activated
receptor (step 5)

Specific change in
cellular response
(cellular function,
metabolic change or
gene expression) (step
6a & 6b)

Removal of the signal to
terminate the cellular
response (step 7)
Different Ways Cells Signal Each Other
• Endocrine signaling
• Paracrine signaling
• Autocrine signaling
• Signaling by plasma
membrane-attached
proteins
Chemical Identity of Signals
•
•
•
•
•
•
•
Peptides & Protein Hormones (most abundant): e.g.,
thyrotropin, Gonadotropin releasing hormone
(GnRH), growth hormone (GH), prolactin (PRL),
Insulin etc.
Amino Acid Derivatives: thyroid Hormone,
epinephrien
Steroid Hormones: testosterone, estrogen, cortisone
etc.
Lipids: prostaglandin & retinoic acid
Nucleotides: cAMP, cytokinins, 1-methylalanine
Oligosaccharides: α-1,4-oligogalaturonide
Gases: CO, ethylene etc.
Receptor Proteins Exhibit Ligand-Binding &
Effector Specificity
Dimers
•
•
Each ligand binds to its specific receptor due to binding specificity and
the receptor-ligand complex in turn will exhibit a specific effect (effector
specificity)
Different receptors of the same class that bind different ligands often
induce the same cellular response in a cell
Characteristics of Hormone Receptors
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Hormone receptor should possess high affinity: higher than 10-8
M (10 nM)
Hormone receptor should have high specificity to prevent cross
binding with un-related hormones
Hormone receptor could be saturated, means that it should
have finite number of binding sites. This character separates
them from non-receptors
Hormone – receptor binding should be reversible
Hormone receptor should have a tissue distribution appropriate
to its action (tissue specificity)
Hormone receptor binding should be correlated with some
biological effects
How is the receptor molecule of a bioregulator (hormone)
identified???
Assumptions Used in Assaying Hormone Receptors
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•
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The labeled bioregulator is biologically identical to the native
bioregulator
The labeled bioregulator is homogenous
 Iodination of hormone can generate a mixture of uniodinated,
monoiodinated, and diiodinated species
The receptor is homogenous
 Many receptors exist in multiple forms such as a- and badrenergic receptors. If both receptors are present in a sample
and their affinity are sufficiently different, Scatchard analysis
will not give a curve-linear plot
The receptor acts independently
 Many cytokine receptors are multimeric. Each subunit has low
to intermediate affinity whereas the intact complex has high
affinity
 In growth hormone, binding of growth hormone to its receptor
will induce homodimer formation that has higher affinity than
monomer
Assumptions Used in Assaying Hormone Receptors
•
•
•
The receptor is unoccupied
 If some of the receptor is already occupied by endogenous
hormone and if the affinity is sufficiently high to be replaced by
the radio-labeled hormone, it will not be detected in the assay,
and thus leading to underestimation of the affinity of the
hormone
The reaction is at equilibrium
 Both hormone and the receptor are stable
 The reaction is reversible
 The equilibrium is not perturbed when F (free) and B (bound)
ligands are separated
 It is necessary to keep the hormone and receptor stable by
using protease inhibitors or run the incubation under low
temperature for long time to reach equilibrium
 Internalization of hormone is a problem in determining binding
of hormone in whole cell
There is no specific nonreceptor binding
Receptor Ligand Interaction
kon
R + L
RL
koff
At equilibrium:
Kd =
koff
kon
[R] . [L]
=
[RL]
Where [R] and [L] are the
concentration of free receptor &
ligand at equilibrium. [RL] is the
concentration of the receptorligand complex. Kd is the
dissociation constant
And
Ka (association constant) = 1/Kd = [RL]/[R].[L]
From this equation, one can see that Ka equals to the ratio of
bound [RL] to free ligand [L]
Receptor Binding Assay
•
•
•
•
Preparation of receptors:
 It is important to prevent destruction of receptors by proteolytic
digestion by proteases
 For cytosol or nuclear receptors: prepare cytosol or nuclear soluble
fraction
 For membrane receptors: using whole cells or cell membrane fraction
Prepare radio-labeled hormone
 3H-labeled hormones or 125-labeled hormones
Setting up reaction for measuring total binding
 Incubate various amount of radiolabeled hormone in an appropriate
buffer with a fixed amount of receptor preparation at 4o C for a fixed
period of time to reach equilibrium
 Separate the bound hormone from the unbound hormone
Setting up reaction for measuring non-specific binding
 Incubate various amount of radiolabeled hormone and 500 to 10000
folds excess of un-labeled hormone in an appropriate buffer with a fixed
amount of receptor preparation for a fixed period of time at 4o C
 Separate the bound hormone with the unbound hormone
Binding Assays Are Used to Detect Receptors
and Determine Their Kd Values
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•
(Free Ligand)
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•
Binding assay is used to
demonstrate the presence
of receptors. Both the
number of the ligandbinding sites per cell and
the Kd value are easily
determined from the binding
assay
Figure in the left shows the
binding of ligand (insulin) to
the receptors with high
affinity
High affinity binding, Kd = 10-8 M or lower; Low affinity binding, Kd = 10-7 M or
higher (larger)
If the Kd is larger than 10-7 M, the bound ligand can easily fall off the
receptors in the process of separating unbound ligand from the bound
ligand. A competitive binding assay can be used instead
Scatchard Plot
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•
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•
Slop= -1/Kd
n = number of
receptors ~ number of
binding sites
From this plot, one
can easily figure out
Kd and number of the
binding site of the
receptor
Scatchard Plot
•
If the plot gives a bi-phasic
line, it means that the
receptor contains multiple
binding sites with different
affinities or the presence of
multiple receptors binding to
the same ligand
Use of a Competitive Binding Assay to Detect
Binding of Low Affinity Ligands to Receptors
•
•
•
One way to
determine weak
binding of a
ligand to its
receptor is in a
competition assay
with another
ligand that binds
to the same
receptor with
higher affinity
Alprenolol, a synthetic high affinity ligand to epinephrine receptor;
Epinephrine, natural hormone; isoproternol, an antagonist to
epinephrene.
The Kd of the competitor can be determined at the 50% competition.
The Kd for epinephrine is 5 x 10-5 M
Insulin-Like Growth Factor (IGF) I
Primary translation product of IGF-I
B
S
C
A
D
E
Post-translational processing
Signal peptide
S
B
C
A
Mature IGF-I
D
E
E-peptide
Multiple Forms of Pro-IGF-I E-Peptide
Mature IGF-I
Human pro-IGF-Ia
Human pro-IGF-Ib
Human pro-IGF-Ic
Trout pro-IGF-I Ea-4
Trout pro-IGF-I Ea-3
Trout pro-IGF-I Ea-2
Trout pro-IGF-I Ea-1
B
C
A
E
D
Anti-Tumor Activities of the
Pro-IGF-I E-peptide
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•
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Induces morphological differentiation and inhibits
anchorage-independent growth in oncogenic
transformed cell lines (Chen et al., 2002; Kuo and
Chen, 2002)
Inhibits tumor cell growth and invasion, and tumorinduced angiogenesis in developing chicken embryos
(Chen et al., 2007)
Induces programmed cell death of cancer cells (Chen
et al., 2012)
Up-regulate fibronectin 1 and laminin receptor genes
and down-regulate uPA, tPA and TIMP1 genes (Siri
and Chen 2006a, 2006b; Chen et al., 2007)
Is there a specific membrane receptor
present on the membrane of cancer cells
that binds to E-peptide?
To answer this question, we used binding assay to
demonstrate the presence of specific receptor molecules on
the membrane cancer cells
Binding of 35S-E-Peptide to SK-N-F1 Cells
Human Eb-peptide
Kd = 2.9 ± 1.8 x 10-11 M
Trout Ea4-peptide
Kd = 2.9 ± 1.8 x 10-11 M
Competitive
Displacement Assay
A. Labeled hEb was
competed out with
unlabeled hEb
B. Labeled rtEa4 was
competed out with
unlabeled rtEa4
Competitive
Displacement Assay
C. Labeled hEb competitive
with unlabeled rtEa4
D. Labeled rtEa4 competitive
with unlabeled hEb
Competitive Binding Assay with
hIGF-I
The data suggest that E-peptide does not bind to the same receptor
that binds IGF-I
Maximal Physiological Response to Many External Signal Occurs When
Only a Fraction of the Receptor Molecules are Occupied by Ligand
•
In all signaling systems, the
affinity for any signaling
molecules to its receptor
must be greater than the
normal physiological level
of the signaling molecule
Take insulin for example,
the kd of insulin to its
receptor is 1.4 x 10-10M, and
the circulating insulin is 5 x
10-12M. By substituting
these number into the equation: Kd= [R][L]/[RL], at equilibrium, about 3% of the
total insulin receptors are bound by insulin. If the circulating concentration of
insulin rises five fold to 2.5 x 10-11M, the number of the receptor-hormone
complexes will rise about 5 fold to 15% of the total receptors are bound by
insulin
In many cases, the maximum cellular response to a particular ligand is
induced when less than 100% of its receptors are bound to the ligand. The
example is shown in the figure above
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•
Sensitivity of a Cell to External Signals is
Determined by the Number of Surface Receptors
•
•
The cellular response to a particular signaling molecular depends
on the number of receptor-ligand complex. The fewer receptors
present on the surface of the cell, the less sensitive is the cell to the
ligand
In the erythroid progenitor cells, the Kd for binding of erythropoietin
(Epo) is 10-10 M. Only 10% of the 1000 cell-surface erythropoietin
receptors must be bound to ligand to induce maximum cellular
response. By following the equation below, we can calculate the [L]
needed to induce the response:
Kd
[L] =
RT/ [RL] - 1
• Where [RT] = [R] + [RL]
•
If the RT=1000, Kd = 10-10 M, [RL] = 100, the [Epo] = 10-11 M will elicit
the maximal response. If RT = 200, 10-10 M of erythropoietin will be
required to occupy 100 receptors to elicit the maximum response
Purification of l Receptor (I)
• Purification of nuclear receptors by conventional
methods
 Ammonium sulfate fractionation
 Chromatography on ion exchange column (DEAE
cellulose or phospho-cellulose)
 Chromatography on size exclusion column (sepharose
gel, agarose gel or polyacrylamide gel)
 Polyacrylamide gel electrophoresis
 Isoelectric focusing
Purification of Receptors (II)
•
Membrane receptors can be purified by:
 Affinity binding method
 Label the ligand with isotope
 Binding of the labeled ligand to cells that may contain the
desired receptor, washing off the unbound ligand and
covalent bound the ligand to the receptor
 Isolate the membrane fraction, dissolve the membrane
protein and purify the receptor
 Affinity Chromatography
•
 Link the ligand to beads (agarose or polyacrylamide) and
pack the beads in a column
 Pass the crude extract of membrane fraction containing
receptors through the column, wash column several times to
remove the contaminants
 Elute the column with excess amounts of ligand and the
receptor will be eluted from the column
These methods are suitable for the isolation of high
affinity membrane receptors
Purification of hEb Binding Component from
MDA-MB-231 Cell Membrane
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•
A Functional Assay to
Confirm the Identity of a
Receptor cDNA
Once a receptor is purified, the partial
sequence of the receptor can be
identified mass spectrometer analysis.
This information can be used to clone
the full-length cDNA of the receptor
The identity of the receptor cDNA can
be confirmed by the method depicted
in the figure on the left of this slide
An expression construct with the fulllength of receptor cDNA is transfected
into a cell line that dose not have the
endogeneous receptor in question.
The transfected cells will express the
desired receptor which can be detected
by receptor binding assay
Reading List VII:
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Isolatiion and characterization of colagen
receptor
Isolation of interleukins by immunoaffinityreceptor affinity chromatography
Isolation, characterization and regulation of the
prolactin receptor
Isolation and characterization of human
prolactin receptor
The General Structure of a Membrane Receptor
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A signal molecule
binds to a
receptor protein,
causing to change
shape
Most signal
receptors are
plasma membrane
proteins
G-protein-coupled
receptor, tyrosine
kinase receptor,
ligand-gated ionchannel receptor
etc.
Receptors Activate a Limited Number of
Signaling Pathways (I)
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There are seven classes of membrane receptors that
can receive external signaling molecules:
 G-protein-coupled receptors, cytokine receptors, receptor
tyrosine kineses, TGFb receptors, Hedgehog receptors, Wnt
receptors, Notch receptor
External signals induces two types of cellular
responses:
 Change in the activity or function of specific pre-existing
proteins (Activating enzymes)
 Changes in amounts of specific proteins produced by a cell
as a result of activation of genes (gene expression)
Signaling from G-protein-coupled receptors often
results in changes in the activity of pre-existing
proteins, but it can also result in activation of gene
expression
Receptors Activate a Limited Number of
Signaling Pathways (II)
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The other classes of receptors operate primarily to
modulate gene expression:
 The activated TGFb and cytokine receptors directly activate a
transcription factor in the cytosol
 The Wnt receptors assemble an intracellular signaling
complex to the cytosol transcription factors
 Tyrosine receptor kinases activate several cytosolic protein
kinases that translocate into nucleus and regulate the activity
of nucleus transcription factors
Some classes of receptors can initiate signaling via
more than one intracellular signal-transduction
pathways, leading to different responses. This is
typical of G-protein-coupled receptors, receptor
tyrosine kinases and cytokine receptors
Only limited number of signal transduction
mechanisms are responsible for signal transduction
Seven Major Classes of Cell-Surface
Receptors
Four Common Intracellular Second Messenger
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Besides signaling molecules from
outside of the cells, there are additional
micromolecules from inside of the cells
that are involved in signal transfer.
These are second messengers
Second messengers carry and amplify
signals from receptors
Binding of the signaling molecules to
many cell surface receptors leads to a
short-lived increase in the
concentration of low molecular weight
intracellular signaling molecules (i.e.
second messengers)
These molecules include cAMP, cGMP,
DAG, IP3, Ca++, and inositol
phospholipids (phosphoinositide
embedded in cellular membranes)
Appropriate Cellular Responses Depend on
Interaction and Regulation of Signal Pathways
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Activation of a single type receptor often leads to
production of multiple second messengers which
have different effects
The same cellular response may be induced by
activation of multiple signaling pathways. Such
interaction of different signaling pathways permits the
fine-tuning of cellular activities required to carry out
complex developmental and physiological processes
Regulation of signaling pathways is critical for the cell
to response to signals properly
Cells down regulate the effects of signal transduction
processes by degrading second messengers,
deactivate signal transduction proteins, desensitizing
the receptors or removing the signaling molecules by
endocytosis etc.
Overview of Cell Signaling
Reception
Transduction
Response
The components of intracellular signal transduction pathways are
highly conserved
Signal Transduction Pathways
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Signal on the membrane receptors will be transduced
by a multi-step pathway in order to amplify a signal
Protein phosphorylation by protein kinase is a major
mechanism of signal transduction
Unlike receptor tyrosine kinases, cytoplasmic protein
kinases do not phosphorylate themselves but
phosphorylate other substrate proteins on
serine/threonine residues (serine/threonine kinase)
About 1% of our genes are thought to code for protein
kinases, indicating the importance of protein kinases in
the cell
The activated protein kinases are quickly reversed by
protein phosphatases
Protein Kinases
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•
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•
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Protein kinases and phosphatases are used in virtually all
signaling pathways
Protein kinases: enzymes add phosphate groups to the OHgroup of tyrosine, serine or threonine of its own or other proteins
Phosphatases: enzymes remove phosphate groups from
proteins
In human genome, there are at least 600 genes encoding for
different protein kinases and 100 genes encoding different
phosphatases
In some of the signaling pathways, receptor itself possesses
intrinc kinase activity. It can phosphorylate itself upon binding to
its ligand
The activity of all protein kinases is opposed by the activity of
protein phosphatases
A Phosphorylation
Cascade
•
G-Protein Coupled Receptors
•
Receptor Tyrosine Kinase
 G-protein-coupled receptors that regulate ion
channels
 G-protein-coupled receptors that activate or inhibit
adenylyl cyclase
 G-protein-coupled receptors that activate
phospholipase C
 Activation of G protein-coupled receptors leading
to gene expression
General Elements of G Protein-Coupled Receptors
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•
•
•
G protein-coupled receptors (GPCRs) are the most numerous
class of receptors found in organisms from yeast to human
All GPCR signaling pathways share the following common
elements:
 A receptor that contains seven membrane-spanning elements
(transmembrane domains)
 A coupled trimeric G protein which functions as a switch by cycling
between active and inactive forms (activator or inhibitory)
 A membrane-bound effector protein
 Feedback regulation and desensitization of the signaling pathway
A second messenger also occurs in many GPCR pathways, and
these components are modular and can be mixed and matched
GPCR pathways have short term effects in cells by quickly
modifying existing proteins or enzymes or ion channels, but also
long term effects involving change in transcription leading to
differentiation
General Structure of G Protein-coupled Receptor
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•
•
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•
G Protein-coupled
receptors are a
large and diverse
families with a
common structure
and function
GPCR activate
exchange of GTP
for GDP on the asubmit of a
Trimeric G protein
G-protein coupled receptors consists of hydrophobic amino acids
that allow proteins to be stably anchored in the hydrophobic core
of the membrane (seven membrane spanning domains)
Loops C3 and C4 are involved in binding to G protein. In some
cases, C 2 is also involved
There are several sub-families of G protein-coupled receptors with
high conservation of amino acid sequence and structure
Switching Mechanism for Monomeric & Trimeric G Proteins
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External signals induce two types of
cellular responses:
 Change the activity or function of
specific enzymes or proteins
 Change the amount of proteins in
the cell via modification of
transcription factors
Trimeric and monomeric G proteins:
GTPase Switch Proteins, belong to
GTPase superfamily proteins. These
guanine nucleotide-binding proteins
are turned “on” when bound to GTP
and turned “off” when bound to GDP.
The signal-induced conversion from
the inactive to active state is
mediated by a guanine nucleotideexchange factor (GEF)
Subsequent binding of GTP induces a conformational change in two
segments of the G protein, switch I and II, allowing the protein to bind to and
activate other downstream signaling proteins
The rate of GTP hydrolysis is enhanced by GTPase-activating protein (GAP)
and a regulator of G protein signaling protein (RGS)
Activation of Effector Proteins Associated with G
Protein-Coupled Receptors
•
•
G proteincoupled
receptors
activate
exchange of
GTP for GDP on
the a subnit of a
trimeric G
protein
A built-in
feedback
mechanism is
present to make
sure that the
effector protein
is only activated
for a short
period of time
•
•
Different G proteins are activated by different GPCRs and in turn
regulate different effector proteins
Adenylyl cyclase and phospholipase C are different effectors
Hormone-Induced Activation and Inhibition
of Adenylyl Cyclase in Adipose Cells
•
•
•
Binding of ligan to Gas or Gai protein activates or inhibits adenylyl cyclase
to synthesize cAMP
cAMP, in turn, activates cAMP-dependent protein kinase that phosphorylate
target proteins
PGE1: postaglandin
cAMP Activates Protein Kinase A by Releasing
Catalytic Submits
•
•
cAMP-dependent protein kinase has regulatory and catalytic submits
Binding of cAMP to the regulatory submit results in release of the
catalytic submits
Synthesis and Degradation of Glycogen Is
Regulated by Hormone-Induced Activation of
Protein Kinase A
• Adding of glucose to
•
•
glycogenolysis
glycogen is catalyzed by
glycogen synthetase, and
removal of glucose moiety
from glycogen is by
glycogen phosphorylase
Glucose-1-phosphate is
converted to G-6-P in the
liver and then dephosphorylated by
phosphatase and released
into blood stream
Epinephrine-stimulated
activation of adenylyl
cyclase resulted in
increase of cAMP which in
turn activates protein
kinase leading to increase
of G-1-P from glycogen
Regulation of Glycogen Metabolism by cAMP in
Liver and Muscle Cells
cAMP-mediated activation of protein kinase A produces diverse responses in
different cell types. It is phosphorylated at ser and thr in a motif: X-Arg(Arg/Lys)-X-(Ser/Thr)-Φ where X denote any AA and Φ, hydrophobic AA
Amplification of an External Signal Downstream
from a Cell-Surface Receptor
Several Mechanisms Down-Regulate Signaling
from GPCR
•
There are several mechanisms contribute to termination of
cellular responses to hormone mediated by b-adrenergic
receptors and the G protein coupled receptors coupled to Gas
 The affinity of the receptor to its ligand decreases when GDP bound
to Gas is replaced with GTP. This increase in Kd of the receptorhormone complex enhances the dissociation of ligand from the
receptors and thereby limits the number of Gas protein that are active
 The intrinsic GTPase activity of Gas converts the bound GTP to GDP,
resulting in inactivation of the protein and decreased adenylyl
cyclase activity
 The rate of hydrolysis of GTP bound to Gas is enhanced when Gas
binds to adenylyl cyclase thus by decrease the duration of cAMP
production
 cAMP phosphodiesterase acts to hydrolyse cAMP
 Receptors can also be down regulated by feedback repression
because the phosphorylated Gas protein can not be activated by
ligand again
 Heterologous desentization
Synthesis of Second Messangers DAG and IP3
•
•
•
Ca++ ions play an essential role in regulating cellular responses to
external signals and internal metabolic changes
A small changes in levels of cytosolic Ca++ ions induces a variety of
cellular respomnses including hormone secretion by endocrine cells,
secretion of digestive enzymes by pancreatic exocrine cells and
contraction of muscle
Acetylcholine stimulates G-protein receptors in secretory cells of
pancreas to rise Ca++ ions
Analogues to
Adenylyl
cyclase,
Phospholipase
C is also an
effector protein
in this system,
and DAG and
IP3 are the
second
messengers
•
IP3/DAP Pathway and the Elevation of Cytosolic Ca++
Protein Kinases
•
There are following protein kinases involved
in signal transduction:
 Protein Kinase A (PKA): PKA is activated by cAMP
 Protein Kinase B (PKB, Akt): PKB is activated by
receptor tyrosine kinase (RTK)
 Protein Kinase C (PKC): Activated by DAG
(diacylglycerol)
 Protein Kinase G (PKG): Activated by cGMP
•
Some Receptors and Signal-Transduction
Proteins Are Localized
Clustering of membrane proteins mediated by adapter
domains
 Distribution of signals release in the presynaptic cells and
receptors in the postsynaptic cells is the best example
clustering of receptors
 Proteins containing PDZ domains play fundamental role in
organizing the plasma membrane of the postsynaptic cell
 The PDZ domain was identified as a common element in several
cytosolic proteins that bind to integral membrane proteins
 The PDZ protein is a small domain containing 90 amino acid
residues, that bind to three-residue sequences at the Cterminus of target proteins. Some PDZ domains bind to the
sequence Ser/Thr-X-Φ, others bind to Φ-X-Φ, where X denotes
any amino acid and Φ denotes any hydrophobic amino acid
 Most receptors contain multiple domains that binds to PDZ.
This interactions permit clustering of membrane proteins into
complexes
Activation of Gene Transcription by G ProteinCoupled Receptors (I)
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•
Intracellular signal pathways (such as GPCR
pathway) can result in short-term effect (seconds to
minutes) to modulate the pre-existing enzymes or
long-term (hours to days) effect to modulate gene
expression leading to cell proliferation or
differentiation
Membrane-localized tubby transcription factor is
released by activation of phospholipase C
 Tubby gene is expressed primarily in certain areas of the
brain involved in control of eating behavior
 Tubby gene encodes a protein that contains a DNA-binding
domain and a transcription-activation domain
 Tubby protein is localized near the plasma membrane which
binds to PIP2
Activation of Gene Transcription by G ProteinCoupled Receptors (II)
•
•
•
Binding of hormone to Go- or
Gq-coupled receptors resulted
in activation of phospholipase
C leading to hydrolysis of PIP2
and release tubby protein into
cytosol
Tubby then enters the nucleus
and activates transcription of
genes
Tubby protein(s) may regulate
the expression of the following
genes:
 Up-regulation: erythroid
diffrentiation factor 1 (erdr 1) and
capase 1 genes
 Down-regulation: tripartite motif
proteinss 3 (Trim 3), cholecystokinin 2 receptor (Cck 2) etc.
Activation of Gene Transcription by G ProteinCoupled Receptors (III)
•
•
•
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Binding of ligand to Gs proteincoupled receptor results in
activation of adenylyl cyclase
leading to production of cAMP
Activation of Protein kinase A by
cAMP
The activated protein kinase A is
translocated into the nucleus
Activated protein kinase A
phosphorylates CREB (c-AMPresponse element binding protein)
CREB and CBP/P300 together
activate the transcription of the
responsive genes (c-fos,
neurotrophin, brian-derived
neutrophic factor (BDNF), tyrosine
hydroxylase genes)
Receptor Tyrosine Kinase
•
•
•
•
Ligands for receptor tyrosine kinases are soluble or membrane
bound peptide or peptide hormones including NGF (Nerve growth
factor), PDGF (plated-derived growth factor), FGF (fibroblast growth
factor), EGF (epidermal growth factor) and insulin
Ligand –induced activation of RTK stimulates tyrosine kinase
activity, which subsequently stimulates the Ras-MAK pathways and
several other signal-transduction pathways
RTK signaling pathways have a wide spectrum of functions
including regulation of cell proliferation and differentiation,
promotion of cell survival and modulation of cellular metabolism
Some studies have indicated that RTKs are involved in human
cancers:
 Constitutively activated Her2 (a receptor for EGF-like protein, a mutant
form) enables uncontrolled proliferation of cancer cells in the absence
of EGF
 Over-production of wild type EGF receptor in certain human breast
cancer results in proliferation of cancer cells at low EGF levels that do
not stimulate normal stimulation
Ligand Binding Leads to Transphosphorylation of
Receptor Tyrosine Kinases
•
•
•
•
RTKs contain an extracellular ligand bind
domain, a single transmembrane domain,
regulatory domain and a cytosolic domain
with a protein kinase activity
Upon binding to one molecule of ligand, the
receptor forms a dimer
Some monomeric ligands, including FGF,
bind tightly to heparin sulfate that enhances
ligand binding to the monomeric receptor
and formation of a dimeric ligand-receptor
complex
Binding of ligand to the receptor will result in
the kinase in one submit to phosphorylate
one or more tyrosine residues in the
activation lip near the catalytic site in the
other submit. This leads to a conformational
change that facilitates binding of ATP
•
•
The resulting enhanced kinase activity then phosphorylates other
sites in the cytosolic domain of the receptor. This ligand-induced
activation of RTK kinase activity is analogous to the activation of
the JAK kinase associated with cytokine receptors
As in signaling by cytokins receptors, phosphotyrosine residues
in the activated RTKs serve as docking sites for proteins
involving in downstream of signal-transduction. These adaptor
proteins contain SH2, PTB or SH3 domains but have no intrinsic
enzymatic or signaling activity
Characteristics of the Common Classes of
Receptor Tyrosine Kinase
Class
I
II
Examples
EGF receptor, NEU/HER2,
HER3
Insulin receptor, IGF-I receptor
III
PDGF receptors, c-Kit
IV
FGF receptors
V
VEGF receptor
VI
HGF and SF receptors
VII
Neurotropin receptor family
and NGF receptor
Structural Features
Cysteine-rich sequence
Cysteine-rich sequences; disulfidelinked heterotetramers
Contain 5 immunoglobulin-like
domains as well as kinase insert
Contain 5 immunoglobulin-like
domains as well as kinase insert;
acidic domain
Contain 7 immunoglobulin-like
domains and the kinase insert
domain
Heterodimeric like the class II
receptors
Contain no or few cysteine-rich domain;
NGFR has leucine rich domain
Down Regulation of RTK Signaling by
Endocytosis and Degradation
•
There are two mechanisms that down regulate RTK
signaling:
 Ligand induced endocytosis: Ligand induced endocytosis of
the ligand-receptor complex
 Sorting of the internalized receptor-ligand to lysosome for
degradation
Ras, a GTPase Switch Protein, Cycles Between
Active and Inactive States
•
•
•
•
•
•
Ras is a monomeric GTP-binding switch protein that alternates
between an active “on” state with a bound GTP and an inactive “off”
state with a bound GDP. This is like the trimeric G proteins in the G
protein coupled receptor system
Ras activation is accelerated by a guanine nucleotide-exchange
factor (GEF) which binds to Ras-GDP complex causing dissociation
of bound GDP from Ras
Due to the presence of high levels of GTP in the cytosol, GTP binds
quickly to the empty Ras to form Ras-GTP
Deactivation of Ras-GTP requires the assistance of GTPaseactivating protein (GAP). GAP binds to specific phosphotyrosine in
the activated RTKs so that it can get close to the Ras-GTP to exert
its accelerating effect on GTP hydrolysis
Both trimeric G proteins and Ras are members of a family of
intracellular GTP-binding switch proteins referred to as GTPase
superfamily
Mutation of Ras oncogene (i.e., gly 12 to any amino acid except Pro)
results in blocking to GDP and thus locks Ras in activated form
Receptor Tyrosine Kinases Are Linked to Ras
by Adapter Proteins
• Cultured fibroblast cells can be
•
•
•
•
induced to proliferate by PDGF and
EGF, and microinjection of anti-Ras
antibody into these cells blocked
proliferation
Injection of RasD, a constitutively
active mutant Ras that hydrolyzes
GTP very inefficiently and thus
perisists in the active state, causes
the cell to proliferate in the absence
of growth factors
GRB2 and Sos provide the key links
with the Ras
SH2 in GRB2 binds to a
phosphotyrosine of the activated
receptor. GRB2 has two SH3
domains which bind and activate
Sos
Sos: son of sevenless protein
•
Sos is a guanine nucleotide-exchange protein (GEF) which catalyzes
conversion of inactive GDP-bound Ras to the activate GTP-bound
form
Key Signal-Transduction Proteins Downstream from RTK
•
•
•
•
The compound eye of Drosophila is composed of about 800
individual eyes called ommatidia. Each ommatidium consists of
22 cells 8 of which are photosensitive neurons called retinula, or
called as R cells designated R1-R8
Sevenless (Sev) encode an RTK that regulate the development of
R7. Mutant of Sev gene fail to development R7 ommatidia
A protein called Boss (Bride of Sevenless) is expressed on the
surface of the R8 cells. This protein is the ligand for the Sev RTK
on the surface of the neighboring R7 precursor
Mutants Boss or Sev RTK that do not express Boss or Sev, RTK
fail to develop R7 cells
Genetic Studies Reveal the Activation of Ras
Induces the Development of R7 photoreceptors
Kinase Cascade That Transmits Signals
Downstream from Activated Ras to MAP Kinase
RTK
Ras
Raf
MEK
MAP kinase
•
•
•
•
•
Raf, a serine/threonine kinase, is activated by RasGTP
Activated Raf activates MEK by phosphorylating MEK
Activated MEK phosphorylates and activates MAP
kinase
Activated MAP phosphorylates another proteins in the
nucleus including transcription factors
MEK: MAP and ERK kinase
Induction of Gene
Transcription by
Activated MAP Kinase
•
•
•
•
MAP kinase induces the
expression of genes including cfos gene by modifying two
transcription factors ternary
complex factor (TCF) and serum
response factor (SRF)
It is done through activating
p90RSK in cytosol and both the
activated MAP kinase and p90RSK
activates TCF and SRF
SRF: c-fos serum response
factor
TCF: ternary complex factor; it
forms complex with SRF
•
•
TGFb Receptors and the Direct
Activation of Smads
Cytokine Receptors and JAK/STAT
Pathways
TGFb Receptors and the Direct Activation of Smads
•
•
•
•
TGFb (Transforming Growth Factor β) superfamily proteins play
important roles in regulating development of vertebrates and
invertebrates
 Bone Morphogenic Protein (BMP) is one of the TGFb
superfamily important in regulating formation of mesoderm
and the earliest blood forming cells
 TGFb-1 is another member of the TGFb superfamily proteins
which can induce a transformed phenotype of certain cells in
culture
There are three human TGFb isoforms known to have potent antiproliferative effects on many types of mammalian cells. Mutation
of TGFb will result in releasing cells from growth inhibition
(frequently occurs in human tumors)
TGFb also promotes expression of cell-adhesion molecules and
extracellular matrix molecules
TGFb can induce some cells to produce growth factor to
overcome TGFb-induced growth inhibition. This is why it was
considered as a growth factor initially
TGFb Is Formed by Cleavage of a Secreted
Inactive Precursor
•
•
•
TGFb consists of three
protein isoforms, TGFb1,
TGFb2 and TGFb3
Each isoform is encoded by a
unique gene in tissue specific
and developmental stage
specific fashion
Each TGFβ is synthesized as
a larger precursor
4 antiparallel β strands
TGFβ Receptor Signaling
• TGFβ: TGFβ-1, -2,
-3.
• TGFβ receptors:
type RI, RII, RIII
• Smad: R-Smad,
Co-Smad, I-Smad
• SnoN & Ski, ISmad: feedback
control
TGFb Signaling Receptors Have Serine/Threonine
Kinase Activity
•
•
•
•
•
•
TGFb signaling receptor is isolated by first conjugating I125-labeled
TGFb to receptors on the cell membrane and then fractionate the
membrane proteins to isolate the membrane protein that
associates with I125-TGFb
Three different polypeptide with apparent molecular weights of 55,
85 and 280 kDa were purified, referred to as types RI, RII and RIII
TGFb receptors
Type RIII TGFb receptor is a cell-surface proteoglycan, also called
b-glycan which bind and concentrate TGFb near the cell surface
Type RI and type RII receptors are dimeric transmembrane proteins
with serine/threonine kinases as part of their cytosolic domains
RII is a constitutively active kinase that phosphorylates itself in the
absence of TGFb
Binding of TGFβ induces the formation of two copies each of RI
and RII. A RII then phosphorylates serine/threonine of RI adjacent
to the cytoplasm and thus activate the RI kinase activity
Activated Type I TGFb Receptors
Phosphorylate Smad Transcription
Factors
•
Smads are transcription factors. There
are three types of Smads, receptorregulated Smads (R-Smads), co-Smads,
inhibitory Smads (I-Smads)
• R-Smads contain two domains, MH1 and
MH2, separated by a flexible linker region.
The N-terminus of the MH1 contains a
specific DNA binding segment and a NLS
sequence
• When R-Smads are in inactive state, the
NLS is masked and the MH1 and MH2
domains associate in a way that they can
not bind to DNA or to a co-Smad
• Phosphorylation of three serine residues
near the C-terminus of a R-Smad (Smad2
or Smad3) by activated type I TGFb
Plasmanogene activator inhibitor: receptors separates the domains,
PAI
allowing binding of importin b to the NLS
•
•
•
•
•
•
Simultaneously a complex containing two molecules of Smad3
(or Smad2) and one molecule of a co-Smad (Smad4) forms in the
cytosol
The complex is stabilized by binding two phosphorylated serines
in both the Smad3 and the Smad4 MH2 domains
The importin b–bound heteromeric R-Smad3/Smad4 complex will
translocate into nucleus
After importin b dissociates from the complex in the nucleus, the
Smad2/Smad4 or Smad3/Smad4 will cooperate with other
transcription factors to turn on specific target gene
In the nucleus, R-Smads are continuously being
dephosphorylated, which results in the dissociation of the RSmad /co-Smad complex and export of these Smads from the
nucleus. Therefore, the concentration of the active Smads in the
nucleus closely reflects the levels of the activated TGFb
receptors on the cell surface
One of the genes that is regulated by this signal transduction
pathway is plasmanogene activator inhibitor (PAI)
Oncoproteins and I-Smads Regulate Smad
Signaling via Negative Feedback Loop
HDAC;
histone
deacetylase
•
•
•
•
•
Smad signaling is regulated
by additional intracellular
proteins including SnoN and
Ski (Ski stands for “SloanKettering Cancer Institute”
These proteins are
oncoproteins since they cause
abnormal cell proliferation
when over expressed in
cultured fibroblasts
SnoN and Ski can bind to Smad2/Smad4 or Smad3/Smad4 complex after
TGFb stimulation
Binding of SnoN and Ski to Smad2/Smad4 or Smad3/Smad4 will block
transcription activation of the target gene and renders cells resistant to the
growth inhibition induced by TGFb
PAI-1 gene: encodes plasminogen activator inhibitor-1
Cytokines Influence Development of Many Cell Types
•
Cytokines form a family of small secreted proteins of about 160
amino acids that control many aspects of growth and
differentiation of specific types of cells
 Prolactin induces epithelial cells lining the immature ductules of the
mammary gland to differentiate into acinar cells to produce milk
proteins secreted into the ducts during pregnancy,
 Interleukin 2 (IL-2) is essential for proliferation and functioning of the
T-cells of the immune system
 IL-4 is essential for formation and function of antibody-producing B
cells
 Interferon α is produced and secreted by many types of cells following
virus infection. Then secreted interferon acts nearby cells to induce
enzymes that render these cells more resistant to virus infection
 Many cytokines induce formation of important blood cells.
Granulocyte colony stimulating factor (G-CSF) induce progenitor cells
in bone marrow to differentiate into granulocyte, thrombopoietin acts
on megakaryocyte progenitors to differentiate into megakaryocytes
which then fragmented into cell pieces called platelets
Cytokine Receptor Signaling
•
•
•
•
•
•
•
•
Similar to Receptor Tyrosine Kinase signaling
Receptor dimerization
Phosporylation and activation of JAK kinase
Binding of STAT to p-Receptor via SH2 domain
Phosphorylation of STAT by JAK kinase
Translocation of p-STAT into nucleus
Activation of transcription
Feedback regulation: SHP1 and SOCS
Cytokine Receptors and Jak-Stat Pathway
•
•
•
•
The cytosolic domain of the
cytokine receptor associates
with a family of cytosolic protein
tyrosine kinase, the JAK kinase
Receptor tyrosine kinases
(RTKs) also contain intrinsic
protein tyrosine kinase activity
in their cytosolic domains
The mechanisms by which
cytokine receptors and receptor
tyrosine kinases become
activated by ligand are very
similar, and there is
considerable overlap by
activation of receptors in both
cases
The figure on the left shows the
dimerization of cytokine
receptor after binding to EGF
Cytokine Receptors and Receptor Tyrosine Kinases
Share Many Signaling Features
•
Autophosphorylation
•
•
Ligand binding to both cytokine
receptors and receptor tyrosine
kinases triggers formation of
functional dimeric receptors
In some cases, the ligand
induces association of two
monomeric receptor subunits
diffusing in the plan of the
plasma membrane; in other
cases, the receptor is a dimer in
the absence of ligand and
ligand binding alters the
conformation of the
extracellular domains of the two
subunits
In either cases, formation of the
functional dimeric receptor
causes the cytosolic kinases to
phosphorylate the second
kinase
The Role of Erythropoietin in the Formation of Red
Blood Cells (Erythrocytes)
•
•
•
Erythroid progenitor cells
[colony-forming units erythroid
(CFU-E) ] are derived from
hematopoietic stem cells,
which also give rise to
progenitor cells of other blood
cell types
Binding of erythropoietin (Epo)
to its receptor on a CFU-E
induces transcription of
several genes encoding
proteins preventing apoptosis
of CFU-E and allow the cells to
go through several rounds of
proliferation
Epo also stimulate expression
of specific genes leading to
differentiation of CFU-E into
red blood cells
Structure of Erythropoietin Bound to the Extracellular
Domains of a Dimeric Erythropoietin Receptor
•
•
•
•
All cytokines have a similar
tertiary structure consisting
of four long conserved a
helicies folded together in a
specific orientation
Similarly, all cytokine
receptors have quite similar
structures, with their
extracellular domains
consisted of two
subdomains, each of which
contains seven conserved b
strands folded together in a
characteristic fashion
One molecule of
erythropoietin binds to two
monomers of EpoR
All cytokines and their receptors have similar structures and activate similar
signal pathways
Overview of Signal-Transduction Pathways Triggered
by Ligand Binding to the Erythropoietin Receptor, a
Typical Cytokine Receptor
GRB2, a linker protein (adaptor protein)
All of these four pathways lead to eventual increase or decrease in
transcription of target genes
Both the Erythropoietin Receptor and JAK2 Are
Essential for Development of Erythrocytes
•
•
Mice embryos in which both alleles of EpoR or JAK2 gene are knocked out,
can develop normally until embryonic day 12 and at which they begin to die
of anemia due to lack of erythrocyte-mediated transport of oxygen to fetal
organ
These results suggest that EpoR and JAK2 are required for erythrocyte
development in early embryonic development
JAK-STAT Signaling Pathway
•
•
•
•
Once the JAK kinases become
activated, they phosphorylate several
tyrosine residues on the cytosolic
domain of the receptor. Some of the
phosphorylated tyrosine residues
serve as binding sites for a group of
transcription factors, STATs
All STAT proteins contain an Nterminal SH2 domain that binds to
phosphotyrosine in the receptor’s
cytosolic domain, a central DNA
binding domain and a C-terminal
domain with a critical tyrosine residue
Once the STAT is bound to the
receptor, the C-terminal tyrosine is
phosphorylated by an associated JAK
kinase
The phosphorylated STAT dissociates
from the receptor, and two activated
STATs form a dimer and then enters
the nucleus
Two Mechanisms for Terminating Signal
Transduction from the Erythropoietin Receptor
Signaling from Cytokine Receptors Is Modulated by
Negative Signals (Feedback Loop) (I)
•
•
•
Signal-induced transcription of target genes can not last for too
long and needs de-sensitized
Signaling from cytokine receptor is usually dampened by two
classes of proteins: short term regulation by SHP1 phosphatase
and long term regulation by SOCS proteins
SHP1 Phosphatase
 Mutant mice lacking SHP1 phosphatase die because of producing
excess amount of erythrocytes and other blood cells. These results
suggest that SHP1 negatively regulates signaling from several types of
cytokine receptors in several types of progenitor cells
 Binding of an SH2 domain SHP1 to a particular phospho-tyrosine in the
activated receptor unmasks its phosphatase catalytic site and position
it near the phosphrylated tyrosine in the lip region of JAK2
 Removal of the phosphate from this tyrosine inactivates the JAK
kinase
Signaling from Cytokine Receptors Is Modulated by
Negative Signals (Feedback Loop) (II)
•
Signal blocking and protein degradation induced by SOCS
proteins:
 STAT proteins induce a class of small proteins termed SOCS
proteins. These proteins terminate signaling from cytokine
receptors. These negative regulators are also known as CIS
proteins
 CIS proteins act in two ways to negatively regulate cytokine
receptor stimulated signaling:
 The SH2 domain in several SOCS proteins bind to
phosphotyrosines on an activated receptor, preventing binding of
other SH2-containing signaling proteins and thus inhibiting receptor
signaling
 SOCS-1 can bind to critical phosphotyrosine in the activation lip of
activated JAK2 kinase thereby inhibiting its catalytic activity
 All SOCS proteins contain a SOCS box that recruits components of
E3 ubiquitin ligases. As a result of SOCS-1 binding, JAK2 becomes
polyubiquitinated and then degraded in proteasomes and thus
terminate the signaling permanently
Components & Modularity of Major
Signaling Pathways
Cross Talk in Signal Transduction Pathways
• For cells to carry out all the cellular functions, different signal
•
transduction pathways may communicate among one another.
This is called signal transduction pathway cross talk
Examples:


There two types of estrogen receptors: (i) nuclear ER; (ii)
membrane bound ER. While nuclear ER activates the expression
of estrogen-responsive gene, membrane bound ER activates
protein kinases to activate steroid receptor co-activator (SRCs)
and CREB binding protein-associated factor by phosphorylation
(Reading list VII: Cross talk between membrane and nuclear
pathways by steroid hormone)
cAMP-responsive genes are modulated by CREB (cAMP
responsive binding protein), CREM (cAMP responsive modulator
protein) and ICER (inducible cAMP early repressor). CREM gene
can encode two isoforms, CERM and ICRE, by differential use of
promoters. While CREB and CREM activate the expression of
cAMP-responsive genes, ICER represses the expression of these
genes. The expression of ICER is regulated NGF (Reading List VII:
Cross-talk in signal transduction: Ras-dependent induction of
ICER by NGF)
More Examples of Cross Talk of Signals
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Win.Wingless and TGF-b/BMP
TGF-b/BMP and Hedgehog
Estrogen receptor and progesterone receptor
Angiotensin II receptor: between AT1 and AT2
receptors
Androgen receptor and vitamin D receptor
Chemokine receptors and epidermal growth factor
receptor
Epidermal growth factor receptor and c-Met
FGF-receptor tyrosine kinase and G-protein
Glucocorticoid receptor, C/EBP, HNF3 and protein
kinase A
GABA receptors and dopamine D5
FGF receptor and N/E-cadherin
RTK-RSK
PKC, cAMP and MAP kinase