[VII]. Regulation of Gene Expression Via Signal Transduction

1
VII. Regulation of Gene Expression Via Signal
Transduction

• Reading List VII
• Signal transduction
• Signal transduction in biological systems

2
External Signal Regulating the Expression of Genes
Signals
Signal transduction cascades
Gene Expression
or
Cytoplasmic mechanism/muscle contraction/etc.
New Proteins
mRNAs
Proteins
3
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

4
Communication among Bacteria
Aggregation in progress
Individual cells
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.
5
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

6
Signaling Via Cell-Surface Receptors (I)

• Synthesis and release of signaling molecules by
  signaling cells (step 12)
• 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)

7
Signaling Via Cell-Surface Receptors (II)

• Initiating one or more intracellular
  signal-transduction 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)

8
Different Ways Cells Signal Each Other

• Endocrine signaling
• Paracrine signaling
• Autocrine signaling
• Signaling by plasma membrane-attached proteins

9
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 a-1,4-oligogalaturonide
• Gases CO, ethylene etc.

10
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

11
Receptor Ligand Interaction
At equilibrium
Where R and L are the concentration of free
receptor ligand at equilibrium. RL is the
concentration of the receptor-ligand complex. Kd
is the dissociation constant
koff
Kd

kon
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
12
Binding Assays Are Used to Detect Receptors and
Determine Their Kd Values

• Binding assay is used to demonstrate the presence
  of receptors. Both the number of the
  ligand-binding 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

(Free Ligand)

• 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

13
Scatchard Plot

• 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

-
14
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

15
Insulin-Like Growth Factor (IGF) I
16
Multiple Forms of Pro-IGF-I E-Peptide
17
Anti-Tumor Activities of the Pro-IGF-I E-peptide

• 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
  tumor-induced 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)

18
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
19
Binding of 35S-E-Peptide to SK-N-F1 Cells
Human Eb-peptide
Trout Ea4-peptide
Kd 2.9 1.8 x 10-11 M
Kd 2.9 1.8 x 10-11 M
20
Competitive Displacement Assay

1. Labeled hEb was competed out with unlabeled hEb
2. Labeled rtEa4 was competed out with unlabeled
   rtEa4

21
Competitive Displacement Assay

• C. Labeled hEb competitive with unlabeled rtEa4
• Labeled rtEa4 competitive with unlabeled hEb

22
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23
Competitive Binding Assay with hIGF-I
The data suggest that E-peptide does not bind to
the same receptor that binds IGF-I
24
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

25
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 RL/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

26
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
• If the RT1000, 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

27
Purification of Membrane Receptors

• 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

28
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 full-length 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

29

• Reading List VII
• Isolatiion and characterization of colagen
  receptor
• Isolation of interleukins by immunoaffinity-recept
  or affinity chromatography
• Isolation, characterization and regulation of the
  prolactin receptor
• Isolation and characterization of human prolactin
  receptor

30
The General Structure of a Membrane Receptor

• 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 ion-channel receptor etc.

31
Receptors Activate a Limited Number of Signaling
Pathways (I)

• 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

32
Receptors Activate a Limited Number of Signaling
Pathways (II)

• 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

33
Seven Major Classes of Cell-Surface Receptors
34
Four Common Intracellular Second Messenger

• 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)

35
Appropriate Cellular Responses Depend on
Interaction and Regulation of Signal Pathways

• 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.

36
Overview of Cell Signaling
Reception Transduction Response
The components of intracellular signal
transduction pathways are highly conserved
37
Signal Transduction Pathways

• 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

38
Protein Kinases

• Protein kinases and phosphatases are used in
  virtually all signaling pathways
• Protein kinases enzymes add phosphate groups to
  the OH-group 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

39
A Phosphorylation Cascade
40

• G-Protein Coupled Receptors
• 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
• Receptor Tyrosine Kinase

41
General Elements of G Protein-Coupled Receptors

• 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
• 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

42
General Structure of G Protein-coupled Receptor

• 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
  a-submit of a Trimeric G protein

• G-protein coupled receptors consists of
  hydrophobic amino acids that allow proteins to be
  stabled 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

43
Switching Mechanism for Monomeric Trimeric G
Proteins

• 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
  nucleotide-exchange 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)

44
Structural Model of Complex Formed between
Epinephrine the b-Adrenergic Receptor

• The amino acids that form the interior of
  different G protein-coupled receptors are
  diverse, allowing different receptors to bind to
  very different small molecules

• b-adrenergic receptor binding to epinephrine
• Three a-helices are involved in binding of
  b-adrenergic receptor to epinephrine
• Examples Binding of epinephrine to
  b-adrenergenic receptor in the liver and adipose
  tissue results in liberation of glucose and fatty
  acids. In heart muscle cells, binding of
  epinephrine to b-adrenergeic receptor results in
  increase of heart contraction rate

45
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46
Studies with Chimeric Adrenergic Receptors
Identifying the Long C3 Loop as Critical to
Interaction with G Proteins

• Although all a- and b-adrenergic receptors bind
  to epinephrine, different receptors coupled to
  different G proteins that induces different down
  stream signaling pathways, leading to different
  responses
• This slide describes an experiment demonstrating
  that the specificity of G protein is determined
  by the cytosol-facing C3 loop between helices 5
  and 6 of the receptor

47
Activation of Effector Proteins Associated with G
Protein-Coupled Receptors

• G protein-coupled 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

48
Activation of G Protein Occurs within Seconds of
Ligand Binding in Amoeba Cells
The experiment shown above is fluorescence
energy transfer experiment
CFP cyan fluorescent protein, excitation at
440nm, emision at 490 nm YFP yellow fluorescent
protein, excitation at 490 nm, emision at 527 nm
cAMP is a ligand of G protein coupled receptor in
Dictyostellium discoideum cells
49

• Different G proteins are activated by different
  GPCRs and in turn regulate different effector
  proteins
• Adenylyl cyclase and phospholipase C are
  different effectors

50
Activation of the Muscarinic Acetylcholine
Reporter and Its Effector K Channel in Heart
Muscle

• G protein-coupled receptors can activate ion
  channels
• Muscarinic acetylcholine receptor is in cardic
  muscle cells which control muscle contraction
• Gbg submits of the G protein activate K channel
  protein by opening the channel

51
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

52
Mammalian Adenylyl Cyclases with Gas.GTP

• Structural studies established how Gas.GTP binds
  to and activate adenylyl cyclase
• Interaction of Gas.GTP with the catalytic domains
  of adenylyl cyclase
• The ligand of the system is epinephrine

53
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

54
Synthesis and Degradation of Glycogen Is
Regulated by Hormone-Induced Activation of
Protein Kinase A

• Adding of glucose to 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 de-phosphorylated 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

glycogenolysis
55
Regulation of Glycogen Metabolism by cAMP in
Liver and Muscle Cells
56
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)-F where X
denote any AA and F, hydrophobic AA
57
A Phosphorylation Cascade
58
Amplification of an External Signal Downstream
from a Cell-Surface Receptor
59
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 receptor-hormone
  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

60
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
  inclusing hormone secretion by endocrine cells,
  secretion of digestive enzymes by pancretic
  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

61
IP3/DAP Pathway and the Elevation of Cytosolic
Ca
62
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

63
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 C-terminus of target proteins.
  Some PDZ domains bind to the sequence
  Ser/Thr-X-F, others bind to F-X-F, where X
  denotes any amino acid and F denotes any
  hydrophobic amino acid
• Most receptors contain multiple domains that
  binds to PDZ. This interactions permit
  clustering of membrane proteins into complexes

64
Clustering of Membrane Proteins Mediated by
Cytosolic Adaptor Proteins Containing Protein
Binding Domains

• Three dimensional surface structure of a PDZ
  domain showing the backbone of the bound peptide
  shown in red
• Regions in the PDZ domain that bind to the COO-
  group and the side chain to the C-terminal
  residues colored in yellow blue

65
Clustering of Membrane Proteins Mediated by
Cytosolic Adaptor Proteins Containing Protein
Binding Domains

• Schematic diagram of protein-protein interactions
  that cluster several different membrane proteins
  in a postsynaptic segment of a nerve cell and
  anchor the resulting complex to cytoskeletal
  actin filaments
• PDZ adaptor protein Ank ankyrin repeats
  Neuroligin adhesive protein that interacts with
  component of the extracellular matrix

66
Activation of Gene Transcription by G
Protein-Coupled Receptors (I)

• 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

67
Activation of Gene Transcription by G
Protein-Coupled 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 a still unknown gene or 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), cholecys-tokinin 2 receptor (Cck 2) etc.

68
Activation of Gene Transcription by G
Protein-Coupled Receptors (III)

• Binding of ligand to Gs protein-coupled 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-AMP-response 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)

69
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

70
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

71

• 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

72
Characteristics of the Common Classes of Receptor
Tyrosine Kinase
Structural Features
Examples
Class
Cysteine-rich sequence
I
EGF receptor, NEU/HER2, HER3
II
Insulin receptor, IGF-I receptor
Cysteine-rich sequences disulfide-linked
heterotetramers
III
PDGF receptors, c-Kit
Contain 5 immunoglobulin-like domains as well as
kinase insert
FGF receptors
IV
Contain 5 immunoglobulin-like domains as well as
kinase insert acidic domain
V
VEGF receptor
Contain 7 immunoglobulin-like domains and the
kinase insert domain
VI
HGF and SF receptors
Heterodimeric like the class II receptors
VII
Neurotropin receptor family and NGF receptor
Contain no or few cysteine-rich domain NGFR has
leucine rich domain
73
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

74
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 GTPase-activating 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

75
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

76

• Sos is a guanine nucleotide-exchange protein
  (GEF) which catalyzes conversion of inactive
  GDP-bound Ras to the activate GTP-bound form

77
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

78
Genetic Studies Reveal that Activation of Ras
Induces Development of R7 Photoreceptor in the
Drosophila Eye
Temperature sensitive mutants were used to
demonstrate the importance of Boss and Sev on R7
neuron development RasD constitutive expression
of an activated Ras
79
Binding of Sos Protein to Inactive Ras Causes a
Conformational Change That Activates Ras

• The adaptor protein GBR2 contains two SH3 domains
  which bind to Sos in addition to SH2 domain
• SH3 domains are present in a large number of
  proteins involved in intracellular signaling
• Figure in the left shows the 3-D structure of SH3
  interaction with a target protein through the
  proline residues

80
Structures of Ras Bound to GDP, Sos Protein and
GTP

• Binding of Sos to inactive Ras causes a large
  conformational change that permits release of GDP
  and binding of GTP, forming active Ras. GAP,
  which accelerates GTP hydrolysis, is localized
  near Ras-GTP by binding to active RTKs

81
Kinase Cascade That Transmits Signals Downstream
from Activated Ras to MAP Kinase
RTK Ras Raf MEK MAP kinase
82

• Raf, a serine/threonine kinase, is activated by
  Ras-GTP
• 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

83
(No Transcript)
84
Induction of Gene Transcription by Activated MAP
Kinase

• MAP kinase induces the expression of genes
  including c-fos 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

85

• TGFb Receptors and the Direct Activation of Smads
• Cytokine Receptors and JAK/STAT Pathways

86
TGFb Receptors and the Direct Activation of Smads

• TGFb (Transforming Growth Factor ß) 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 anti-proliferative 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

87
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 TGFß is synthesized as a larger precursor

4 antiparallel ß strands
88
TGFß Receptor Signaling

• TGFß TGFß-1, -2, -3.
• TGFß receptors type RI, RII, RIII
• Smad R-Smad, Co-Smad, I-Smad
• SnoN Ski, I-Smad feedback control

89
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
  phosphrylates itself in the absence of TGFb
• Binding of TGFß induces the formation of two
  copies each of RI and RII. A RII then
  phophorylates serine/threonine of RI adjacent to
  the cytoplasm and thus activate the RI kinase
  activity

90
Activated Type I TGFb Receptors Phosphorylate
Smad Transcription Factors

• Smads are transcription factors. There are three
  types of Smads, receptor-regulated 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 receptors separates the
  domains, allowing binding of importin b to the
  NLS

Plasmanogene activator inhibitor API
91

• 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 bbound 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 R-Smad /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 (API)

92
Oncoproteins and I-Smads Regulate Smad Signaling
via Negative Feedback Loop

• Smad signaling is regulated by additional
  intracellular proteins including SnoN and Ski
  (Ski stands for Sloan-Kettering Cancer
  Institute
• These proteins are oncoproteins since they cause
  abnormal cell proliferation when over expressed
  in cultured fibroblasts

HDAC histone deacety-lase

• 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

93
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 a 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

94
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

95
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

96
Cytokine Receptors and Receptor Tyrosine Kinases
Share Many Signaling Features

• 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

Autophosphorylation
97
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

98
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

99
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
100
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

101
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 N-terminal SH2
  domain that binds to phosphotyrosine in the
  receptors 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

102
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

103
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

104
Two Mechanisms for Terminating Signal
Transduction from the Erythropoietin Receptor
105
Components Modularity of Major Signaling
Pathways
106
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
  prompters. 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)

107
More Examples of Cross Talk of Signals

1. Win.Wingless and TGF-b/BMP
2. TGF-b/BMP and Hedgehog
3. Estrogen receptor and progesterone receptor
4. Angiotensin II receptor between AT1 and At2
   receptors
5. Androgen reeptor and vitemin D receptor
6. Chemokine receptors and epidermal growth factor
   receptor
7. Epidermal growth factor receptor and c-Met
8. FGF-receptor tyrosine kinase and G-protein
9. Glucocorticoid receptor, C/EBP, HNF3 and protein
   kinase A
10. GABA receptors and dopamine D5
11. FGF receptor and N/E-cadherin
12. RTK-RSK
13. PKC, cAMP and MAP kinase