[VII]. Regulation of Gene Expression Via Signal Transduction

Reading List VII: Signal transduction Signal transduction in biological systems

Gene Expression

mRNAs

Proteins

Signals

Signal transduction cascades

External Signal Regulating the Expression of Genes

Cytoplasmic mechanism/muscle contraction/etc.

or

New 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 and

• 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 cellsAggregation in progressSpore 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

• 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 intra-cellular 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

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 (I)

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)

Signaling Via Cell-Surface Receptors (II)

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

• 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

Dimers

Characteristics of Hormone Receptors

• 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

• 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 - and -

adrenergic 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

R + L RLkon

koff

Kd = [R] . [L]

[RL]

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

At equilibrium:

kon

koff=

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

• 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

• 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

(Free Ligand)

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

-

• 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

Scatchard Plot

Use of a Competitive Binding Assay to Detect Binding of Low Affinity Ligands to Receptors

• 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

• 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

Insulin-Like Growth Factor (IGF) I

B C A DS E

Signal peptide

Mature IGF-I E-peptide

Post-translational processing

EB C A DS

Primary translation product of IGF-I

Multiple Forms of Pro-IGF-I E-Peptide

Human pro-IGF-Ia

Human pro-IGF-Ib

Trout pro-IGF-I Ea-3

Trout pro-IGF-I Ea-1

Trout pro-IGF-I Ea-2

Trout pro-IGF-I Ea-4

EMature IGF-I

B C A D

Human pro-IGF-Ic

• 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)

Anti-Tumor Activities of the Pro-IGF-I E-peptide

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 Trout Ea4-peptide

Kd = 2.9 ± 1.8 x 10-11 M 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

• 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

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

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

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

Reading List VII:

• Isolatiion and characterization of colagen receptor

• Isolation of interleukins by immunoaffinity-receptor affinity chromatography

• Isolation, characterization and regulation of the prolactin receptor

• Isolation and characterization of human prolactin receptor

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.

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, TGF 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

• The other classes of receptors operate primarily to modulate gene expression: The activated TGF 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

Receptors Activate a Limited Number of Signaling Pathways (II)

Seven Major Classes of Cell-Surface Receptors

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)

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.

Overview of Cell Signaling

Reception Transduction Response

The components of intracellular signal transduction pathways are highly conserved

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

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

A Phosphorylation Cascade

• 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

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 (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

• 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

• 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 -submit of a Trimeric G protein

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)

Activation of Effector Proteins Associated with G Protein-Coupled Receptors

• G protein-coupled receptors activate exchange of GTP for GDP on the 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 Gs or Gi 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

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

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 -adrenergic receptors and the G protein coupled receptors coupled to Gs

The affinity of the receptor to its ligand decreases when GDP bound to Gs 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 Gs protein that are active

The intrinsic GTPase activity of Gs 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 Gs is enhanced when Gs 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 Gs 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 C-terminus 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 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

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 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.

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)

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 Examples Structural Features

I EGF receptor, NEU/HER2,HER3

Cysteine-rich sequence

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

IV FGF receptors 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

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 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

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 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

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

• SRF: c-fos serum response factor

• TCF: ternary complex factor; it forms complex with SRF

• TGF Receptors and the Direct Activation of Smads

• Cytokine Receptors and JAK/STAT Pathways

TGF Receptors and the Direct Activation of Smads • TGF (Transforming Growth Factor β) superfamily proteins play

important roles in regulating development of vertebrates and invertebratesBone Morphogenic Protein (BMP) is one of the TGF

superfamily important in regulating formation of mesoderm and the earliest blood forming cells

TGF-1 is another member of the TGF superfamily proteins which can induce a transformed phenotype of certain cells in culture

• There are three human TGF isoforms known to have potent anti-proliferative effects on many types of mammalian cells. Mutation of TGF will result in releasing cells from growth inhibition (frequently occurs in human tumors)

• TGF also promotes expression of cell-adhesion molecules and extracellular matrix molecules

• TGF can induce some cells to produce growth factor to overcome TGF-induced growth inhibition. This is why it was considered as a growth factor initially

TGF Is Formed by Cleavage of a Secreted Inactive Precursor

• TGF consists of three protein isoforms, TGF1, TGF2 and TGF3

• 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, I-Smad: feedback control

TGF Signaling Receptors Have Serine/Threonine Kinase Activity

• TGF signaling receptor is isolated by first conjugating I125-labeled TGF to receptors on the cell membrane and then fractionate the membrane proteins to isolate the membrane protein that associates with I125-TGF

• Three different polypeptide with apparent molecular weights of 55, 85 and 280 kDa were purified, referred to as types RI, RII and RIII TGF receptors

• Type RIII TGF receptor is a cell-surface proteoglycan, also called -glycan which bind and concentrate TGF 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 TGF

• 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 TGF 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 TGF receptors separates the domains, allowing binding of importin to the NLS

Plasmanogene activator inhibitor: PAI

• 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 –bound heteromeric R-Smad3/Smad4 complex will translocate into nucleus

• After importin 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 TGF 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

• 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

• SnoN and Ski can bind to Smad2/Smad4 or Smad3/Smad4 complex after TGF 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 TGF

• PAI-1 gene: encodes plasminogen activator inhibitor-1

HDAC; histone deacety-lase

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

• 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

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 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 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 N-terminal 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. Win.Wingless and TGF-/BMP2. TGF-b/BMP and Hedgehog3. Estrogen receptor and progesterone receptor4. Angiotensin II receptor: between AT1 and AT2

receptors5. Androgen receptor and vitamin D receptor6. Chemokine receptors and epidermal growth factor

receptor7. Epidermal growth factor receptor and c-Met8. FGF-receptor tyrosine kinase and G-protein9. Glucocorticoid receptor, C/EBP, HNF3 and protein

kinase A10. GABA receptors and dopamine D511. FGF receptor and N/E-cadherin12. RTK-RSK13. PKC, cAMP and MAP kinase