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Cellular Signaling Mechanisms

Receptor Tyrosine KinasesCytokine Receptors Nuclear ReceptorsDeath Receptors

Phar 735/590Winter 2006

Mark LeidOffice: Pharmacy 407

Tel: 737-5809E mail: Mark.Leid@oregonstate.edu

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Objective

To understand the structural and mechanistic basis for cellular signaling involving:

• Receptor Tyrosine Kinases (e.g., insulin receptor)

• Cytokine receptors (e.g., interleukin receptors)

• Nuclear receptors (e.g., steroid hormone receptors)

• Death receptors (receptors mediating apoptosis)

This material will form the cornerstone for understanding the pharmacological basis of therapeutics with regard to agents acting in the above pathways.

…so keep your mind open.

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Funkadelic: Free Your Mind… (1971; Westbound Records)

The purpose of this handout is to free your hand and mind so that you may participate in class more

fully.

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Signaling on an Intermediate Time Scale

I. Receptor tyrosine kinases (RTKs)A. Examples

1. Insulin Receptor

2. Insulin-like GFRs

3. Platelet-derived GFR

4. Epidermal GFR

5. Fibroblast GFR

B. RTKs span plasma membrane only once

C. Receptor harbors intrinsic tyrosine kinase activity that is activate by GF binding

D. The signaling pathway involves phosphorylation of cytoplasmic substrates by carboxyl terminus of RTK

E. Phosphosphorylation alters substrate's activity

• Required for growth and development

• Involved in metabolic and mitogenic processes

• Implicated in pathological processes

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RTK Superfamily

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Regulation of glucose homeostasis

• Insulin production: B cells of islets of Langerhans (60-80% of cells there are B cells).

• Species differences in islet architecture

• In general, four peptides with hormonal activity are secreted by the islet cells:

Insulin (beta or B cells; stimulates glucose uptake)

Glucagon (A cells; stimulates glycogenolysis and gluconeogenesis primarily in liver, both of which increase BS)

Somatostatin (D cells; negatively regulates A and B cell secretions)

Pancreatic polypeptide (F cells)

• Insulin secretion is stimulated by glucose (alters the ATP/ADP ratio in a cell, blocks ATP-sensitive K+ channels (Kir6.2/SUR1), depolarizes the cell, opens voltage-gated Ca++ channels causing exocytosis).

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ATP-sensitive K+ channel (Kir6.2/SUR1)

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Intermediate Signaling--RTKs

F. Signaling cascade

1. Growth factor binding to extracellular domain leading to;

2. Conformational change in protein resulting in;

3. Activation of the tyrosine kinase on the cytoplasmic face of the receptor, leading to;

4. Receptor autophosphorylation.

I

Y Y

Y Y* *

ITyrosineKinase

ATP

PY YP* *

I

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A. Glipizide opens ATP-dependent K+ channels in pancreatic cells and thereby enhances insulin secretion.

• Glipizide blocks ATP-dependent K+ channels in pancreatic cells and thereby enhances insulin secretion.

• Glipizide directly increases expression of GLUT4, the insulin-sensitive glucose transporter in target tissues.

D. Glipizide blocks absorption of carbohydrates from GI tract.

E. Glipizide activates insulin receptors in target tissues

Sample Question

Which of the following best describes the mechanism of action of Glipizide (Glucotrol, a sulfonylurea) in the treatment of non-insulin-dependent diabetes mellitus?

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Intermediate Signaling--RTKs

G. Signaling cascade5. Cytoplasmic

substrates bind to phosphotyrosine on RTK and are phosphorylated by the activated tyrosine kinase, which;

6. Alters the activity of the substrates (positively or negatively)

7. Signal terminated by receptor internalization

PY YP* *

I

Y

ATP

PY YP* *

I

YP

PY YP* *

I

YPSubstrate with altered

activity

PY YP* *

I

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RTK-Mediated Transcriptional

Regulation

Glucose

Insulin Secretion

IR Activation

Substrate Phosphorylation

GLUT4-mediated glucose uptake

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A. Inducing translocation of GLUT-4, the insulin-sensitive glucose transporter, to the plasma membrane

B. Blocking ATP-dependent K+ channels in pancreatic cells and thereby enhances its own secretion

C. Activating MAP kinase

D. Inducing insulin receptor internalization

E. Inducing insulin receptor translocation to the nucleus

Sample Question

Insulin stimulates glucose uptake in sensitive tissues by:

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RTK Signaling Pathways

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Intermediate Signaling

II. Cytokine Receptors

A. Family members

1. Interleukins

2. Interferons

3. Erythropoietin

4. GM-CSF

5. TNF

6. Leptin

7. Growth Hormone

B. Play key roles in immune system and hematopoiesis

HSC

CLPT CellsB CellsNK Cells

CMP

RBCMegakary.Mono-MacNeutroMastEosinophil

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Interleukins CSFs Interferons

IL-1 GM-CSF IFN-IL-1 G-CSF IFN-IL-2 M-CSF IFN-IL-3 EPOIL-4 TNF-IL-5 TNF- Growth factorsIL-6 LIF EGFIL-7 Steel factor TGF-IL-8 aFGFIL-9 bFGFIL-10 TGF-family KGFIL-11 TGF-1 PDGF-A

MCP-1 TGF-2 PDGF-BTGF-3 NGF-

Chemotactic factors Inhibin IGF-ILeptin Activin IGF-II

Cytokines: mediators of immune system signaling

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http://www.kidneycancerassociation.org/images/Maars_Image2.gif

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CFU-BasIL3, IL5, GM-CSF,

SCF

Totipotent / PluripotentStem Cell

IL1, IL, IL6, SCF, SCPFG-CSF, IL11, IL12,

thymosin 4MP1, TGF

Common Myeloid PrecursorIL1, IL3, IL6, SCF, GM-CSF, IL12

BFU-GEMMIL3, GM-CSF, EPOSCF, LIF, IL5, IL6,

IL9, IL11, IL12, bFGF, IGF, E-CSF, M-CSF, Activin A, Chemokines, LIF,

Epo, IFNMIP, NRP, AcSDKP, RA, inhibin, TNF,

TGF

BFU-MegIL3, GM-CSF

Epo, Meg-CSF, IL1, IL6, IL7, IL11, IL12,

SCF, LIF, bFGF, Endothelins, TGF

CFU-EIL3, GM-CSF, EPOIL11, IGF, E-CSF,

SCF, Activin A, IL5, IFN, IP, NRP, AcSDKP, RA

ProerythroblastEPO

Erythrocyte

CFU-MegIL3, GM-CSF

Epo, Meg-CSF, IL1, IL6, IL7, IL11, SCF,

LIF, bFGF, Endothelins, TGF

MegakaryoblastIL3, GM-CSF

Epo, Meg-CSF, IL1, IL6, IL7, IL11, SCF,

LIF, bFGF

MegakaryocyteTPO, IL6, Epo,

Meg-CSF, IL1, IL6, IL7, IL11, SCF, LIF,

bFGF

Thrombocyte

CFU-EoIL3, IL5, GM-CSF, IL4, IL7

MyeloblastIL3, IL5, GM-

CSF, IL4

Eosinophilic MyelocyteIL3, IL5, GM-

CSF, IL4

Eosinophil

MyeloblastIL3, IL4, GM-

CSF

BasophilicMyelocyteIL3, IL4, GM-

CSF

Basophil

CFU-MCSCF,

MCOP,IL3

MastCell

CFU-GM (CFU-C)IL3, GM-CSF, G-CSF

IL1, IL3, IL4, IL5, IL6, IL9, IL11, IL12, TGF , SCF, LIF, bFGF, M-

CSF, RA, Chemokines, AcSDKP, pEEDCK, TNF, Inhibin, Activin A, RA, IL4

CFU-GIL3, GM-CSF, G-

CSFIL4, IL6, IL12,

IFN, RA, CFU-G inhibitory factor,

IFN, IL4

MyeloblastIL3, GM-CSF, G-

CSF, IL4

Neutroophilic myelocyte

GM-CSF,G-CSF, IL4

PMN

CFU-MIL3, GM-CSF, G-

CSFIL4, IL6, IL12, IFN, RA, IL4

MonoblastIL3, GM-CSF, G-

CSF, IL4

PromonocyteIL3, GM-CSF, M-

CSF, IL4

MonocyteGM-CSF, M-CSF, IL13

Macrophage

CFU-GEMMIL3, SCF, GM-CSF, IL5, IL6, IL11, IL12, LIF, Epo, IFN, IL4, Inhibin, TGF

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Common Lympoid PrecursorIL1, IL2, IL6, IL7

Pro B cellIL1, IL2, IL3, IL4

IL5, IL6, IL7, IL10

Pre B cellIL3, IL4, IL7, SCF, IFN

Immature B cellIL1, IL4, IL5, IL6

Mature B cellIL1, IL4, IL6, 13

Antibody-producingIgM secreting B cell

Switched plasma cellSecreting non-IgM

Memory B cell(activated by rechallenge)

LGL(Null cells)

NK cellsIL1, IL2, IL4, IL7,IL12, IL13, TNF

CD4-CD8- TCR-

IL2, IL4, IL7, IL9,IL10, TSTGF, Thymic hormones

CD4+CD8+ TCRlo

T HelperCD4+CD8-

TCR+

IL10

T SuppressorCD4-CD8+

TCR+

CD4-CD8-

TCR+

IL10

IL6, IL11, IL12, G-CSF, LIF, and SCF are required to maintain B cell potential

IL2IL7

IL12

IL2IL5IL7

IL12

Isotype Switch SignalsIL1, IL2, IL4, IL6, IL10, IL13

IFN, TGF

Totipotent / PluripotentStem Cell

IL1, IL, IL6, SCF, SCPFG-CSF, IL11, IL12,

thymosin 4MP1, TGF

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Cytokine Receptor-mediated Txn Regulation

C. Cytokine receptors (CR) are very similar to RTKs, both in terms of overall structure and cytoplasmic substrates that are ultimately tyrosine phosphorylated.

D. CRs do not possess intrinsic tyrosine kinase activity.

E. Therefore, CRs must rely on a second or intermediary protein(s) that functions as a surrogate tyrosine kinase.

F. The proteins that function as surrogate tyrosine kinases for CRs are the JAKs family of tyrosine kinases (Tyk1, Tyk2, Jak1, Jak2, and Jak3).

Kinase-like DomainEA B C D

DOMAIN STRUCTURE OF JAKS KINASES

H2N - - COOHKinase Domain

Cytokine Receptor Interaction Domain

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G. Cytoplasmic substrates for JAKs are the STAT (Signal Transducers and Activators of Transcription; STAT1, 2,3, 4, 5a, 5b, 6) proteins

H. Following phosphorylation by JAKS kinases, STAT proteins:1. Physically interact (homo- or heterodimers)2. Translocate to nucleus as a complex3. Bind to a specific DNA sequence4. Activate transcription of the corresponding

(target) gene

SH2Dimerization DBD SH3 TAF

Y

H2N - - COOH

DOMAIN STRUCTURE OF STAT PROTEINS

Cytokine Receptor-mediated Txn Regulation

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Jak/Stat Signaling: IL-6 Receptor Family

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Cytokine Receptor Activation and Signaling Pathway

1. Receptor binds cytokine.

2. Receptors dimerize, JAKs kinases associated with cytoplasmic tails interact, are activated, and phosphorylate STAT proteins (and also themselves and the receptor on tyrosines).

3. STAT proteins dimerize

4. STAT protein dimers translocate to the nucleus.

5. In the nucleus, STAT dimers bind to specific DNA sequences (response elements; 5´-TTN5-6AA-3´) located in the promoter region of a target gene and regulate expression of that gene.

5

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Specificity in Cytokine Signaling

Cytokine Receptor

IFNIFN/

IL2IL3IL4IL6

IL10IL12

JAK

JAK1/2JAK1/Tyk2

JAK1/3JAK2

JAK1/3JAK1

JAK1/Tyk2JAK2/TYK2

STAT

STAT1STAT2STAT5STAT5STAT6STAT3STAT3STAT4

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Cytokine Receptor Activation and Signaling Pathway

• Cytokine target genes include those encoding other cytokines, growth factors, transcription factors

• Other cytokine target genes are SOCS (suppressors of cytokine signaling) and PIAS (protein inhibitor of activated STAT) family members, which serve to down regulate the cytokine responsiveness of the cell.

Pseudosubstrates

Direct binding to/inhibition of STAT dimers

Covalent modifications that target STATs for degradation

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Cytokine receptors play a major role(s) in:

I. Glucose homeostasis II. Immune system functionIII. Hematopoiesis

A. I onlyB. III onlyC. I and II onlyD. II and III onlyE. I, II and III

Sample Question

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Slow/Persistent Signaling Pathways

III. Nuclear Hormone Receptor Superfamily

A. Background

1. 48 members of the family in humans.

2. Play diverse roles in regulation of growth, development and homeostasis.

3. Based on importance in biology/medicine and the simple mechanism of regulation, NRs are one of the best studied and understood classes of TXN factors.

4. Soluble, non-membrane-associated proteins that function as ligand-dependent transcription factors

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NUCLEAR RECEPTOR SUPERFAMILY

STEROID HORMONE STEROID HORMONE RECEPTORSRECEPTORS

VITAMIN D RECEPTORVITAMIN D RECEPTOR

ECDYSONE RECEPTORECDYSONE RECEPTOR

OXYSTEROL RECEPTORSOXYSTEROL RECEPTORS

ORPHAN RECEPTORSORPHAN RECEPTORS

XENOBIOTIC RECEPTORSXENOBIOTIC RECEPTORS

RETINOIC ACID RECEPTORSRETINOIC ACID RECEPTORS

THYROID HORMONE RECEPTORSTHYROID HORMONE RECEPTORS

PEROXISOME PROLIFERATOR- PEROXISOME PROLIFERATOR- ACTIVATED RECEPTORSACTIVATED RECEPTORS

FATTY ACID RECEPTORSFATTY ACID RECEPTORS

BILE ACID RECEPTORSBILE ACID RECEPTORS

ANDROSTANE RECEPTORANDROSTANE RECEPTOR

H2N - - COOH

CC DD EEA/BA/B

DNA LIGAND

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ORPHAN NUCLEAR RECEPTORS

SF1SF1

LRH-1LRH-1

DAX-1DAX-1

SHPSHP

TLXTLX

PNRPNR

GCNFGCNF

HNF4HNF4

TR2, 4TR2, 4

NGFI FAMILYNGFI FAMILY

ROR FAMILYROR FAMILY

RVR FAMILYRVR FAMILY

COUP-TFCOUP-TFFAMILYFAMILY

H2N - - COOH

CC DD EEA/BA/B

DNA LIGAND

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DNA

mRNA processing

ExonExonExonExonMature mRNA

Function

Nuclear mRNA Exon Exon Exon Exon

Transcription(Gene Expression)

N

C

Protein

Translation

HRHR

Positive or Negative Regulation of Transcription

PromoterRegion

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DNA

Nuclear mRNA Exon Exon Exon Exon

Transcription(Gene Expression)

HRHR

Positive or Negative Regulation of Transcription

Hormone Receptors Are Transcription Factors

• Bind directly to DNA (promoter region of gene)• Regulate transcription in a hormone-dependent

manner

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Hormone Receptors Are Transcription Factors

• Bind directly to DNA (promoter region of gene)• Regulate transcription in a hormone-dependent

manner

What are the mechanisms by which hormone receptors bind DNA and regulate transcription in a

hormone-dependent manner?

DNA

Nuclear mRNA Exon Exon Exon Exon

Transcription(Gene Expression)

HRHR

Positive or Negative Regulation of Transcription

HH

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DNA Binding by Nuclear Receptors

Alpha helix sits in major groove of DNA

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APO and HOLO LIGAND BINDING DOMAINS STRUCTURES AGONIST and ANTAGONIST CONFORMATIONS

ER / DiethylStilbestrol(Shiau & al., 1998)

apo-RXR(Bourguet & al.,1995)

holo-ER/4 hydroxyTamoxifen(Shiau & al., 1998)

AGONISTCOACTIVATOR BINDING

ANTAGONISTCOREPRESSOR BINDING

H12

H11

H3

H4

H5H4 H4

H5 H5

H3

H12

H11H11

H12

H3

H1 H1H1

H2

H6

H6

H7H7

H7

H6

H10

H10

H10

H9

H9

H9H8

H8H8

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Activation of NR LBD

BottomlineStructurally unique drugs push receptors into unique

conformations that have unique activities…

No Ligand Ligand A Ligand B Ligand C

NoActivity

ActivityX

ActivityY

ActivityZ

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Chromosomal DNA is Highly Compacted

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Condensation: Deacetylation, Methylation, Phosphorylation

Decondensation: Acetylation, Methylation, Remodeling, Proteolysis

Transcriptional Repressors

Transcriptional Activators

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Activation of Transcription

A. Receptor-DNA interaction (DNA binding)

B. Receptor-Drug interaction (Ligand binding)

C. Regulation of transcription involves recruitment to the template of:

1. Enzymes that covalently modify the protein components (histones) of the nucleosome that decrease their affinity for DNA.

2. "Nucleosome remodeling complexes" that push nucleosomes around and enhance the access of RNA polymerase II to the template.

D. Once the above have been recruited and have acted, proteases are then recruited that degrade the receptor, and most likely other proteins, to clear the template and enhance initiation of RNA polymerase II-mediated transcription.

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THE HISTONE CODE

N C

Tail Fold

• General structure of histones

• Modifications generally occur in the tails

H2A

H3H2B

H2A

H2B

H3

H4

H4

Octamer

M

P

M A/M A MA A MM

P

P

M A A A A M

A A A A A A

= A= M= P

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A. Pioglitazone opens ATP-dependent K+ channels in pancreatic cells and thereby enhances insulin secretion.

• Pioglitazone blocks ATP-dependent K+ channels in pancreatic cells and thereby enhances insulin secretion.

• Pioglitazone directly increases expression of GLUT4, the insulin-sensitive glucose transporter in target tissues.

D. Pioglitazone blocks absorption of carbohydrates from GI tract.

E. Pioglitazone binds to and activates insulin receptors in target tissues

Sample Question

Which of the following best describes the mechanism of action of Pioglitazone (Actos, a thiazolodinedione that activates the nuclear receptor PPAR) in the treatment of non-insulin-dependent diabetes mellitus?

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Death Receptors

• Death receptors are cell surface receptors that transmit apoptosis signals initiated by specific ligands, and can activate a caspase cascade within seconds of ligand binding resulting in a rapid cell death

APO-1L(FasL/CD95L)

APO-2L(TRAIL) APO-3L TN

F

Death Domain

Death Effector Domain

Procaspase 8

Procaspase 8

Procaspase 8

Procaspase 8

Ligand

Receptor

Coupler

Transducer

Receptor Intracellular death domain Adaptor Procaspase 8 Caspase 8 Caspase 3

BID

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Decoy receptors antagonize TRAIL-mediated induction of apoptosis

• Decoy receptors that compete for binding of TRAIL with the DR4 and DR5 receptors.

• The decoy receptors are called DcR1and DcR2

• Both of these receptors are capable of competing with DR4 or DR5 receptors for binding to the ligand (TRAIL)

• However, ligation of these receptors does not initiate apoptosis since DcR1 does not possess a cytoplasmic domain, while DcR2 has a truncated death domain lacking 4 out of 6 amino acids essential for recruiting adaptor proteins.  

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Death Receptor Signaling Apoptosis Receptor

Intracellular death domain

Adaptor

Procaspase 8

Caspase 8

Caspase 3 Bid

MitochondrialStability

Cell &DNA

Integrity

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1. Multiple signaling pathways have evolved that allow multicellular organisms to respond to environmental signals• Rapid response: LGICs (TC, JI)• Intermediate response: GPCRs, RTKs, CRs (TF,

ML)• Slow/Persistent Response: NRs, Death

Receptors (ML)2. A given cell receives hundreds, if not thousands,

signals at any given time.3. This cell must integrate these signals and come up

with a response that is contextually appropriate.4. There are multiple therapeutic targets (current and

future) in each of these signaling pathways.5. Death receptors represent an exciting new avenue

to exploit in the Tx of proliferative disease.

Summary

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Which of the following protein(s) is/are localized in the plasma membrane of mammalian cells?

I. Insulin receptorII. Interleukin 6 receptorIII. Estrogen receptor

A. I onlyB. III onlyC. I and II onlyD. II and III onlyE. I, II and III

Sample Question

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Which of the following protein(s) is/are localized in the nucleus of mammalian cells and regulate(s) transcription DIRECTLY?

I. Insulin receptorII. Interleukin 6 receptorIII. Estrogen receptor

A. I onlyB. III onlyC. I and II onlyD. II and III onlyE. I, II and III

Sample Question

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Which of the following most accurately distinguishes decoy receptors from Death Receptors DR4 and DR5? A. DR4 and DR5 bind TRAIL but decoy receptors do not B. Decoy receptors bind TRAIL but DR4 and DR5 do not C. DR4 and DR5 harbor a death domain but decoy receptors do not D. Decoy receptors harbor a death domain but DR4 and DR5 do not E. Decoy receptors interact with procaspase-8 but DR4 and DR5 do not

Sample Question

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Sample Question

Which of the following would be most useful for induction of apoptosis in cancer cells that express Fas?

A. Synthetic FAS agonistsB. Synthetic FAS antagonistsC. FLIP inhibitorsD. Caspase 8 inhibitorsE. Caspase 3 inhibitors

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Sample Question

Which of the following may underlie the resistance of some cancer cells to TRAIL-mediated apoptosis?

I. Cancer cells that are sensitive to TRAIL-induced apoptosis express decoy receptors that compete with Death Receptors DR4 and DR5 for binding to TRAIL

II. Cancer cells that are insensitive to TRAIL-mediated apoptosis express decoy receptors that compete with Death Receptors DR4 and DR5 for binding to TRAIL

III. Cancer cells that are insensitive to TRAIL-mediated apoptosis express FLIP, which is an inhibitor of caspase 8

A. I onlyB. III onlyC. I and II onlyD. II and III onlyE. I, II, and III