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Cellular Signaling Mechanisms Receptor Tyrosine Kinases Cytokine Receptors Nuclear Receptors Death Receptors Phar 735/590 Winter 2006 Mark Leid Office: Pharmacy 407 Tel: 737-5809 E mail: [email protected]. Objective. - PowerPoint PPT Presentation
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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: [email protected]
2
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.
3
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.
4
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?
10
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
18
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
19
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
23
Specificity in Cytokine Signaling
Cytokine Receptor
IFNIFN/
IL2IL3IL4IL6
IL10IL12
JAK
JAK1/2JAK1/Tyk2
JAK1/3JAK2
JAK1/3JAK1
JAK1/Tyk2JAK2/TYK2
STAT
STAT1STAT2STAT5STAT5STAT6STAT3STAT3STAT4
24
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
5
25
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
26
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
27
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
28
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
29
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
31
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
32
DNA Binding by Nuclear Receptors
Alpha helix sits in major groove of DNA
33
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
34
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
35
Chromosomal DNA is Highly Compacted
36
Condensation: Deacetylation, Methylation, Phosphorylation
Decondensation: Acetylation, Methylation, Remodeling, Proteolysis
Transcriptional Repressors
Transcriptional Activators
37
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.
38
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
39
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?
40
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
41
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.
42
Death Receptor Signaling Apoptosis Receptor
Intracellular death domain
Adaptor
Procaspase 8
Caspase 8
Caspase 3 Bid
MitochondrialStability
Cell &DNA
Integrity
43
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
44
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
45
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
46
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
47
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
48
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