Cellular Signaling Mechanisms

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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 Mark.Leid_at_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)
• Examples
• Insulin Receptor
• Insulin-like GFRs
• Platelet-derived GFR
• Epidermal GFR
• Fibroblast GFR
• RTKs span plasma membrane only once
• Receptor harbors intrinsic tyrosine kinase
  activity that is activate by GF binding
• The signaling pathway involves phosphorylation of
  cytoplasmic substrates by carboxyl terminus of
  RTK
• 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
• Growth factor binding to extracellular domain
  leading to
• Conformational change in protein resulting in
• Activation of the tyrosine kinase on the
  cytoplasmic face of the receptor, leading to
• Receptor autophosphorylation.

Tyrosine Kinase
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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?

• 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

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Intermediate Signaling--RTKs

• G. Signaling cascade
• 5. 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

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RTK-Mediated Transcriptional Regulation
Glucose
Insulin Secretion
IR Activation
Substrate Phosphorylation
GLUT4-mediated glucose uptake
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Sample Question
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
Insulin stimulates glucose uptake in sensitive
tissues by
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RTK Signaling Pathways
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Intermediate Signaling

• II. Cytokine Receptors
• Family members
• Interleukins
• Interferons
• Erythropoietin
• GM-CSF
• TNFa
• Leptin
• Growth Hormone
• Play key roles in immune system and hematopoiesis

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Cytokines mediators of immune system signaling
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Totipotent / Pluripotent Stem Cell IL1, IL, IL6,
SCF, SCPF G-CSF, IL11, IL12, thymosin b4 MP1a,
TGFb
BFU-Meg IL3, GM-CSF Epo, Meg-CSF, IL1, IL6, IL7,
IL11, IL12, SCF, LIF, bFGF, Endothelins, TGFb
CFU-Bas IL3, IL5, GM-CSF, SCF
BFU-GEMM IL3, GM-CSF, EPO SCF, LIF, IL5, IL6,
IL9, IL11, IL12, bFGF, IGF, E-CSF, M-CSF, Activin
A, Chemokines, LIF, Epo, IFNg MIP, NRP, AcSDKP,
RA, inhibin, TNFa/b, TGFb
CFU-Eo IL3, IL5, GM-CSF, IL4, IL7
CFU-MC SCF, MCOP, IL3
CFU-Meg IL3, GM-CSF Epo, Meg-CSF, IL1, IL6, IL7,
IL11, SCF, LIF, bFGF, Endothelins, TGFb
CFU-M IL3, GM-CSF, G-CSF IL4, IL6, IL12, IFNg,
RA, IL4
Myeloblast IL3, IL4, GM-CSF
CFU-G IL3, GM-CSF, G-CSF IL4, IL6, IL12, IFNg,
RA, CFU-G inhibitory factor, IFNg, IL4
Myeloblast IL3, IL5, GM-CSF, IL4
CFU-E IL3, GM-CSF, EPO IL11, IGF, E-CSF, SCF,
Activin A, IL5, IFNg, IP, NRP, AcSDKP, RA
Monoblast IL3, GM-CSF, G-CSF, IL4
Eosinophilic Myelocyte IL3, IL5, GM-CSF, IL4
BasophilicMyelocyte IL3, IL4, GM-CSF
Megakaryoblast IL3, GM-CSF Epo, Meg-CSF, IL1,
IL6, IL7, IL11, SCF, LIF, bFGF
Myeloblast IL3, GM-CSF, G-CSF, IL4
Promonocyte IL3, GM-CSF, M-CSF, IL4
Megakaryocyte TPO, IL6, Epo, Meg-CSF, IL1, IL6,
IL7, IL11, SCF, LIF, bFGF
Neutroophilic myelocyte GM-CSF, G-CSF, IL4
Proerythroblast EPO
Monocyte GM-CSF, M-CSF, IL13
Mast Cell
Erythrocyte
Thrombocyte
Eosinophil
Basophil
PMN
Macrophage
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Totipotent / Pluripotent Stem Cell IL1, IL, IL6,
SCF, SCPF G-CSF, IL11, IL12, thymosin b4 MP1a,
TGFb
Common Lympoid Precursor
IL1, IL2, IL6, IL7
IL6, IL11, IL12, G-CSF, LIF, and SCF are
required to maintain B cell potential
Pro B cell IL1, IL2, IL3, IL4 IL5, IL6, IL7, IL10
LGL (Null cells)
CD4-CD8- TCR- IL2, IL4, IL7, IL9, IL10, TSTGF,
Thymic hormones
CD4CD8 TCRablo
Pre B cell IL3, IL4, IL7, SCF, IFNg
Immature B cell IL1, IL4, IL5, IL6
IL2 IL5 IL7 IL12
IL2 IL7 IL12
Mature B cell IL1, IL4, IL6, 13
NK cells IL1, IL2, IL4, IL7, IL12, IL13, TNF
T Helper CD4CD8- TCRab IL10
T Suppressor CD4-CD8 TCRab
CD4-CD8- TCRgd IL10
Antibody-producing IgM secreting B cell
Switched plasma cell Secreting non-IgM
Isotype Switch Signals IL1, IL2, IL4, IL6, IL10,
IL13 IFNg, TGFb
Memory B cell (activated by rechallenge)
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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).
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Cytokine Receptor-mediated Txn Regulation

• 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
• Physically interact (homo- or heterodimers)
• Translocate to nucleus as a complex
• Bind to a specific DNA sequence
• Activate transcription of the corresponding
  (target) gene

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Jak/Stat Signaling IL-6 Receptor Family
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Cytokine Receptor Activation and Signaling Pathway

• Receptor binds cytokine.
• Receptors dimerize, JAKs kinases associated with
  cytoplasmic tails interact, are activated, and
  phosphorylate STAT proteins (and also themselves
  and the receptor on tyrosines).
• STAT proteins dimerize
• STAT protein dimers translocate to the nucleus.
• 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.

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Specificity in Cytokine Signaling
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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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Sample Question
Cytokine receptors play a major role(s)
in I. Glucose homeostasis II. Immune system
function III. Hematopoiesis A. I only B. III
only C. I and II only D. II and III only E. I, II
and III
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Slow/Persistent Signaling Pathways

• III. Nuclear Hormone Receptor Superfamily
• Background
• 48 members of the family in humans.
• Play diverse roles in regulation of growth,
  development and homeostasis.
• 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.
• Soluble, non-membrane-associated proteins that
  function as ligand-dependent transcription factors

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NUCLEAR RECEPTOR SUPERFAMILY
STEROID HORMONE RECEPTORS VITAMIN D
RECEPTOR ECDYSONE RECEPTOR OXYSTEROL
RECEPTORS ORPHAN RECEPTORS XENOBIOTIC RECEPTORS
RETINOIC ACID RECEPTORS THYROID HORMONE
RECEPTORS PEROXISOME PROLIFERATOR- ACTIVATED
RECEPTORS FATTY ACID RECEPTORS BILE ACID
RECEPTORS ANDROSTANE RECEPTOR
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ORPHAN NUCLEAR RECEPTORS
COUP-TF FAMILY
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DNA
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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?
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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
H9
H9
H9
H1
H8
H1
H1
H10
H8
H8
H4
H10
H10
H4
H4
H5
H7
H7
H5
H5
H12
H3
H7
H2
H11
H12
H3
H3
H6
H11
H11
H6
H6
H12
apo-RXR? (Bourguet al.,1995)
ER? / DiethylStilbestrol (Shiau al., 1998)
holo-ER?/4 hydroxyTamoxifen (Shiau al., 1998)
AGONIST COACTIVATOR BINDING
ANTAGONIST COREPRESSOR BINDING
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Activation of NR LBD

• Bottomline
• Structurally unique drugs push receptors into
  unique conformations that have unique activities

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Chromosomal DNA is Highly Compacted
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(No Transcript)
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Activation of Transcription

• Receptor-DNA interaction (DNA binding)
• Receptor-Drug interaction (Ligand binding)
• Regulation of transcription involves recruitment
  to the template of
• Enzymes that covalently modify the protein
  components (histones) of the nucleosome that
  decrease their affinity for DNA.
• "Nucleosome remodeling complexes" that push
  nucleosomes around and enhance the access of RNA
  polymerase II to the template.
• 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
Octamer

• General structure of histones

H4
H3
H2A
H2B
H3

• Modifications generally occur in the tails

H2B
H2A
P
P
H4
M
M
M
M
A/M
A
M
A
A
M
A
A
A
A
M
P
A
A
A
A
A
A
A
M
P
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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?

• 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

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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-2L (TRAIL)
APO-1L (FasL/CD95L)
Ligand
APO-3L
TNF
Receptor
Coupler
Transducer
Procaspase 8
Procaspase 8
Procaspase 8
Procaspase 8
Death Effector Domain
Death Domain
BID
?
Receptor ? Intracellular death domain ? Adaptor ?
Procaspase 8 ? Caspase 8 ? Caspase 3
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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
Mitochondrial Stability
Cell DNA Integrity
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• Summary

• 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)
• A given cell receives hundreds, if not thousands,
  signals at any given time.
• This cell must integrate these signals and come
  up with a response that is contextually
  appropriate.
• There are multiple therapeutic targets (current
  and future) in each of these signaling pathways.
• Death receptors represent an exciting new avenue
  to exploit in the Tx of proliferative disease.

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Sample Question
Which of the following protein(s) is/are
localized in the plasma membrane of mammalian
cells? I. Insulin receptor II. Interleukin 6
receptor III. Estrogen receptor A. I
only B. III only C. I and II only D. II and III
only E. I, II and III
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Sample Question
Which of the following protein(s) is/are
localized in the nucleus of mammalian cells and
regulate(s) transcription DIRECTLY? I. Insulin
receptor II. Interleukin 6 receptor III. Estroge
n receptor A. I only B. III only C. I and II
only D. II and III only E. I, II and III
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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 agonists B. Synthet
ic FAS antagonists C. FLIP inhibitors D. Caspase
8 inhibitors E. 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 only B. III only C. I and II
only D. II and III only E. I, II, and III