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Cell Signaling Systems
1. General Principles of Information Propagation
2. Signaling Pathways vs Networks Mechanisms and Consequences of Networking: Information Processing
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SignalEpinephrineGlucagonLH
Receptor-ARGR, LHR
EffectorAdenylylCyclase
cAMP ATPSecondMessenger
Protein Kinase A
Phosphorylated Proteins Change in activity of Enzymes, channels, transcription factors
TransducerGs heterotrimer
cAMP
+
A Linear Signaling Pathway
Glucose Metabolism
Cardiac Contractility
Gene Expression
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Epinephrine
-AR
AdenylylCyclase
cAMP ATP
Phosphorylated Proteins Change in activity of :Enzymes, channels,transcription factors
Gs
Ligand-Protein Interaction
Enzymatic Reaction
Protein Kinase A
Ligand-Protein Interaction
ATP
ADP
Protein P
Enzymatic Reaction
Protein-Protein Interaction
Chemistry of Signaling
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Signaling through non-covalent interactions
•Outside the cell
Ligand – receptor interactions
•Inside the cell
1) Interactions between second messengers (small molecules) and targets
cAMP with protein kinase A or cAMP-GEF
IP3 with the IP3 receptor (ER Ca2+ channel)
2) Protein-protein interactions
GRB and SOS (Ras-GEF) with Ras
Ras with Raf
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Signaling through enzymatic activity
• GTPases Heterotrimeric G proteins
Small GTPases: Ras, Rho/Cdc42, Rap and Rab families
• Protein Kinases and PhosphatasesSer-Thr Kinases: Protein kinases A and C CaMK-II
Tyr-Kinases: EGFR , Src and JAK
Phosphatases: PP2A , PP1, Calcineurin , SHP and PTP-1
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Mechanism of Information Transfer
Change in activity state of the upstream component leads to change in activity of the down-stream component.
• Change is generally vectorial in nature
• Change can be either activating or inhibiting. This is interaction specific. (e.g. phosphorylation of the target can either increase or decrease its activity)
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Pathways vs Networks
Generally pathways involve simple cascade of reactions leading to information flow.
Examples of pathways
1) G protein Pathways
2) RTK–Ras–MAPK pathway
3) Cytokine Receptor –JAK-Stat Pathway
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The Gq-PLC-pathway
Ram and IyengarSTKE Connections Map
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Cytokine Receptor JAK-STAT Pathway
Aaronson and HorvathSTKE ConnectionsMap
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JohnsonSTKE ConnectionsMap
The Growth Factor Receptor-Ras-MAPK pathway
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Pathways vs Networks
Networks arise from interactions whereby
a component of one pathway regulates the activity of a second pathway
An example of simple network:
Interactions between RTK–Ras–MAPK and RTK-PLC-PKC pathways
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DAG
Ca2+
RTK
c-Raf
MAPK-1,2
MEK-1,2
Grb2SOS Ras
Transcription Factors,Other substrates
PLC-
PKC
AA
cPLA2
IP3
GAP
A simple signaling network
Ability of PKC to regulate Ras/Rafand MAPK to regulate PKC through Phospholipase A2 leads to networking between the two pathways
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DAG
Ca2+
RTK
c-Raf
MAPK-1,2
MEK-1,2
Grb2
SOS Ras
Stimulation of Proliferation
PLC-
PKC
AA
cPLA2
IP3
PersistentActivation
GAP
Consequence of Networking: A feedback loop that displays bistability
Bhalla and Iyengar(1999)
Science 283:381
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0
0.05
0.1
0.15
0 15 30 45 60
Act
ivat
ed M
AP
K (M
)
Time (min)
Sustained phospho-MAPK levels after brief PDGF stimulus
P-MAPK 25 15 30 40 6050
Time after wash (min)
-
Time after wash (min)
Pho
spho
-MA
PK
2(a
rbitr
ary
units
)0 10 20 30 40 50 60
StimWash Assay
Bhalla , Ram and Iyengar (2002) Science297:1018
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Summary-I
1. Signaling pathways allow for linear flow of information: Such information may processed (i.e. amplified, or dampened).
Many important physiological processes are regulated by linear signaling pathways, e.g. Adrenaline regulation of glucose metabolism, visual transduction.
2. Networking arises from the ability of a component of one pathway to interact with and regulate another pathway, or by the same component participating in multiple pathways.
Networking results in signal processing so that information is transferred across spatial and temporal domains.
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General Themes in Heterotrimeric G protein Pathways
Most ligands interact with more than one receptor isoform
All pathways involve small G proteins
Small molecule diffusible messengers are key components in several pathwaysImportance of Spatial Domains
Rockman H.A. et al (2002) Nature 415:206
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Table 1: Phenotypes of Mice deficient in adrenergic receptor subtypes
From Philpp M And Hein L (2004)Pharmacol Ther 101: 65-74
Most ligands interact with more than one receptor isoform
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Heterotrimeric G protein Pathways
Coupling to different receptor isoforms lead to different G protein pathways and different biological effects
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This abridged table from the human Genome Sequence Paper shows only the subset of relevant proteinsVenter et al (2001) Science 291:1304
Such isoform diversity is found for many signaling components.
#s of isoforms generally increase with the evolution.
Mammals have the most isoforms and the most complex networking
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Isoforms of cellular componentscan promote signal integrationby having partially overlapping connectivity
Jordan , Landau, Iyengar (2000) Cell 103:193
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Connections from heterotrimeric to small GTPases increase the number of pathways G proteins can regulate and thus increase the effects GPCRs have on cellular function
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Jordan , Landau , Iyengar (2000) Cell 103:193
The same small GTPase regulates multiple functions throughdifferent effectors
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SignalEpinephrineGlucagonLH
Receptor-ARGR, LHR
EffectorAdenylylCyclase
cAMP ATPSecondMessenger
Protein Kinase A
Phosphorylated Proteins Change in activity of Enzymes, channels, transcription factors
TransducerGs heterotrimer
cAMP
+
A Linear Signaling Pathway
Glucose Metabolism
Cardiac Contractility
Gene Expression
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Small Molecule Messengers and Spatial Domains
Signal
ReceptorAdenylylCyclase
cAMP ATPGs
cAMP is a diffusible second messenger ( ~600 Da)
Does it diffuse through the cell or are there selected regions in the cell with high concentrations of cAMP?
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A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell Thomas C. Rich*, Kent A. Fagan , Tonia E. Tse*,
Jerome Schaack , Dermot M. F. Cooper , and Jeffrey W. Karpen*,
( 2001) PNAS 98:13049
Study compares local levels of cAMP by using the cyclic nucleotide gated channel as the a cAMP biosensor and compares cAMP levels near the plasma membrane by measurement with the biosensor to
cAMP levels in the whole cell by biochemical (radioisotope) labeling
measurements
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Copyright ©2001 by the National Academy of Sciences
Rich, Thomas C. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13049-13054
Fig. 1. Distinct cAMP signals measured in different subcellular compartments
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Copyright ©2001 by the National Academy of Sciences
Rich, Thomas C. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13049-13054
Fig. 4. A quantitative description of the localized transient cAMP response and the total cellular cAMP accumulation
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Discrete Microdomains with High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes
Manuela Zaccolo* and Tullio Pozzan(2002) Science 295:1711
Live Cell Imaging of cAMP using Using FluorescenceResonance Energy Transfer (FRET)
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Figure 2 fromManuela Zaccolo and Tullio Pozzan(2002) Science 295:1711
cAMP microdomains visualized by FRET in rat cardiac myocytes
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Calcium Microdomains in Aspiny Dendrites
Jesse H. Goldberg Gabor Tamas Dmitriy Aronov and
Rafael Yuste (2003) Neuron 40: 807-821
Imaging of Ca2+ in the dendrites of interneurons in tissues slices using fluorescent probes (Fluo-4) by two-photon microscopy
The neurons are spontaneously activated or electrically stimulated and imaged using fluorescent probes for Ca2+.
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The Ca2+ microdomains are localized and dynamic
Fig 2Jesse H. Goldberg Gabor Tamas Dmitriy Aronov and Rafael Yuste (2003) Neuron 40: 807-821
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The Ca2+ microdomains in the dendrites of the interneurons used in the Yuste study are due to:
1) Calcium inflow through the calcium permeable AMPA channels
2) Extrusion of Ca2+ by the Na+/Ca2+ exchanger
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Summary II
1.Through different receptor isoforms, ligands for GPCRs couple to different signaling pathways. These pathways lead to different physiological effects.
2. The coupling between the heterotrimeric G proteins and small GTPases can lead to signal routing to multiple effectors and thus evoke several responses.
3. Diffusible small molecules are often constrained to spatial domains and thus allow the stimulus to achieve specificity of physiological effects.