Cell Signaling Systems

1. General Principles of Information Propagation

2. Signaling Pathways vs Networks Mechanisms and Consequences of Networking: Information Processing

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

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

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

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

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)

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

The Gq-PLC-pathway

Ram and IyengarSTKE Connections Map

Cytokine Receptor JAK-STAT Pathway

Aaronson and HorvathSTKE ConnectionsMap

JohnsonSTKE ConnectionsMap

The Growth Factor Receptor-Ras-MAPK pathway

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

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

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

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

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.

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

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

Heterotrimeric G protein Pathways

Coupling to different receptor isoforms lead to different G protein pathways and different biological effects

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

Isoforms of cellular componentscan promote signal integrationby having partially overlapping connectivity

Jordan , Landau, Iyengar (2000) Cell 103:193

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

Jordan , Landau , Iyengar (2000) Cell 103:193

The same small GTPase regulates multiple functions throughdifferent effectors

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

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?

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

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

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

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)

Figure 2 fromManuela Zaccolo and Tullio Pozzan(2002) Science 295:1711

cAMP microdomains visualized by FRET in rat cardiac myocytes

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+.

The Ca2+ microdomains are localized and dynamic

Fig 2Jesse H. Goldberg Gabor Tamas Dmitriy Aronov and Rafael Yuste (2003) Neuron 40: 807-821

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

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.