mechanism of signal transmissionbiochemie.lf2.cuni.cz/anglicky/biox2zimni/prednasky/mechanism of...
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Mechanism of Signal Transmission into the Cells
Magdaléna Fořtová
Department of Medical Chemistry and Clinical Biochemistry, Charles University,
2nd Faculty of Medicine and University Hospital Motol
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Signal transduction pathways
Allow the cell to sense and respond to signals in the environment.
Signal Receptor Transducer Effector Response
Upstream Downstream
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Signals
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Receptors
• Soluble receptors (cytoplasmic or nuclear receptors)
• Transmembrane receptors (varies with the signal that
is generated inside the cell upon binding of the extracellular signal molecule to the receptor)
– Enzyme coupled receptors
– G-protein coupled receptors (GPCRs)
– Ion-channel coupled receptors
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Examples of lipophilic signaling molecules
Cortisol (glucocortikoid) Androsteron (steroid)
All-trans retinol acid (retinoid)
3,3',5-trijodo-L-thyronine (thyroid)
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Soluble receptors
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Ion channel coupled receptors
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Enzyme coupled receptors • Tyrosine kinases phosphorylate protein tyrosine
residues using ATP • Phospholipase C cleaves PIP2 into IP3 and DAG
GNRP: guanine nucleotide-releasing protein GAP: GTPase-activating protein, GTPase-accelerating protein
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Phospholipase C
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Tyrosine kinases / RAS MAP kinases
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GPCRs Characteristic receptor structure
Heptahelical receptors (7 transmembrane α-helixes)
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GPCRs
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Second messengers
Second messenger Substrate Enzyme Effector Degradation
cAMP ATP adenylate cyclase (AC) protein kinase A (PKA) phosphodiesterases (PDE)
cGMP GTP guanylate cyclase (GC) protein kinase G (PKG) phosphodiesterases (PDE)
calcium cations (Ca2+) - release from ER after IP3 stimulation
calmodulin ER re-uptake by Ca2+ATPase
inositol-1,4,5-trisphosphate (IP3) phosphatidylinositol-4,5-bisphosphate (PIP2)
phospholipase C (PLC) protein kinase C (PKC) phosphatases – dephosphorylation to inositol
diacylglycerol (DAG) phosphatidylinositol-4,5-bisphosphate (PIP2)
phospholipase C (PLC) protein kinase C (PKC) lipases – creation of glycerol and free fatty acids
phosphatidylinositol-3,4,5-trisphosphate (PIP3)
phosphatidylinositol-4,5-bisphosphate (PIP2)
phosphatidylinositol-3-kinase (PI3K)
protein kinase B (PKB)
phosphatase PTEN – cleavage of the phosphate at position 3
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Calmodulin + CaM kinase II
Calmodulin + Ca2+ → change of conformation → activation of CaM kinase
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MAP kinase pathway
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MAPK - simplified scheme
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Signalling molecules – vesicle stored and released using SNAP-SNARE proteins
“SNARE”: stands for Soluble N–ethylmaleimide sensitive factor (NSF) attachment protein receptor
SNARE proteins are essential for membrane fusions during all trafficking steps of eukaryotic secretory pathways
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Signal termination
• The chemical messenger itself (acetylcholine esterase, insulin degradation in liver)
• The reaction itself (when GTP in G-protein is used, G-protein GDP complex forms the original structure)
• Degradation of second messenger (phosphodiesterase cleavage of cAMP)
• Phosphatases
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Arrestins
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Arrestins
• important intracellular proteins, multifunctional regulators of G-protein-coupled receptor signaling
• they form complexes with most GPCRs (following agonist binding and phosphorylation of receptors) and play a central role in the processes of homologous desensitization, sequestration and downregulation of receptors, which lead to termination of G-protein activation
• control of cell differentiation, proliferation, migration and apoptosis organism growth and development, oncogenesis
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ARRESTINS
α-arrestins Visual/β-arrestins
Visual arrestins Rod arrestin
Cone arrestin
β-arrestins β-arrestin 1
β-arrestin 2
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Arrestin functions
• Regulation of cell responses to ligand binding
– homologous desensitization, sequestration and downregulation
of receptors (GPCRs)
• Transfer of signals in a cell (-arrestins)
– promotion of the formation of signaling complexes with tyrosine
kinase Src and mitogen-activated protein kinase cascades allowing G-
protein-coupled receptors to signal independently from G-protein
– scaffold and adaptor proteins in these cascades
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Role of β-arrestins in the desensitization, sequestration and intracellular trafficking of GPCRs
dynamin
AP2
clathrin
arrestin
agonist
G-protein
effector
GPCR-specific
protein phosphatase phosphate
GRK2
Slow
recycling
degradation degradation
rapid
recycling
“class A” GPCR
“class B” GPCR
desensitization
se
qu
estratio
n
clathrin
coated
pit
clathrin
coated
vesicle
endosomal vesicle
endosomal vesicle
acidified vesicle compartment
recycling vesicle
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Roles of β-arrestin-dependent recruitment of Src kinases in GPCR signaling
dynamin
-arrestin
agonist
G-protein
effector
GRK
Dynamin
phosphorylation
Ras
Raf-1
MEK
ERK1/2
Src-TK
ERK1/2 activation
Cell proliferation
Neutrophil
degranulation
Exocytic granule endosomal
vesicle
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β-arrestins as "scaffolds" of MAP kinase cascades
• MAPKKK (MKKK, MAP3K) • MAPKK (MKK, MEK) • MAPK
• Raf-1, B-Raf • MEK1, MEK2 • ERK1/2
Activated MAP kinases phosphorylate membrane, cytoplasmic,
nuclear, cytoskeletal substrates; phosphorylate and activate nuclear transcription factors
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Roles of β-arrestins in the activation and targeting of MAP kinases
-arrestin
agonist
G-protein
effector
GRK
Raf-1
MEK
ERK1/2
phosphorylation
of cytosolic
substrates
other kinases
nucleus
endosomal vesicle
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• β-arrestin 1 is involved in the formation of a nuclear complex in the promotor regions of p27 and c-fos genes
• Complex: transcription factor CREB (cAMP response element-binding protein), histon acetyltranferase p300, β-arrestin 1 (event. other proteins)
• β-arrestin 1 acts as a nuclear "scaffolding" protein recruiting p300 to CREB
increased histone H4 acetylation, chromatin reorganization, increased gene transcription of p27 and c-fos
Nuclear function of arrestins
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Eicosanoids
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Eicosanoids - biosynthesis
arachidonic acid
CYP450
DiHETEs
19-, 20-, 8-, 9-, 10-, 11-, 12-, 13-, 15-, 16-, 17-, 18-HETE
cyklooxygenases
prostacyklins
prostaglandins
tromboxanes
lipoxygenases
5-, 8-, 12-, 15-HETE
lipoxins
hepoxilins
leukotrienes
EETs (epoxides)
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Main eicosanoid production sites
• Endothelial cells • Leukocytes • Platelets • Kidneys
• Unlike e.g. histamin, eicosanoids are not synthesized in
advance and stored in granules • In case of an emergent need, these are rapidly produced
from a released arachidonate • Eicosanoids biosynthesis takes place in every cell type
except red blood cells
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Cytokines
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Cytokines
• Group of proteins and peptides (glycopeptides)
• Influence cell growth (growth factors)
• Signal transmission from a cell to another cell
• Important group - lymphokines (also interleukins), proteins released from activated cells of immune system which coordinate immune response of the organism
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Cytokine nomenclature
• Lymphokines - produced by activated T-lymphocytes, they control the response of immune system by signalization between immunocompetent cells
• Interleukins (IL) - target cells for IL are leukocytes
• Chemokines - specific class, mediating chemotaxis between cells; stimulate leukocyte movement and regulate their migration from blood into tissues
• Monokines - produced mainly by mononuclear cells, such as macrophages
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Main functions of cytokines
• Hematopoiesis (e.g. CSF - colony stimulating factor)
• Inflammatory reactions (e.g. IL1 - interleukin, TNF - tumor
necrosis factor)
• Chemotaxis (e.g. IL8, MIP1- macrophage inflammatory protein 1, BLC – B-lymphocyte chemoatractant)
• Imunostimulation (e.g. IL12, IFNg - interferon)
• Imunosupression (e.g. IL10)
• Angiogenesis (e.g. VEGF- vascular endothelial growth factor)
• Embryogenesis (e.g. TGF-, LT – lymphotoxin)
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Literature
• R.K. Murray et al.: Harper's Illustrated Biochemistry, twenty-sixth edition, McGraw-Hill Companies, 2003
• Allan D. Marks, MD: Basic Medical Biochemistry a Clinical Approach, Lippincott Williams & Wilkins, 2009
• Ernst J. M. Helmreich, The Biochemistry of Cell Signalling, Oxford University Press, USA, 2001
• Geoffrey M. Cooper, Robert E. Hausman, The Cell: A Molecular Approach, Fourth Edition, Sinauer Associates, Inc., 2006
• Michael J. Berridge, Peter Lipp and Martin D. Bootman, The versatility and universality of calcium signalling, Nature Reviews | Molecular Cell Biology (1), 2000
• Luttrell L. M., Lefkowitz R. J. The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals, Journal of Cell Science 2002, 115(3): 455-465