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

Signal transduction pathways

Allow the cell to sense and respond to signals in the environment.

Signal Receptor Transducer Effector Response

Upstream Downstream

Signals

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

Examples of lipophilic signaling molecules

Cortisol (glucocortikoid) Androsteron (steroid)

All-trans retinol acid (retinoid)

3,3',5-trijodo-L-thyronine (thyroid)

Soluble receptors

Ion channel coupled receptors

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

Phospholipase C

Tyrosine kinases / RAS MAP kinases

GPCRs Characteristic receptor structure

Heptahelical receptors (7 transmembrane α-helixes)

GPCRs

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

Calmodulin + CaM kinase II

Calmodulin + Ca2+ → change of conformation → activation of CaM kinase

MAP kinase pathway

MAPK - simplified scheme

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

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

Arrestins

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

ARRESTINS

α-arrestins Visual/β-arrestins

Visual arrestins Rod arrestin

Cone arrestin

β-arrestins β-arrestin 1

β-arrestin 2

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

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

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

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

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

• 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

Eicosanoids

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)

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

Cytokines

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

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

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)

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