1

Second-messenger

amplification

Second messengers are readily diffusible

They can serve to amplify a signal

Amplification: an example of how amplification could take place in a signal transduction cascade

Amplification

Amplification

Amplification

Amplification

Amplification

epinephrineSecond Messengers

Molecules whose presence is a signalSynthesized or released from storageAct as intracellular ligandsCommonly used in G protein-coupled receptor signaling

– Made or released by effector proteins– (Ligand for GPCR is 1st messenger)

General characteristics– Low amounts in resting state– Regulated synthesis (or release)– Regulated destruction (or removal)– Act on intracellular proteins as agonists

Signal is required to produce cellular change– Signal generated by receptor (or effector)– Signal size related to → of active receptors– (dose response!)

2

Flour common intracellular second messengers

-Many receptors,few second messengers

Second messengers carry signals from many receptors

Binding of ligands (1st messenger) to surface receptors– Short-lived increased of

signaling molecules – Signaling molecules activate

kinases or phosphatases = amplify signal

Second messenger needs amplification

An extracellular signaling molecule is termed “first messenger”First messenger → production of small and transient signaling molecule

on the inside of the cell → referred to as second messenger →intracellular response

What is second messenger ?The term second messenger was originally coined (錢幣) to describe the signalling molecules which are found to be produced by cells in response to the perception (感知) of the first message (extracellular ligand). They are not the second component in the signaling pathway and term is now confusing and misleading, and should be avoided.

One very important feature of the production of intracellular messenger need amplification.

For example, one molecule bind to the exterior surface of the cells →activation of hundreds or thousands of enzyme molecules inside the cell → catalyze many rounds of reaction → produced amplification of signal ; It do not need a large concentration of extracellular signals or the vast amount of ligand binding.

adenylyl cyclase cyclic AMP from ATP

guanylyl cyclase cyclic GMP from GTP

phospholipase C (PLC) cleaves phosphatidylinositol bisphosphate (PIP2)

diacylglycerol (DAG) — diffuses in membrane inositol trisphosphate (IP3) — released into cytosol

Primary amplifier enzymes:

Secondary amplifier enzymes:protein kinase A (PKA)protein kinase G (PKG)protein kinase C (PKC)calcium-calmodulin kinase (Ca-CM-K)

cyclic AMP protein kinase A (PKA); also ion channels

cyclic GMP protein kinase G (PKG); also ion channels

protein tyrosine kinase (PTK) serine/threonine kinases

diacylglycerol (DAG) protein kinase C (PKC)

inositol trisphosphate (IP3) Ca2+ release calcium-calmodulin kinase (Ca-CM-K)

Five modes of ligand-triggered second messengers:

3

Cyclic AMP as a Second Messenger

In many cases, a G protein is activated which then activates an enzyme, adenylyl cyclase which converts ATP to cyclic AMP (cAMP).

cAMP then serves as a second messenger which activates another enzyme in the cell, often a protein kinase (an enzyme that phophorylates a protein, activating it).

cAMP initiates a chain of events (the signal transduction pathway) that results in some specific response of the cell to the first messenger (hormone).

Most water-soluble hormones do not readily enter the target cell - they bind to a surface receptor.

cAMP pathway

PKA phosphorylatesvoltage-gated Ca2+

channel, enhancing response to depolarization.

Inactivation of the pathway is partly by phosphodiesterase, which converts cAMP to AMP, and also by various protein phosphatases, which dephosphorylate the activated downstream proteins.

Example

GS activated

epinephrine binds cardiac muscle receptor

cAMPCyclic AMP is most common second

messenger in animal cells; identified by Earl Sutherland in the 1960s

Hormones bind receptors, and membrane-bound adenylyl cyclase is activated via the G protein

Adenylyl cyclase catalyzes the conversion of ATP to cAMP

cAMP activates protein kinasesProtein kinases catalyze the

phosphorylation of a specific protein, which triggers a chain of reactions leading to the particular metabolic effect of the hormone

Protein kinases are very specific in action

cAMP is rapidly inactivated and converted to AMP

ATP → cAMP , by adenylyl cyclase (AC)

Integral membrane proteinsImportant in many cellular signals and pathwaysCatalyses the conversion of ATP to cAMP

- cAMP functions as second messengerActivated or inhibited by G-proteins9 known adenylyl cyclases in mammals

4

GTP bound Gsα interacts with adenylyl cyclase

The structural changes that are induced are not known, but the result is active enzyme

Many signals, through different receptors, can activate Gsα, resulting in a higher concentration of GTPGsα and the production of higher levels of cAMP

Forskolin, applied to cells, will activate pathways mediated by cAMP

Gsα stimulates adenylyl cyclase Adenylyl cyclase is stimulated and inhibited by different receptor-ligand complexes

Isoforms of mammalian adenylyl cyclase

Sunahara RK, Taussig R. Isoforms of mammalian adenylyl cyclase: multiplicities of signaling.Mol Interv. 2002 Jun;2(3):168-184.

cAMP activated protein kinase A mediates various responses in different cells

Activation of cAMP-dependent protein kinase (PKA)

cAMP binds to the PKA regulatory subunits conformational changes, which causes their dissociation from the catalytic subunits kinaseactivation. Release of the catalytic subunits requires the binding of more than two cyclic AMP molecules greatly sharpening the response of the kinase to changes in [cAMP].

PKA is a Ser/Thr kinase with discrete substrate specificity, thus facilitating a cascade of highly regulated protein phosphorylations.

5

Intracellular signaling was first elucidated by studies of the action of epinephrine Signaling from GPCRs

Regulation of glycogen metabolism in liver and in muscle cells正腎上腺素或cortisol

Glycogen metabolism is regulated by hormone induced activation of protein kinase A

PKA

cyclic AMP

ATP cAMP AMPadenylylcyclase

phosphodiesterase

GS ( subunit)+

agonist + receptor

cAMP-dependent protein kinase (PKA)+

• Binding of agonist to receptor cascade: 1 agonist 102 cAMP 104 phosphorylated enzyme

106 products

• Inhibitory regulation of adenylyl cyclase via receptors linked to Gi cAMP levels

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How does coffee perk you up?

caffeine inhibits phosphodiesterase so cAMP levels remain high

adrenalin’s effect on heart is prolonged– more O2 to brain & tissues

gives a feeling of increased vitality and energy– a moderate “fight or flight” rush

cAMP AMPphosphodiesterase

caffeine

activation of gene transcription by G-protein coupled receptor

Synthesize cAMPcAMP activates PKACatalytic subunits of PKA translocate to

the nucleusActivate the cAMP response element

binding protein (CREB transcription factor)

CREB binds to CRE site and activates gene

cAMP-response element (CRE) links cAMP to transcription

CREB: cAMP-response element binding protein; a transcription factor

Cyclic GMPMade from GTPGuanylate cyclase

– Membrane or soluble– Acts as receptor

Hydrolysis of cGMP to GMP terminates signal

Regulates several proteins :Ion channel, protein kinase GImportant in smooth muscle relaxation

Viagra interferes with cGMPhydrolysis

Increases [cGMP]Prolongs cGMP signal

cGMPThe cGMP Signal Transduction Pathway

Two forms of guanylate cyclaseMembrane-bound (particulate; pGC )

• Activated by ANF (atrial natriuretic factor)– ANF released when BP elevated

Cytosolic (soluble; sGC)• Activated by nitric oxide• NO produced from arginine by NO synthase

– Nitroglycerine slowly produces NO, relaxes cardiac and vascular smooth muscle, reduces angina

cAMP activates Protein Kinase G– Phosphorylates smooth muscle proteins

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Production of the 2nd Messenger – cGMP

The membrane-bound formof guanylate cyclase is acti-vated directly by hormonebinding to its receptor, e.g.atrial natriuretic peptides.

The cytosolic form is a different protein and is activated by nitric oxide (NO).

Protein kinase G (PKG) is activated by cGMP.

atrial natriuretic peptide (ANP), endotoxin

GTP

cGMP +PPi

Soluble guanylyl cyclase

NO

NO – receptor - soluble guanylyl cyclase

Binding NO to the heme cofactor in the N-terminal domain

Dimerisation of the C-terminal domain

Activation of C-terminal domain and catalyses of GTP to cGMP conversion

cGMP – second messenger in the cellscauses smooth muscle relaxation

In endothelial cells, acetylcholine leads to an increase in Ca++/calmodulin and NO synthesis. NO diffuses to smooth muscle cells, where it binds to the NO receptor and leads to relaxation of the muscle cell and vasodilation.

Signal-induced relaxation of vascular smooth muscle is mediated by cGMP activated protein kinase G

Viagra的作用機制

勃起訊息

勃起訊息

Guanylatecyclase

cGMP

PDE5

GTP

GMP

NO Endothelial cells

Penile erectionRelax

NO

XViagra

PDE: phosphodiesterase

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Http://www.kumc.edu/research/medicine/biochemistry/bioc800/sig02-11.htm

10-7 M

10-3 M

„Second messenger from phospholipid“DAG, IP3 and Ca++

calmodulin

Three classes of lipids are found in biomembrane

1. Phosphoglycerides; most abundant2. Sphingolipids3. Steroid: stable lipid bilayerThere are amphipathic

hydropholic

PE: phosphatidylethanolaminePC: phosphatidylcholinePS: phosphatidylserinPI: phosphatidylinositolSM: sphingomyelinsGlcCer: glycolipid glucosylcerebroside

磷酸甘油酯

Acyl group:c16 or c18,0, 1 or 2 double bond

鞘酯

choline head膽鹼

補充PC vs. alzheimer's disease

phosphatidyl- groupglycerol c c c

fatty acids entire molecule is “phosphatidyl{R}”(e.g., phosphatidylcholine).

P

P

P

Phosphatidylinositol-4,5-bisphosphate

OH|

diacylglycerol(DAG)

P

P

P

inositol-1,4,5-trisphosphate

(IP3)

phospholipase C cleaves here

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Phosphatidylinositol Signal Cascades

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OPO32

HH

OPO32H

OH

H

O

H OH

1 6

5

43

2

PIP2 phosphatidylinositol- 4,5-bisphosphate

Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol(PI).

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OH

HH

OHH

OH

H

O

H OH

1 6

5

43

2

phosphatidyl-inositol

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OPO32

HH

OPO32H

OH

H

O

H OH

1 6

5

43

2

PIP2 phosphatidylinositol- 4,5-bisphosphate

cleavage by Phospholipase C

Kinases sequentially catalyze transfer of Pifrom ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4,5-bisphosphate (PIP2).

PIP2 is cleaved by the enzyme Phospholipase C.

Different isoforms of Phospholipase C have different regulatory domains, & thus respond to different signals.

Cleavage of PIP2, catalyzed by Phospholipase C, yields 2 second messengers: inositol-1,4,5-trisphosphate (IP3) diacylglycerol (DG or DAG).

Diacylglycerol, with Ca++, activates Protein Kinase C, which catalyzes phosphorylation of several cellular proteins, altering their activity.

OHH2C

CH

H2C

OCR1

O O C

O

R2

diacylglycerol

OH

H

OPO32

HH

OPO32H

OH

H

H OH

OPO32

1 6

5

43

2

IP3 inositol-1,4,5-trisphosphate

Different isoforms of Phospholipase C have different regulatory domains, & thus respond to different signals.

This enzyme hydrolyzes the ester linkage between a fatty acid and the OH at C2 of the glycerol backbone, releasing the fatty acid & a lysophospholipid as products

Phospholipase Cyields diacylglycerol(and IP3).

蛇毒 vs. 溶血

Phospholipase C activates 2 signaling pathways

1. PLC converts inositollipid to IP3 + DAG

2. IP3 binds to calcium cannels on ER

3. Ca+2 is released from the ER into cytoplasm

4. Ca+2 and DAG recruit protein kinase C to plasma membrane

5. PKC is activated to phosphorylate various cellular enzymes and receptors

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PLC — DAG — IP3 pathway

+

CaMkinase

Contractile proteins

SER Ca2+-ATPase (SERCA)

IP3 receptor channel (IP3R)

Plasma membrane Ca2+-ATPase (PMCA)

Ca2+

[Ca2+]in ≈ 0.1M

[Ca2+]out ≈ 2mM

or IP3

CaMKMLCK

NOS

Ca2+, from internal stores or plasma membrane channels, binds to various Ca2+-binding proteins, which in turn activate other proteins or enzymes.

Ca2+-calmodulin activates CaMK, which activates myosin light-chain kinase in smooth muscle and nitric oxide synthasein blood vessel walls.

Ca2+ binds troponin C (related to calmodulin) in skeletal muscle cells to allow actin-myosin interaction.

Ca2+ binds proteins involved in fusion of vesicles with cell membrane, as in release of neurotransmitter at nerve terminal. Myosin light chain kinase

有時Ca2+被認為second messenger??

DAG activated Protein Kinase C

Binding of DAG in membrane– C2: Ca2+ and phosphatidyl serine– pulls psuedosubstrate from active site– positive feedback between IP3 activity ,

Phospholipase C activity, and PKC activityActivation of serine/threonine phosphorylation on

target proteins (growth/division pathways)

Phorbol Ester involved in Phorbol Ester involved in potentiation of tumorogenesis.potentiation of tumorogenesis.Mimics DAG and binds PKCMimics DAG and binds PKC

Molecular Interactions of signal transduction

Protein-protein interactions– Binding or unbinding (formation or breaking of

complex)– Covalent modification:

phosphorylation (tyr, thr, ser)– Conformation changes– Translocation– Targeting for degradation

Small molecule regulated events– Binding or unbinding, resulting in conformation

change: Steroid ligand, nucleotide binding– Production of second messengers (e.g. Ca+2)

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Intracellular target

Determining the “end” of a signaling pathway is often difficult

For example, after transcription, a phosphatase may be synthesized that dephosphorylates one of the enzymes in the pathway

One approach is to consider an event that is “biochemically different” (e.g. transcription, metabolism) as the intracellular target

Intracellular Endpoint

Three major molecular targets

– Regulation of gene expression (e.g. activate a

transcription factor and translocate it to the nucleus)

– Changes in the cytoskeleton (e.g. induce movement or

reorganization of cell structure)

– Affect metabolic pathways

Many critical processes can occur in response to external

signals, without any new synthesis of RNA or proteins. The

most well known one is “cell suicide”, termed apoptosis

Change in the cell

An animal cell depends on multiple extracellular signals

Multiple signals are required to survive, additional to divide and still others to differentiate

When deprived of appropriate signals most cells undergo apoptosis

DIFFERENTIATE

F G

Change in the cell

The same signal molecule can induce different responses in different target cells, which express different receptors or signaling molecules

For example, the neurotransmitter acetylcholine induces contraction in skeletal muscle cells, relaxation in heart muscle cells and secretion in salivary gland cells

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Regulating proteins

How much protein is created?

Transcription, splicing,

degradation, translation

Change in conformation

by ligand binding. Only bound protein can bind DNA

Change in conformation by

protein phosphorylation. Only phospho-

protein can bind DNA

Only dimercomplex of two

proteins can bind DNA

Binding site is revealed only

after removal of an inhibitor

In order to bind DNA, the

protein must first be

translocated to the nucleus