1

Signal molecule

ActiveMAPKKK

1

ActiveMAPKK

2

ActiveMAPK

3

InactiveMAPKKK

1

InactiveMAPKK

2

InactiveMAPK

3

Inactiveprotein

Activeprotein

Cellularresponse

Receptor

P

P

P

ATPADP

ADP

ADP

ATP

ATP

PP

PP

PP

Activated Ras-GTPA G-Protein

i

Phosphorylationcascade

P

P

i

i

P

ProteinPhosphatases

BCOR 011 BCOR 011 Cell Communication IICell Communication II LectLect 1919

Chapt 11

10/17/05

And they tell 2 friendsAnd they tell 2 friends

And they tell 2 friends…

2

Lecture OutlineLecture Outline

1. Finish Trimeric G-Protein: Phospholipase C-DAG, IP3 second messengers

Protein Kinase C, Ca++ release

2. Nitric Oxide, cGMP activation- consequences of failing to inactivate

3. Tyrosine Kinase Receptor- RAS G-protein, MAP Kinase Cascade- Cell Cycle Control, Gene Control

4. Internal Receptors – Steroid Receptor- Gene Regulation

3

TWO subclasses of trimeric G-protein-activated signal transduction pathways:

A. target protein adenylate cyclasecAMP-> PKA

B. target protein phospholipase C

4

target effector enzyme is Phospholipase C

PLC cleaves a membranephospholipid (Phoshatidyl inositol) to

two 2nd Messengers:

Inositol-1,4,5-Trisphosphate(InsP3) &

Diacylglycerol (DAG)

5

InsP3WaterSoluble

DAGLipid

Soluble

PIP2

6

DAG ActivatesProteinKinase C

(StartsCascade)

InsP3Ligandfor ER

ligand-gatedCa++

channels↑ Ca++ levels

7

Figure 11.12

321

IP3 quickly diffuses throughthe cytosol and binds to an IP3–gated calcium channel in the ERmembrane, causing it to open.

4 The calcium ionsactivate the nextprotein in one or moresignaling pathways.

6Calcium ions flow out ofthe ER (down their con-centration gradient), raisingthe Ca2+ level in the cytosol.

5

DAG functions asa second messengerin other pathways.

Phospholipase C cleaves aplasma membrane phospholipidcalled PIP2 into DAG and IP3.

A signal molecule bindsto a receptor, leading toactivation of phospholipase C.

EXTRA-CELLULARFLUID

Signal molecule(first messenger)

G protein

G-protein-linkedreceptor

Variousproteinsactivated

Endoplasmicreticulum (ER)

Phospholipase CPIP2

IP3(second messenger)

DAG

Cellularresponse

GTP

Ca2+

(second messenger)

Ca2+

IP3-gatedcalcium channel

8

Protein Kinase C phosphorylatestarget proteins (ser & thr)

cell growthregulation of ion channelscytoskeletonincreases cell pH Protein secretion

Ca++Binds & activates calmodulin

Calmodulin-binding proteins activated(kinases & phosphatases)

Response:

9

Shut OffShut Off- remember, signal needs to be transient

- Must shut off cascade: removal of ligand, (self)-hydrolysis of GTP,remove IP3, protein phosphatases, Ca++ ion pumps

10

Direct activation -signal: nitric oxide (NO) It’s a gas!lipid soluble, binds directly to activate enzyme

made by: endothelial cells (line blood vessels) NO synthasetarget: guanylyl cyclase GTP->cGMP

arginine citrulline

+NOresponse: relaxes smooth muscle

vessels dilate, blood flow

11

Shut offby cGMPphosphodiesterasetarget

12

1977 –Ferid MuradNitroglycerine acts to elict release of NO,relaxes cardiac muscle

Nitroglycerine – taken to relieve angina

1998 Nobel Prize – Murad, Furchgott, Ignarro

13

Shut offBy cGMPphosphodiesteraseVI

AGRA

Nitroglycerine

14

Sildenafil citrate (VIAGRA)

15

b. G-protein-linked

cell-surface receptors

a. ion-channel-linked

c. Protein-kinase associated

(enzyme-linked)

16

MitogenMitogen ActivatedActivatedProteinProtein--KinaseKinase associated receptorsassociated receptors

receptor has enzymatic activity- only when ligand binds

ligand:allosteric effector -> conformational change

receptor tyrosine kinasesGrowth & differentiationGrowth & differentiationcontrol of cell cyclecontrol of cell cyclecontrol of gene expressioncontrol of gene expressionbad news when messed up bad news when messed up -- cancercancer

17

Reception

Transduction

Response

mRNANUCLEUS

Gene

P

Activetranscriptionfactor

Inactivetranscriptionfactor

DNA

Phosphorylationcascade

CYTOPLASM

Receptor

Growth factor

Figure 11.14

Growth FactorGrowth Factor

““MitogenMitogen--activated”activated”

Signaling CascadeSignaling Cascade

18

•Monomer receptor1 transmembranesegment

EGFR – epidermal growth factor receptor

EGFEGF – growth factoror mitogenmitogen“Mitosis-generator”

•GDP/GTP exchange•Activate Ras G-protein

••Scaffold to Bind/activateScaffold to Bind/activateTarget proteinsTarget proteins

•Activates Tyr kinase•autophosphorylate

•Ligand binds –receptors dimerizedimerize

19

Receptor tyrosine kinasesSignalmolecule

Signal-binding site

CYTOPLASM

Tyrosines

Signal moleculeαHelix in the

Membrane

Tyr

Tyr

Tyr

Tyr

Tyr

TyrTyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

Tyr

DimerReceptor tyrosinekinase proteins(inactive monomers)

P

P

PP

P

P Tyr

Tyr

Tyr

Tyr

Tyr

TyrP

P

P

P

P

PCellularresponse 1

Inactiverelay proteins

Activatedrelay proteins

Cellularresponse 2

Activated tyrosine-kinase regions(unphosphorylateddimer)

Fully activated receptortyrosine-kinase(phosphorylateddimer)

6 ATP 6 ADP

Figure 11.7

20

Receptor tyrosine kinases can activate rasras is a monomeric G-protein“molecular switch”

21

A phosphorylation cascadecascade

Figure 11.8

Signal molecule

Activeproteinkinase

1

Activeproteinkinase

2

Activeproteinkinase

3

Inactiveprotein kinase

1

Inactiveprotein kinase

2

Inactiveprotein kinase

3

Inactiveprotein

Activeprotein

Cellularresponse

Receptor

P

P

P

ATPADP

ADP

ADP

ATP

ATP

PP

PP

PP

Activated relaymolecule

i

Phosphorylationcascade

P

P

i

i

P

A relay moleculeactivates protein kinase 1.

1

2 Active protein kinase 1transfers a phosphate from ATPto an inactive molecule ofprotein kinase 2, thus activatingthis second kinase.

Active protein kinase 2then catalyzes the phos-phorylation (and activation) ofprotein kinase 3.

3

Finally, active proteinkinase 3 phosphorylates aprotein (pink) that brings about the cell’s response tothe signal.

4Enzymes called proteinphosphatases (PP)catalyze the removal ofthe phosphate groupsfrom the proteins, making them inactiveand available for reuse.

5 MAPMAPKinaseKinase

MAPMAPKinaseKinase

MAPMAPKinaseKinaseKinaseKinase

MAPMAPKinaseKinaseKinaseKinase

MAPMAPKinaseKinaseKinaseKinaseKinaseKinase

MAPMAPKinaseKinaseKinaseKinaseKinaseKinase

Ras-GTP

22

Ras activation sets off a phosphorylation cascade

MitogenActivatedProteinKinasesMAPKs

Controls:-TranscriptionFactors

-TranslationFactors

-Cell Division

MAPK

MAPKKK

MAPKK

10 100

1,000

100,000

23

How do you turn it off?How do you turn it off?

phosphatasesGTPase(GTP->GDP + P)

molecular switch on internal timerIf timer broken If timer broken –– on all the timeon all the time 24

PROBLEMS IN CANCER:PROBLEMS IN CANCER:

- Broken receptor – thinks ligand thereeven when it isn’t

- broken ras – won’t shut off

-broken MAPK – on all the time, even whennot phosphorylated

RESULT: continuous signal for cell to divideRESULT: continuous signal for cell to divide

25

1. receptor-mediated cell-surface

2.2.receptorreceptor--mediated mediated intraintracellularcellular

Specific signal transduction cascades:

Lipid soluble things:steroid hormonessteroid hormones

26

Hormone(testosterone)

EXTRACELLULARFLUID

Receptorprotein

DNA

mRNA

NUCLEUS

CYTOPLASM

Plasmamembrane

Hormone-receptorcomplex

New protein

Figure 11.6

The steroidhormone testosteronepasses through theplasma membrane.

The bound proteinstimulates thetranscription ofthe gene into mRNA.

The mRNA istranslated into aspecific protein.

Testosterone bindsto a receptor proteinin the cytoplasm,activating it.

The hormone-receptor complexenters the nucleusand binds to specific genes.

Steroid HormoneSteroid Hormone

Lipid solubleCrosses membranes

Binds intracellular receptorintracellular receptorIn cytosol

steroid receptor Complex steroid receptor Complex Changes shape Changes shape Releases from tether proteinTravels to Nucleus

steroid receptor steroid receptor Binds DNATurns genes ON

27

The Specificity of Cell SignalingThe Specificity of Cell Signaling

• The different combinationsdifferent combinations of proteins in a cell– Give the cell great specificity in both the

signals it detects and the responses it carries out

Same hormone can give different responsesSame hormone can give different responsesin different cells in different cells

28

Pathway Pathway “cross“cross--talk”talk”

Response 1

Response 4 Response 5

Response

2

Response

3

Signalmolecule

Cell A. Pathway leads to a single response

Cell B. Pathway branches, leading to two responses

Cell C. Cross-talk occurs between two pathways

Cell D. Different receptorleads to a different response

Activationor inhibition

Receptor

Relaymolecules

Figure 11.15

29

Specific pathways1. Cell-surface receptor mediated

a. ion-channel-linked

b. trimeric G-protein-linked

(i) adenylyl cyclase cAMP ->protein kinase A

(ii) phospholipase C

InsP3, DAG, Ca++, protein kinase C

Direct activation - NO

c. protein kinase-associated (enzyme-linked)

Receptor tyrosine kinase

monomeric G-protein (ras), MAPKs

2. Intracellular receptors – steroid hormones, dioxin