Cell Biology interactive media ”video” or ”interactive” 1

Lecture 4 & 5:

Cell biology 2014 (revised 29/1 -14)

Cell communication

Differentiate Proliferate

xx

DieSecreteMove

Signal molecules/proteins

2

All diseases involve changes of normal cells. In some cases, these changes may affect other cells of the individual

Events during cell communication

Regulated synthesis…..….... or regulated release of a signaling molecule

(Transport of signaling molecule to target cells)

Binding of the signaling molecule to a specific receptor on/in a target cell

Activation of a transduction chain

1.

2.

3.

4.

Target cell response5.

Termination of signal6.

1.

2.

Target cell

3.

4.

5.

Producer cell

3

6.“hormone" = to urge on/impulse

Signaling receptor diversity

The mammalian genome encodes for thousandsof signaling receptors - Many of these are targets for drugs

Tissue specific expression: Each individual animal cellexpress only some of these receptors

4

Membrane permeability

O• Hydrophobic molecules

Na+

• Charged

molecules

IonsAmino acids

Cl-

• “Large” uncharged polar molecules

Glucose

5

Cortisol TestosteroneCholesterol

O N O

O

ON C C

Localization of signaling receptors

Receptor on plasma membrane

Receptor in cytosol Receptor in nucleus

Hydrophilic molecule

(and proteins)

Hydrophobicmolecules

Other compounds than the natural ligand may interact with a receptor – some are used as drugs (legal & illegal)

“natural ligand” = an endogenous receptor binding molecule

6

hydrophobic lipohilic non-polar (often used as synonyms)

Receptor agonists and antagonistsOther compounds than the natural ligand may bind a receptor

Agonists: mimic completely, or partially, the action of the endogenous ligand

Antagonists: bind to receptor without activating it block the action of the “natural” ligand

OHOH

CHCH2NHCH3

OH

OH

CHCH2NHCH3

OH

Adrenalin Phenylephrine(natural) (selective

agonist)

One of the action of adrenalin is to cause a dry mouth in the fight-or-flight reflex. Phenylephrine is used in many “cold-relief” drugs to prevent excessive nasal mucous secretion 7

A

B

C

D

E

Five modes of cell communication

Neuron

Bloodstream

B Paracrine C AutocrineD Endocrine E Neuronal/synaptic

Signaling by secreted ligands:

Contact dependent signaling: Ligands on the cell surfaceA

8

Surface receptorIntra-cellular receptors

"crinis" = secrete

Signaling cell Target cell

Receptor/ligand

Contact-dependent signaling uses ligands and receptors that are plasma membrane-bound:- Persistent signals (uni- or bidirectional) - Directed toward neighboring cells

A Contact-dependent signaling

9

A Distinct types of contact-dependent cell signaling

Cell surface receptors that mediate cell-to-cell adhesion (cadherins) and cell-to-ECM interaction (integrins) are also involved in signaling. Important for: Development

Growth controlSurvival

Gap Junctions permit free passage of smallmolecules between adjacent cells

Important for e.g., synchronous heart contraction

Cadherin

Gap junction

Integrin10

Paracrine signaling involves secretion of a ligand that act locally on cells with the appropriate receptors:

Local effect "para" = near

Signaling cell

Adjacent target cells

B Paracrine signaling

11

Signaling and target cell

Autocrine signaling impliesthat a cell secretes a ligand that it responds to itself

C Autocrine signaling

?

12

"autos" = self

Endocrine signaling involves a signal molecule (poly-peptide or steroid hormone) produced by an endocrine cell. "endo" = inside/within "crinis" = secrete Each endocrine cell secrete only one type of signal molecule!

The hormone travels through the blood system:Global signaling with long-term effect

Relatively slow responses - the signaling molecule have to travel through the blood systems before reaching a target cell

Endocrine cellsecreting

Distant target cells

D Endocrine signaling

13

Signaling cell

"syn" = together "haptein" = hold onto

Neuronal/synaptic signaling is mediated by neurotransmitters released at the interface between the signaling and the target cell, called synapse. The release of neurotransmitters at the synapse is controlled from the cell body through electrical signals. Neurotransmitters bind cell surface receptors.

- Acts rapidly and transiently on the target cells

Target cell

Cell body of a neuronAxon

Synapse

Release of neurotransmittor

E Neuronal/synaptic signaling

14

kidney

*Gland

Neuroendocrine integration

Hormone secreting glands in the brain link neuronal signals and peripheral endocrine glands.

Fight-or-flight reflex: the Hypothalamic- Pituitary-Adrenal (HPA) system

The adrenal gland responds to both the hormone (ACTH) and a nerve signal

ACTH Adrenal Cortex cortisolIncreased blood levels of lipids etc. etc

Nerve signal adrenal medulla adrenaline Increased blood levels of lipids & glucose etc. etc.

Endocrine cell: a cell within an endocrine gland that release a hormone into the circulating blood in response to a neural (synaptic) or hormonal stimulus 15

*Gland

Signaling molecules

Molecules typically produced and released by one cell and recognized by another cell

Signaling molecules are chemically diverse:

- Gases: nitric oxide, carbon monoxide - Steroids: testosterone, cortisol, etc. - Proteins: insulin, glucagon, etc.

- Amines: catecholamines, acetylcholine

“Ryss 5a”: A mix of synthetic anabolic steroids ( muscle growth)

Membrane permeable

Membraneimpermeable

16

Fast versus slow signal transduction events

Altered protein function

Cell response

An altered cytoplasmic signaling protein

DNA

mRNA

Altered gene expression

Alteredproteinlevel

mRNA

Protein

Slow (minutes to hours)

Fast (<seconds)

17

Signal

Signaling with nitric oxide gas

CH2 CH2 CH2

O

NO2

O

NO2

O

NO2

• Nitric oxide (NO) acts as a paracrine signal, only affecting local area, due to its short t1/2 (1-5 seconds)

• Produced by nitric oxide synthase through the deamination of the amino acid arginine

• Nitric oxide is a very potent vasodilator (blood vessel dilatation)

18

Nitroglycerin is converted in blood to NO (used totreat coronary artery disease since 1878)

• Skeletal muscle

• Cardiac muscle

• Smooth muscle cells: i) surrounds hollow organs –

intestines and blood vessels ii) arrector pili muscles

attached to hair follicles

Three types of cells dedicated to contraction

19

All three muscle cell types contains filaments consisting of actin and myosin, which may

contract and slide apart

Endothelial cell

Smooth muscle cell

Blood vessel

Neuron

Arginine

NO (Nitric oxide)

Vasodilatation through nitric oxide signaling

Acetylcholine

Relaxation of smooth muscle cell

Diffusion to adjacentsmooth muscle cell

Increased blood flow20

”2nd messengers”

Cytosolic signal mediators: second messengers

1st messenger: the external signaling molecule (e.g. Nitric oxide)2nd messenger: the molecule that transfer the signal in the cytosol

cAMP, cGMP and Ca2+ are the classical 2nd messengers

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Video 15.1-calcium_signaling 21

Adenylylcyclase

Guanylyl cyclase

Ca2+

=1 mM

=10 nM

Effect of nitric oxide on smooth muscle cells

P P P

P PP +

Cyclic GMP

GTP

Guanylyl cyclase

P

GMP

Cyclic-GMP phosphodiesterase

(constitutively active)

Viagraä

Activation of an “in-ward” Ca2+-pump in membranes of

intra-cellular Ca2+-stores

Nitric oxide

Low [Ca2+] makes contractile filaments (actin and myosin)

slide apart

Relaxation of smoothmuscle cells and

increased blood flow

22

Signaling by intracellular receptors – part I

Target genes

NLS

NLS

NLSCombined receptor/transcription factor

Hydrophobic ligand (e.g. Cortisol)

Plasma membrane

Binding displaces a protein that masks an NLS on the cortisol receptor

1.

2.

Receptor translocationinto the nucleus specific transcription

3.

1.

2.

3.

Cortisol diffuse through the plasma membrane

23

= DNA

Signaling by intracellular receptors – part II

Plasma membrane

Target genes

Target genes

Inhibitor

Inhibitor

1.

3.

2.

3.

2.

1. The DNA-binding receptor/transcription factor is inactive

The ligand (e.g. sex hormones) diffuses into the nucleus

The ligand displaces the inhibitor

24

Hydrophobic ligand

General principle of cell surface receptor signaling

1.

3.

Reception

2. Signal transductioncascade comprising:i. molecular switchesii. 2nd messengers

Response Metabolicenzyme

Gene regulatoryprotein

Etc.

Receptor

Signal molecule(Ligand)

25

P. M.

Cytosol

I. Molecular switches in signal transduction

A signal that can be switched on, also needs to be switched off (all signals are more or less transient)

1. Protein phosphorylationThe most common ‘on-off’ switch is provided by protein phosphorylation

OH

Serine, threonineor tyrosine

Serine, threonineor tyrosine

O O

O -P

O

Kinase

-

Phosphatase

26

+ ATP

Kinase : ~1000 protein kinase genes in vertebrates. Some have only a single substrate. Others are “multi-functional” and may have >10 substrates

GTP

GDP

Guanine-nucleotide Exchange Factor (GEF)

GTPase Activating Protein (GAP)

Inactive Active

P

GDP

II. Molecular switches in signal transduction2. GTP binding proteins (G-proteins)Another ‘on-off’ switch is provided byregulatable GTP-binding and hydrolysis

GDPGTP >>GTP

27

Molecular_models 15.5-Ras (one PO4 makes the diff.)

Signal transduction cascades

MetabolismGene regulation Etc.

P

GTP

A single cell surface receptor may activate several signal transduction pathways

This involves various G-proteins, 2nd messengers and protein kinases

Protein kinases at the end of a cascade mayhave many substrates

Response:

28

P P

Kinase

Ca2+cGMPcAMP

P. M.

Three main classes of cell-surface receptors

G-protein coupled receptors

Receptors with intrinsic enzymatic activity

ZZZ ZZZ

ZZZ

Ion channel coupled receptors

Ion

Ion

Ion

Ligand

29

3. G-proteins may regulate enzymes or ion channels

G-protein coupled receptors (GPCR)

A hallmark of GPCR´s is 7 trans-membrane spanning regions

ZZZ

1. Ligand binding conformational change

2. A specific G-protein is recruited and activated G

30

Down-stream effectors of various G-proteins

Cyclic AMPAdenylylcyclase

ATPGuanylyl cyclase

Cyclic GMP

GTP

Phospholipase C

Ca2+

Increase incytosolic and activation of protein kinase C

Ion

Ion

Ion

1. 2.

3. 4. Ion channels

31

I. Regulation of hetero-trimeric G-proteins

GTP

GDP

Inactive Active

P

GDP

GTP

a abg

b

g+

a-subunit and/or b,g-subunit can activate or suppress

different downstream targets

Complex dissociate upon GTP binding

=GAP

GDPGTP >>

32

RGS

=GEF

RGS: Regulator of G-protein Signaling

P.M.

b g

P.M. GDPab g

GTPab

g +

GDP GTP

Ligand binding causes a conformational change

The G-protein is recruited to the receptor, which acts as a GEF the a-subunit exchanges GDP for GTP dissociation of an active a-subunit

GDPa

No ligand (default state)

33

II. Regulation of hetero-trimeric G-proteins

GTPa

P.M.b

gGDP

a

P

b g

GDPa+

The intrinsic GTP hydrolysis is slow but RGS, an a-subunit specific GAP, catalyzes hydrolysis. This terminates the signal

RGS

GTPas

GTPai

Phospholipase C-b(PLC-b)

GTPaq

Adenylyl cyclase

A family of a-subunits with distinct functions

Anim. 15.3-G-protein_signaling

34III. Regulation of hetero-trimeric G-proteins

Alberts et al: Table 15-3 (tissue specificity)

Adenylyl cyclase activation by the as-subunit of G-proteins

GTPas

P.M.

P P P

P PP +

Cyclic AMP

ATP

P

AMP

Adenylyl cyclase

Cyclic-AMP phosphodiesterase

(constitutively active)

Caffeine

35

Inactive PKA

Cyclic AMP

Active PKA

Target genes

CREB

CREB

P

Cyclic AMP second messenger signaling

Glycogen phosporylase

PGlycogen

phosporylase

Cyclic AMP activates Protein kinase A (PKA), which can regulate: Metabolism Gene transcription 1.2.

1.

2.

Glucose-1- phosphate

Glycogen36

Summary of the cyclic AMP signaling cascade

Cyclic AMP

CREB

P

Adenylylcyclase

PKA

Regulates metabolismATP

PRegulates transcription

Glycogen breakdown

P

GTPas

Anim. 15.4-cAMP_signaling 37

-Regulated DNA binding

P

Glycogen:- Stored in muscles and liver- Rapidly available energy sourceWork/stress adrenalin cAMP PKA Glycogen breakdown

Alberts et al: Table 15-1 (tissue specific response)

GEF (GPCR)

Signal induced cleavage of phospholipids

Variable

Phosphate

GlycerolFatty acid

Fatty acidPhospholipase A1

Phospholipase A2

Phospholipase C

Phospholipase D

External signals may activate distinct phospholipases that cleave phospholipids at specific sites and thereby catalyze the formation of various molecules with signaling properties

Soluble compounds release into the

cytosol38

Precursors for various signaling

substances

Phospholipase C activation generates two 2nd messengers

aq

Fatty acid

Fatty acid

Glycerol

Fatty acid

Fatty acidGlycerol

OH

Diacylglycerol (DAG)

Phosphatidylinositol 4,5- bisphosphate, PI (4,5)P2

Inner leaflet ofplasma membrane

Phospho-lipase C-b(PLC-b) P

PP

PP

P

Inositol 1,4,5-triphosphate, IP3

aq-subunit activates PLC1.

2. PLC cleaves PIP2, generating thetwo 2nd messengers DAG and IP3

1.

2.

GTP

39

PP

P

IP3

OH

Inner leaflet ofplasma membrane

Ca2+ Ca2+

IP3 regulated Ca2+ channel

PKCCa2+

Ca2+

PKC Ca2+

CalmodulinCa2+

Ca2+

Ca2+

Ca2+

Calmodulin

Calmodulin regulated Ca2+ pump in ER

Ca2+

DAG

1.

2.

DAG recruits PKC to plasmamembraneIP3 mediate release of Ca2+ from ER

3. DAG and Ca2+ activates PKC

1.

2.

3.

4. Ca2+ activates calmodulin to terminate signal by pumping Ca2+

back into ER

4.

Role of the 2nd messengers IP3 and DAG

40

Ca2+/calmodulin dependent protein kinase (CaMK)

P

CalmodulinCa2+

Ca2+

Ca2+

Ca2+

Calmodulin

Ca 2+

Ca 2+

Ca 2+

Ca 2+

Calmodulin

Ca 2+

Ca 2+ Ca 2+

Ca 2+

Autophosphorylation

DephosphorylationCatalytic

Inhibitory

Calmodulin

ActivatedFully active

P

Partially active

Resting state

Inactive

Ca2+

Increasedcytosolic

41

Molecular_models 15.6-calmodulin

IP3

OH

Ca2+

PKC

CalmodulinCa2+

Ca2+

Ca2+

Ca2+

DAGPLC-b

PP

P

PP

P

CaMK

Summary of G-protein signaling through PLC-β

aq

GTP

Other regulated enzymes

Ca2+

STOP

Termination of signalCa2+

Etc!

42

Both PKC and CaMK have many potential (tissue specific) substrates

GEF (GPCR)

Enzyme linked receptors

Tyr P Ser/Thr

Many variants on this theme – here we focus on:Receptor tyrosine kinasesReceptor serine/threonine kinases

PHomo-dimers Hetero-dimers

Single pass transmembrane receptors. Ligand binding cause dimer formation and consequent “auto”-phosphorylation

43Jenkinson : RTK - dimerizationAlberts et al: Table 15-4 (tissue specific RTK’s)

Signaling through Receptor Tyrosine Kinases

Tyr

Kinase domain

Tyr

Kinase domain

Tyr

Kinase domain

Tyr

Kinase domain

Inactive receptor monomers

Active receptor dimer

Ligand binding causes receptor dimerization

P P

P. M.

Trans-phosphorylation of tyrosine residues

Single pass transmembrane protein

TyrTyr

TyrTyr TyrTyrP P

TyrTyrP P

44

Cis- prefix means "on this side"Trans- prefix means "across"

TyrTyrP P

TyrTyrP P

Tyr

Kinase domain

Tyr

Kinase domain

P P SH2 SH3

GTPRas

Ras GEF (Sos)

Regions containing phospho-Tyr may serve as specific docking sites for SH2 domain-containing signaling proteins (SH = Src Homology domain)

SH2-proteins binds at specific phospho-tyrosines

P3

These can be enzymes….

Phosphatidyl-inositol (PI) Monomeric G-protein

GDPRas

PI-3 Kinase

45

………….or they act as adaptors forsignaling proteins

Fig. 15-55

Phosphorylation cascade downstream of RasGTP

Ras

MekMek

Raf

P

Erk (MAPK)

P. M.

Erk P

Target genes

P

2.

Erk P

PCytosolic

target proteins

1.

1. Altered protein function

Altered gene expression2.

46

Termination of RTK/Ras/MAPK pathwayReceptor and ligand internalization

GDP GTPRas Ras

Erk (MAPK) Erk

P

1. 2. Ras GTP hydrolysis

Ras GAP

3. Dephosphorylation

Phosphatase

Note: Signalingreceptors arerarely recycled

47Anim. 13.3-receptor_endocytosis (Note: vesicle fusion with endosome)

Fusion withendosome

Fusion withprimary lysosome degradation

3

4

5P

PI kinase PIP kinasePI(4,5)P2

P

PI – phosphorylation cycles on inositol ring position 4 & 5

P PP

P

PI(4)PInositol

Extracellular space

CytosolPhosphatidylinositol (PI)

I. PI-kinases act at specific positions of the inositol ring

PhosphateGlycerol

Fatty

aci

d

Fatty

aci

d

Inositol

48

II. PI-3 kinase completes a PH-domain binding site

PI(3,4,5)P3

P3 3

PI(4,5)P2

PTEN

PI-3 Kinase

Activated receptor recruits and activates PI-3 kinase 1.

2.

PI-3 kinase

PI-3 kinase phosphorylates PI(4,5)P2 to generate PI(3,4,5)P3,which will serve as a docking-site for a family of signaling proteins with a “PH-domain” (PH= Pleckstrin Homology)

1.2.

3. PTEN removes phosphorylation on position 3 on PI(3,4,5)P3 to terminate signal

3.PP

PP

P

Phosphatidyl-inositol (PI)

49

P P

III. PKB/Akt activation downstream of PI-3 kinase

P PPDK1 PKB/Akt

P

3 3

PH-domainsPKB/AktP

P P3 3

PDK1

PKB/Akt

PP P PPP PP

PDK1 phosphorylates PKB/Akt thereby mediating its activation

2.

1. PI(3,4,5)P3 brings PDK1 and PKB/Aktinto proximity through their PH-domains

1.2.

50

PP P P

IV. Different signaling pathways – same target

PPI-3 K

PP

3

GDPa

b g

GTPa b

g+

PI-3 K

Both G-protein- and RTK signaling may result in generation of PI(3,4,5)P3

There are two distinct PI-3 kinases which differ in their regulatory domains

Thus, a PI-3 kinase may be recruited to the plasma membrane via a bg-subunit binding domain or a SH2 domain 51

P

I. Transcriptional regulation by TGF-b / BMP

P.M.

Target genes

P PSmad 2/3 P

Smad 4

Smad 2/3 PSmad 4

Type I receptor

Smad 7 Negative feedback loop

Type II receptor: Ser/Thr kinase

52

TGF-b TGF-bTGF-b

II. Transcriptional regulation by Wnt/winglessWnt

DishevelledLRP Frizzled

Target genesTCFb-catenin

GSK-3b

b-cateninP

Ub

UbUb

Target genes

TCF

Groucho

AxinAPC

GSK-3b

b-cateninAxinAPC

G1myc

MMP7

Dishevelled

ZZZ

53

54

Signal transducing proteins are oftentargets of therapeutic drugs or infections agents

My own favorite protein!

"All science is either physics or stamp collecting"

Ernest Rutherford(1871-1937, Nobelprize1908)

Recommended readingChapter 15879-941946-954

Alberts et al. 5th edition