Haploid a- and -cells form shmoos in response to chemical signals

Shmoos mate to form diploid a/ cell

Examples of:- “differentiated” cell types (a-, -, and a/-cells) cell-cell adhesion-cell-cell signaling

Human body consists of trillions of cells, 200+ specialized cell types that must differentiate (next time) and communicate (today) with one another

Cell-cell communication required to coordinate:- physiology and metabolism- behavior-growth, proliferation, and differentiation

ECB 16-1

Mating dance of a budding yeast (S.cerevisiae)…

Multicellularity: From cells to tissues to organisms

shmoos

“Neuronal”

Cell body of neuron

Post-synaptictarget (muscle,

neuron, etc)

Axon

Synapse

Action potential

Basic categories of cell-cell signaling in animals

ECB 16-3

“Paracrine” (local)ex. inflammation

Signaling cell

Target cells

“Autocrine”

“Contact-mediated” (short range)ex. - nerve cell production

Signaling cell

Target cell

(ex.-hormones)“Endocrine” (long distance)

Endocrine (signaling) cell

Target cells

Bloodstream

hormone

Cellular response depends on specific combination of signals

ECB 16-6

No signal often results in activation of apoptosis

Common features of cell-cell signaling pathways

Other signals

ECB 16-7

Receptors for diffusible signals can be intracellular or surface

Small non-polar molecules Large polar molecules

Plasma membran

e

cross plasma membrane by simple diffusion

And bind to intra- cellular receptors

…cannot cross membrane

They bind cell surface receptors

Membrane receptors for hydrophilic signaling molecules activate a wide variety of intracellular “signal transduction” pathways, including gene regulation

Most receptors for hydrophobic signaling molecules act in nucleus to regulate gene transcription

ECB 16-9

Transcription

Transcription

Intracellular signals

Intracellular receptors Cell surface receptors

A few examples of hydrophobic hormonesECB 16-11

Responses mediated by a conserved family of “steroid” receptors

HO

OH

Estradiol

OH

O

Testosterone

HO O COO-CH2 C

H

NH3+

II

II

Thyroid hormone

HO

Cholesterol

CH2OH

O

HO

C=O

OH

Cortisol

(not hormone)

Responses to hydrophobic hormones are mediated by intracellular receptors

ECB 16-12

Transcription

Translation

Cytoplasm

Nucleus

Nuclear envelope

Plasma membrane Lipophilic hormone carried

in blood

Hormone binds intracellular receptor inducing receptor dimerization and activation

Complex is imported into nucleus

Binds to “hormone response element” to regulate gene expression

Intracellular receptor

Promoter Target gene“Hormone response element”

Target cell

Receptor G-protein(inactive)

Target(inactive)

G-protein linked receptor

Cell-surface receptors - three classes

ECB 16-14

Receptor(active)

G-protein(active)

Target(inactive)

Signaling ligand

Signaling ligand

Catalytic domain(active)

Signaling ligand

Ions

Catalytic domain(active)

Enzyme-linked receptor

Ion channel-linked receptor

Receptor(active)

G-protein(active)

Target(active)

Activation of surface receptor can cause fast (cytoplasmic) or slow (transciptional) changes

Review: phosphorylation and GTPases as molecular switches

ECB 16-15

ADP

ATP Pi

PhosphataseKinase

Pi

GAPGEF

GTP

On

P

On

Energy (in the form of ATP or GTP hydrolysis) used to activate (or inactivate) signaling molecules

Energy use allows transient, high affinity/specificity interactions

Signaling with GTPasesSignaling with phosphorylation

Signal in Signal in

Signal activates protein kinase

Signal activates GEF

Signal out

Signal out

GTP

GDP

GDP

Off

Signaling GTPase

Off

Signaling protein

“Heterotrimeric G-proteins” mediate many cell signals

GDP

See ECB 16-17

G, G subunits

G binds guanine nucleotide

Receptor acts as GEF, activating G-protein

Activated G- and G regulate targets

G inactivated by GTP hydrolysis, subunits reassociate

GTP

+

GTP

GDPPi

G

(inactive GDP form)

ActiveG and G

(GTP form)

Heterotrimeric G-proteins

Downstream targets

Multiple G-proteins with distinct -, -, and -subunits (>20 known)

“Gs” stimulates or activates effectors

“Gi” inhibits effectors

“Gq” mediates Ca2+ signaling

G-protein –GDP(inactive)

GDP

Plasma membrane

Cytoplasm

Extracellular space

See ECB 16-16

“Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane

receptors

Inactive receptor

Seven transmembrane domains (-helices)

Extracellular ligand-binding domain (N-terminal)

Cytoplasmic “effector” domain

Activated receptor acts as GEF to activate “heterotrimeric G-protein”

Ligand binding domain

Effector domain

1 2 3 4 5 6 7

Seven-pass

receptor

“Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane

receptors

ECB 16-18 thru 16-18

Binding of ligand activates receptor

G-protein –GDP(inactive)

Inactive target

GDP

GTP

Active receptor

“Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane

receptors

Binding of ligand activates receptor

Heterotrimeric G-protein binds activated receptor

Activated receptor acts as GEF for heterotrimeric G-protein

Activated components (- and /-) regulate downstream targets

GTP hydrolysis inactivates G-protein, subunits reassociate (switches off)

Activated target

GTP

GDP

ECB 16-18 thru 16-18

Activated target can be enzyme that makes “intracellular messenger”

ECB 16-20

Ephinephrine (adrenaline) acts via heterotrimeric G protein and cAMP (intracellular messenger)

Activated adenylate cyclase forms cAMP

cAMP activates protein kinase A (PKA)

PKA enters nucleus and

phosphorylates a gene regulatory

protein

Result: altered transcription (slow)

ECB 16-24

Adenylate cyclase converts ATP to 5’,3’ cAMP

P O CH2

O

OH OH

1’

2’3’

4’

5’-O A

-O

O

P O P O P O CH2

O

OH OH

1’

2’3’

4’

5’-O A

-O-O-O

OOO

P

O

OH

1’

2’3’

5’

O

A

-O O

CH2

O

PPi 2Pi

ATP

Adenosine 3’,5’ cyclic monophosphate

(cAMP)

cAMP phosphodiesterase

“Adenylate cyclase”

AMP

Methylated xanthines (caffiene, theophylline, and theobromine ) inhibit cAMP PDE

ECB 16-21

cAMP levels rise rapidly in response to extracellular signal

ECB 16-22Serotonin is a neurotransmitter

5 X 10 -8 M cAMP

Assay fluorescence of protein that binds cAMP

10 -6 M cAMP

G-protein coupled receptors also activate IP3 and Ca2+-mediated signaling pathways

Activate receptor acts as GEF

Activated G activates phospholipase C (PLC)

Active PLC cleaves PIP2 to IP3 and diacylglycerol (DAG)

IP3 opens Ca2+ channels in ER releasing Ca2+ to cytoplasm

DAG and Ca2+ activate protein kinase C (PKC)

Active PKC phosphorylates target proteins…

Other Ca2+-dependent responses are regulated by “Calmodulin” (CaM) and “CaM kinases”

Ca2+-calmodulin activates CaM kinases, which phosphorylate and regulate target proteins

CaM contains 4 Ca2+ binding domainsCa2+

Ca2+

Ca2+-CaM binds to regulatory domains of effector proteins (e.g. CaM kinases)

see ECB 16-27

Ca2+

Phosphorylates target proteins in cytoplasm

Inactive CaM kinase

P

Active CaM kinase

ATPADP

Autophosphorylation

Calmodulin (CaM)

Catalytic domainInhibitory domain

Cells carefully regulate “free” Ca2+ levels in their cytoplasm

[Ca2+] >1 mM

[Ca2+ ]free ~0.2 M

In a resting cell, intracellular [Ca2+]free is low relative to external Ca2+…

Ca2+ is pumped into the ER (plant vacuole)

Ca2+ is pumped out of the cell by a Ca2+ ATPase and antiport with Na+ (antiport with H+ in plants/fungi)

Intracellular [Ca2+]free may increase 10-30-fold during signaling…

Moves in through channels and is released from internal stores (mostly from the ER, vacuole)

ATP ADP + Pi

Ca2+

Ca2+

2Na+ATP ADP + Pi

Ca2+

ER

Receptor G-protein(inactive)

Target(inactive)

2. G-protein coupled receptor

Last class of cell surface receptors

Receptor(active)

G-protein(active)

Target(inactive)

Signaling ligand

Signaling ligand

Catalytic domain(active)

Signaling ligand

Ions

Catalytic domain(active)

3. Enzyme-linked receptor

1. Ligand gated ion channel

Receptor(active)

G-protein(active)

Target(active)

Many growth factors bind to receptor tyrosine kinases (enzyme-linked receptor)

Receptor binds growth factor and dimerizesKinase activity activated and receptor autophosphorylates

Signaling proteins bind phosphotyrosine, activating signaling cascades

EGF and other growth factors activate Ras signaling

“GTPase Activating Protein” (Ras-GAPs) promote GTP hydrolysis by intrinsic GTPase

Pi

GAP

GTP Exchange Factors (GEFs) promote GDP/GTP exchange

Downstream effectors

Ras found to be mutated in ~30% of human tumors!

GEF

GDP

GTP

“On”RAS

GTP

“Off”

RAS(inactive)

GDP

Active Ras activates downstream signaling proteins…

MAPKKKinactive

Receptor tyrosine kinases activate intracellular Ras signaling cascades

ECB 16-31, 16-32

P

P

P

P

P

P

DRK

MAPKKinactive

MAP kinase inactive

Ras GEF

ADP

ATP

ADP

ATP

ADP

ATP

Transcriptionfactors

POther

proteinsP

Receptor kinase(active)

Growth Factors

Downstream of Receptor Kinaseactivates Ras GEF

“Mitogen-activ. protein kinase”MAP kinase

MAP kinase kinase(MAPKK)

MAPKKK active

MAPKKactive

MAP kinase active

P

PP

Regulate gene expression and protein

activity

RAS(inactive)

GDP

RAS

GTP MAP kinase kinase kinase(MAPKKK)

Mutations in Ras signaling pathway cause uncontrolled cell proliferation: cancer

MAPKKKactive

MAPKKactive

MAP kinaseactive

P

P

P

P

P

P

DRK

ADP

ATP

ADP

ATP

ADP

ATP

Transcriptionfactors

POther

proteinsP

Receptor kinase(active)

Downstream of Receptor Kinase

P

PP

Regulate gene expression and protein

activity…

RAS

GTP

RAS(inactive)

GDP

Ras GEFGTP

The Ras pathway activates expression of G1 cyclins that stimulate cell proliferation

Constituitive activation of pathway components results in uncontrolled cell proliferation = “cancer”

Cancer causing genes = “Oncogenes”

Predict effects of Ras mutations?

Signal transduction cascades are complex and interconnected

ECB 16-38

Why?• Integration

Multiple inputs to a single response…

• Divergence

Single input to multiple responses

• Amplification

• Regulation

P

P

P

P

P

P

G-protein coupled receptors

G-protein

Adenylate cyclase

cAMP

Protein kinase A

Phospholipase C

IP3

Ca2+

Calmodulin

CaM kinase

Diacylglycerol

Protein kinase C

G-protein Adapter

Ras activator

Ras

Kinase I

Kinase II

Kinase III

Receptor tyrosine kinases

Gene regulatory proteins Cytoplasmic target proteins

Communication by direct cytoplasmic continuity between cells

Cytoplasmic bridges and cell junctions

Communication via cell junctions: some embryonic cells and/or tissues are “dye-coupled”

Membrane-impermeant dye injected into on cell passes into neighbors

Cytoplasmic coupling is limited to small molecules (<1000 Da)

100 Da

1,000 Da

10,000 Da

“Gap junctions” are responsible for cytoplasmic coupling of animal cells

Membranes of coupled cells closely apposed, separated by 2-4 nm “gap”

ECB figure 19-28 MBoC figure 19-16

Large“gap jnctn”

Common in developing embryo, cardiac muscle, liver, and lens

TEM/Freeze fracture of gap junctions reveals “plaques” of intra-membrane particles

Gap junctions are composed of “connexons” made of “connexin”

hexamers

Cytoplasm of cell #2

Cytoplasm of cell #1

“Connexon”(2 per channel)

= “connexin” x 6

ECB 21-28

Plasma membrane of cell

#2

Plasma membrane of cell

#1

Extracellular “gap”(2-4 nm)

Channel is ~ 1.5 nm (~1000 Da

cutoff)

Two connexons in register form channel coupling cytoplasm of adjacent cells

The cytoplasm of plant cells is coupled by “plasmadesmata”

Membranes continuous from cell to cell

ER continuous from cell to cell thru “desmotubule”

Limited to small molecules (<800 Da), but can open to let through 20,000 Da

Primarily (but not exclusively) formed during cell division

ECB 21-30

Nucleus

Nucleus

NucleusVacuole

Plasmadesmata

Cell wall

Cytoplasm

100 nm

Cell wallPlasma membrane of adjacent cells

Cytoplasm

Cytoplasm

Desmotubule

Endoplasmic reticulum