MCB- Signal Transduction Lecture 1 - PowerPoint Presentation

Slide1

MCB- Signal Transduction Lecture 1

General Concepts of Signal Transduction

Cell Communication

Types of Receptors

Molecular Signaling

Receptor Binding

Scatchard Analysis

Competitive Binding

Second MessengersSlide2

Signaling throughout Evolution

Bacteria

Sense nutrients

Lac operon--bacteria turn on gene expression of 3 genes necessary to metabolize lactose (Jacob & Monod, Nobel 1965)

Chemotaxis- che proteins that couple nutrient receptors to flagellar motors

Quorum sensing

Yeast

Pheromone signaling for haploid yeast mating

Multicellular Organisms

Many signaling pathways (G proteins, channels, kinases)Slide3

The Integration of Biochemical Networks

Cell cycle and

DNA repair

Cytokines

Growth factors

Cell suicide

(Apoptose)

Pathogenic virusSlide4

Can a biologist fix a radio?

First step: obtain grants to purchase large number of functioning radios

Perform comparative analysis: take out all the pieces, classify them and give them names

Lazebnik,

Cancer Cell

2002

Begin

“

genetic analysis

”

by bombarding functioning radio with small metal objects: misfunctioning radios will display

“

phenotypes

”Slide5

Can a biologist fix a radio?

Lucky postdoc discovers Serendipitously Recovered Component (Src) that connects to the extendable object Most Important Component (Mic).

Another lab identifies Really Important Component (Ric) in radios where Mic does not play important role.

Undoubtedly-Mic (U-mic) controls Src & Ric (AM/FM switch)Slide6
Slide7

Cell Communication

Lodish, 20-1Slide8

Intracellular Receptors

Ligands need to be lipophilic

Steroids

Thyroid hormone

Retinoids

Cell surface receptors

Ligands can be either water soluble or lipophilic--but bind at the surface

Lodish, 20-2Slide9

Four classes of cell-surface receptors

Lodish, 20-3Slide10

Transmission of signals from

one molecule to another

3 basic modes (may be combined)

1.

Allostery

2.

Covalent

modification

3.

Proximity

(= regulated recruitment)

P

Shape change, often induced by binding a protein

or small molecule

Switching can be

very

rapid

Modification itself changes molecule

’

s shape

Memory device; may be reversible (or not)

Regulated molecule

may already be in

“

signaling mode;

”

induced proximity to a

target

promotes transmission

of the

signal

P

PSlide11

How quickly do you need your message to arrive?

VERY FAST (milliseconds)

Nerve conduction, vision

Ion channels

FAST (seconds)

Vision, metabolism, cardiovascular

G protein-coupled receptorsSLOW (minutes to hours)Cell division, proliferation, developmental processesGrowth factor receptorsSteroid hormonesSlide12

General types of protein-protein interfaces

A. Surface-string: examples include SH2 domains, kinase-substrate interactions

B. Helix-helix: also called coiled-coil, found in several families of transcription factors

C. Surface-surface: most common, often involve extended complementary surfaces, such as growth factor receptors.

Alberts 5-34Slide13

Plasticity of Protein-protein interfaces

Recent concept

: Many hormones can bind to different receptors, and a single receptor can bind multiple different hormones. The common protein uses essentially the same contact residues to bind multiple partners.

Example

: The hinge region of Fc portion of IgG antibodies can bind to Staph A, Staph G, RF, and neonatal FcR. Co-crystallization of the hinge region with these four proteins reveals the plasticity of the interaction surface.

Delano, et al.

Science

2000Slide14

Specific binding of insulin to cells

Saturation Binding studies

Can be performed in intact cells, membranes, or purified receptors

1. Add various amounts of labeled ligand (drug, hormone, growth factor)

2. To determine specific binding, add an excess of unlabeled ligand to compete for specific binding sites.

QU: Why is there non-specific binding?

3. Bind until at equilibrium

4. Separate bound from unbound ligand

5. Count labeled ligand

[Adapted from A. Ciechanover et al., 1983,

Cell

32:

267.]

Receptor: ligand binding must be specific, saturable, and of high affinitySlide15

Reversibility & Timing

Activity of a signaling machine often depends on its

association with another molecule

If the association is

reversible

, we can talk about . . .

Equilibrium binding

(A) + (B)

(AB)

k

1

= association rate

= dissociation rate

At equilibrium, the forward reaction

goes at

exactly

the same rate as

the backward reaction

Forward reaction rate = (A)(B)

Backward reaction rate = (AB)

So . . .

(A)(B) = (AB)

k

2

k

1

k

2

k

1

k

2

k

1

k

2Slide16

Reversibility & Timing

If . . .

(A)(B) = (AB)

k

1

k

2

=

Kd =

(A)(B)

(AB)

k

1

k

2

k

1

k

2

=

Define

So . . .

Equilibrium binding is saturable

1.0

0.5

(AB)

(A)

Kd = conc of A at which

half of B binds A

dissociation

constant

Kd =

Bmax

KdSlide17

Reversibility & Timing

Kd =

k

1

k

2

k

1

= association rate constant

= dissociation rate constant

k

2

Units

(M

-1

)(sec

-1

)

(sec

-1

)

k

1

k

2

usually ~ 10

8

M

-1

sec

-1

(diffusion-limited)

just a time constant (sec

-1

)

Thus, knowing the Kd and assuming a

“

usual

”

rate

of association, you can calculate . . .

k

2

, and therefore the duration (or half-life*) of the

(AB) complex

*Half-life = 0.69 ÷ k

2Slide18

Reversibility & Timing

Kd

k

2

*Half-life = 0.69 ÷ k

2

Half-life

of (AB)

(sec)

(M)

(sec

-1

)

Acetylcholine

Norepinephrine

Insulin

10

2

10

0

10

-2

0.007

0.7

70

10

-6

10

-8

10

-10

LIGANDSlide19

Scatchard Analysis

Slope = - 1/Kd

X intercept = # rec

(Bound Lig)

(Bound Lig)

(Free)

For an excellent discussion of principles of receptor binding, and

practical considerations, see http://www.graphpad.com; also posted on MCB website.Slide20

Scatchard Analysis

(Bound Lig)

(Bound Lig)

(Free)

Negative cooperativity: binding of ligand to first subunit decreases affinity of subsequent binding events.

Positive cooperativity: binding of ligand to first subunit increases

Affinity of subsequent binding events. Example: hemoglobin binding O

2Slide21

Cooperative binding

The Hill equation accounts for the possibility that not all receptor sites are independent, and states that

Fractional occupancy = L

f

n

/ (K

d

+ L

f

n)

n= slope of the Hill plot and also is the avg # of interacting sites

For linear transformation, log [B/(R

t

- B)] = n(log L

f

) - log K

d

log [B/(R

t

- B)]

log L

f

Slope= n

If slope = 1, then single class of binding sites

If slope > 1, then positive cooperativity

If slope < 1, then negative cooperativitySlide22

Competitive binding

How many different types of ligands can a receptor bind? Are some ligands more avid for a receptor than others?

You can use the ability of a compound (could be agonist or antagonist) to competitively displace the binding of a fixed amount of a different compound (usually a labeled antagonist).

BIG ADVANTAGE: You only need one labeled compound.

Example

. Adrenergic agonists: isoproterenol (

ISO

), epinephrine (

EPI

)

Adrenergic antagonists: phentolamine (PHEN)

100%

[competitor]

100%

[competitor]

a

-adrenergic receptor

b

-adrenergic receptor

ISO

ISO

PHEN

PHENSlide23

So that

’

s the theory:

How do we

know

whether or not it is true?

1.

Theory is internally consistent

(necessary, not sufficient for belief)

2.

Binding experiments

Stop binding reaction quickly, measure bound complex, (AB)

Assess

k

1

=

“

on-rate

”

Assess

k

2

=

“

off-rate

”

Compare vs. Kd

3.

Seeing is believing

:

Watch

behavior of fluorescent-tagged single molecules

of ligand bound to receptorsSlide24

Seeing is believing* . . .

Assess duration of ligand-GPCR complexes, during chemotaxis

of living

Dictyostelium

cells

Question

: Does GPCR signaling differ at front vs. back of the cell?

Experimental system:

Dictyostelium discoideum

,

a soil amoebaSlide25

Seeing is believing, Total Internal Reflection Fluorescence

http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfintro.html

Question: Does GPCR signaling differ at front vs. back of the cell?

Approach: Tag cAMP ligand

with a fluorescent dye

Bound

cAMP stays in one place on cell surface;

unbound

tagged cAMP diffuses rapidly away

Evanescent wave excites only

tagged cAMP near slideSlide26

Seeing is believing* . . .

*Ueda et al., Science 294:864,2001

0

5

10

20

15

25

0

400

Time (sec)

Pseudopod

k

2

= 1.1 and 0.39 s

-1

k

2

= 0.39 and 0.16 s

-1

Tail

cAMP-R complexes dissociate

~2.5 x faster at the front than

at the back!

True for cells in a ligand gradient

and also

in a uniform concentration of the ligand

Off & On

:

cAMP-R complexes

(movie: 7 sec total)

Cy3-cAMP

bound

Cell surface facing the slide

Each point is a

separate cAMP/R

complexSlide27

Seeing is believing* . . .

*Ueda et al., Science 294:864,2001

Each spot = 1 cAMP/R complex

Spots move ~1-2

m

/sec

# spots per m2 of surface

area equal at front and

back of the cell

(like

receptor density)Slide28

Seeing is believing* . . .

*Ueda et al., Science 294:864,2001

Inferences

Questions

Receptors at the front differ biochemically from those

in the back

Because receptor density and the #

bound

receptors

are the same, faster dissociation (k

2

) at the front must

be matched by faster association (k

1

) as well

What biochemical mechanism underlies this difference?

(Probably reflects residence of the GPCRs and

G proteins in different macromolecular complexes)

The functional difference is

not

created by the gradient,

but instead reflects some difference between the front

and back of the cellSlide29

Other methods of measuring binding

Surface plasmon resonance (BiaCore)

Can measure

“

on

”

rates and “off” rates to calculate binding affinitiesIsothermal calorimetry

Very accurate, requires lots of protein and expensive equipmentEquilibrium dialysis

Useful for binding of small ligands to large proteins

Fluorescence anisotropyExcite fluorescent protein with polarized light. Anisotropy refers to the extent that the emitted light is polarized--the larger the protein/complex, the slower the tumble rate and the greater the anisotropy

Co-immunoprecipitation

Yeast two-hybridSlide30

Second messengers

Cyclic nucleotides: cAMP, cGMP

Inositol phosphate (IP)

Diacylglycerol (DAG)

Calcium

Nitric oxide (NO)

Reactive oxygen species (ROS)

Molecular mediators of signal transduction. Cells carefully, and rapidly, regulate the intracellular concentrations. Second messengers can be used by multiple signaling networks (at the same time).Slide31

Earl Sutherland

1971 Nobel laureate

Rall, et al. JBC 1956Slide32

Fischer & Krebs, Nobel 1992

Discovered that phosphorylase activity was regulated by the reversible step of phosphorylation. Identified PKA and some of the first phosphatases.Slide33

cAMP regulates PKA activity

Alberts 15-31,32

Positive cooperativity--binding of increases affinity for second cAMP

PKA targets include Phosphorylase kinase and the transcription regulator, cAMP response element binding (CREB) proteinSlide34

Diacylglycerol and Inositol Phosphates as second messengers

Alberts, 15-35Slide35

Calcium acts as second (third?) messenger

Lodish, 20-39Slide36

Calmodulin transduces cytosolic Ca

2+

signal

Alberts, 15-40

Calmodulin, found in all eukaryotic cells, and can be up to 1% of total mass. Upon activation by calcium, calmodulin can bind to multiple targets, such as membrane transport proteins, calcium pumps, CaM-kinasesSlide37

CaM-kinase II regulation

Alberts, 15-41Slide38

Frequency of calcium oscillations influences a cell

’

s response

High frequency Ca

2+

oscillations

Low frequency Ca

2+

oscillations

CaM-kinase II activity

CaM-kinase II activity

CaM-kinase uses memory mechanism to decode frequency of calcium spikes.

Requires the ability of the kinase to stay active after calcium drops. This is accomplished by autophosphorylation.

Alberts 15-39,42Slide39

Calcium signaling also occurs in waves

Alberts, 15-37

0 sec

10 sec

20 sec

40 sec

Calcium effects are local, because it diffuses much more slowly than does InsP

3

Sperm binds

InsP

3

receptor is both stimulated and inhibited calcium

[Ca

2+

]

Sensitivity of

InsP3 R to Ca

2+

InsP

3Slide40

NO signaling

Lodish, 20-42

NO effects are local, since it has half-life of 5-10 seconds (paracrine).

NO activates guanylate cyclase by binding heme ring (allosteric mechanism)

Gases can act as second messengers!Slide41

Discovery of NO signaling

Robert F Furchgott showed that acetylcholine-induced relaxation of blood vessels was dependent on the endothelium. His "sandwich" experiment set the stage for future scientific development. He used two different pieces of the aorta; one had the endothelial layer intact, in the other it had been removed.

Louis Ignarro reported that EDRF relaxed blood vessels. He also identified EDRF as a molecule by using spectral analysis of hemoglobin. When hemoglobin was exposed to EDRF, maximum absorbance moved to a new wave-length; and exposed to NO, exactly the same shift in absorbance occurred! EDRF was identical with NO.

Furchgott, Ignarro, Murad, Nobel Prize 1998

http://www.nobel.se/medicine/laureates/1998/illpres/index.htmlSlide42

Reactive Oxygen Species (ROS) Signaling

Finkel & Holbrook, Nature (2000)

ROS important in cell

’

s adaptation to stress

Many of longevity mutations map to ROS pathways

Mutations in Superoxide Dismutase (SOD) cause amyotrophic lateral sclerosis (ALS, Lou Gehrig

’

s Disease)

Unfortunately, no great clinical data showing that anti-oxidants will help us live longer!Slide43

ROS activates multiple pathways

Finkel & Holbrook, Nature (2000)

Activation mechanisms ????

Mimic ligand effect for GF receptors

Oxidants enhance phosphorylation of RTKs and augment ERK/Akt signaling

Inactivation of phosphatases

Hydrogen peroxide inactivates protein-Y phosphatase 1B

Redox sensors

Thioredoxin (Trx) binds and inhibits ASK1, an upstream activator of JNK/p38 pathways. ROS dissociates Trx-ASK1 complex

HSF1, NF-kB, and ERK activities change with age (Pink boxes)