mcb- signal transduction lecture 1

MCB- Signal Transduction Lecture 1

General Concepts of Signal TransductionCell CommunicationTypes of ReceptorsMolecular Signaling

Receptor BindingScatchard AnalysisCompetitive Binding

Second Messengers

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)

The Integration of Biochemical Networks

Cell cycle andDNA repair

Cytokines

Growth factors

Cell suicide(Apoptose)

Pathogenic virus

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”

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)

Cell Communication

Lodish, 20-1

• Intracellular ReceptorsLigands 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, 20-2

Four classes of cell-surface receptorsLodish, 20-3

Transmission of signals from one molecule to another3 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 moleculeSwitching can be very rapid

Modification itself changes molecule’s shapeMemory 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 P

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 receptors

• SLOW (minutes to hours)Cell division, proliferation, developmental processes– Growth factor receptors– Steroid hormones

General types of protein-protein interfacesA. Surface-string: examples include SH2 domains, kinase-substrate interactionsB. Helix-helix: also called coiled-coil, found in several families of transcription factorsC. Surface-surface: most common, often involve extended complementary surfaces, such as growth factor receptors.

Alberts 5-34

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 2000

Specific binding of insulin to cells

Saturation Binding studiesCan be performed in intact cells, membranes, or purified receptors1. 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 equilibrium4. Separate bound from unbound ligand5. Count labeled ligand

[Adapted from A. Ciechanover et al., 1983, Cell 32:267.]

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

Reversibility & TimingActivity 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) k1= 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)

k2

k1

k2

k1

k2

k1 k2

Reversibility & Timing

If . . . (A)(B) = (AB) k1 k2

= Kd =(A)(B)(AB)k1

k2

k1

k2=

Define

So . . .

Equilibrium binding is saturable

1.0

0.5

(AB

)

(A)

Kd = conc of A at which half of B binds A

dissociation constantKd =

Bmax

Kd


Kd = k1

k2 k1= association rate constant

= dissociation rate constantk2

Units

(M-1)(sec-1)

(sec-1)

k1

k2

usually ~ 108M-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 . . .

k2, and therefore the duration (or half-life*) of the (AB) complex

*Half-life = 0.69 ÷ k2


Kd k2

*Half-life = 0.69 ÷ k2

Half-life of (AB)

(sec)(M) (sec-1)

Acetylcholine

Norepinephrine

Insulin

102

100

10-2

0.007

0.7

70

10-6

10-8

10 -10

LIGAND

Scatchard Analysis

Slope = - 1/Kd

X intercept = # rec

(Bound Lig)

(Bound Lig)(Free)

For an excellent discussion of principles of receptor binding, andpractical considerations, see http://www.graphpad.com; also posted on MCB website.

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 increasesAffinity of subsequent binding events. Example: hemoglobin binding O2

Cooperative binding

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

Fractional occupancy = Lfn/ (Kd + Lf

n)

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

For linear transformation, log [B/(Rt - B)] = n(log Lf) - log Kd

log [B/(Rt - B)]

log Lf

Slope= n

If slope = 1, then single class of binding sites

If slope > 1, then positive cooperativity

If slope < 1, then negative cooperativity

Competitive bindingHow 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

PHEN

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 k1 = “on-rate”

Assess k2 = “off-rate”

Compare vs. Kd

3. Seeing is believing: Watch behavior of fluorescent-tagged single molecules of ligand bound to receptors

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 amoeba

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 ligandwith a fluorescent dye

Bound cAMP stays in one place on cell surface; unbound tagged cAMP diffuses rapidly away

Evanescent wave excites onlytagged cAMP near slide


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

0 5 10 2015 250

400

Time (sec)

Pseudopodk2 = 1.1 and 0.39 s-1

k2 = 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

-cA

MP

b

ound

Cell surface facing the slide

Each point is a separate cAMP/R complex



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)



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 (k2) at the front must

be matched by faster association (k1) 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 cell

Other methods of measuring binding• Surface plasmon resonance (BiaCore)

Can measure “on” rates and “off” rates to calculate binding affinities• Isothermal calorimetry

Very accurate, requires lots of protein and expensive equipment• Equilibrium dialysis

Useful for binding of small ligands to large proteins• Fluorescence anisotropy

Excite 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-hybrid

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).

Earl Sutherland1971 Nobel laureate

Rall, et al. JBC 1956

Fischer & Krebs, Nobel 1992

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

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) protein

Diacylglycerol and Inositol Phosphates as second messengers

Alberts, 15-35

Calcium acts as second (third?) messenger

Lodish, 20-39

Calmodulin transduces cytosolic Ca2+ 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-kinases

CaM-kinase II regulation

Alberts, 15-41

Frequency of calcium oscillations influences a cell’s response

High frequency Ca2+ oscillationsLow frequency Ca2+ oscillations

CaM

-kin

ase

II ac

tivity

CaM

-kin

ase

II ac

tivity

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 15-39,42

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 InsP3

Sperm binds

InsP3 receptor is both stimulated and inhibited calcium

[Ca 2+]

Sen

sitiv

ity o

f In

sP3

R to

Ca

2+

InsP3

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!

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.html

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!

ROS activates multiple pathways

Finkel & Holbrook, Nature (2000)

Activation mechanisms ????

Mimic ligand effect for GF receptorsOxidants enhance phosphorylation of RTKs and augment ERK/Akt signaling

Inactivation of phosphatasesHydrogen peroxide inactivates protein-Y phosphatase 1B

Redox sensorsThioredoxin (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)

mcb- signal transduction lecture 1

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