mcb cell signaling lectures 1 and 2 362-1668 lecture...

MCB Cell Signaling Lectures 1 and 2

Ken Blumer

Dept. of Cell Biology & Physiology

506 McDonnell Sciences

[email protected]

362-1668

Lecture 1

General Concepts of Signal TransductionCell CommunicationTypes of ReceptorsMolecular Signaling

Receptor BindingScatchard AnalysisCompetitive Binding

Second Messengers

G proteins

Signaling throughout Evolution

• Bacteria– Sense nutrients

• Lac operon--bacteria turn on gene expression of 3 genesnecessary to metabolize lactose (Jacob & Monod, Nobel1965)

• Chemotaxis- che proteins that couple nutrient receptorsto flagellar motors

– Quorum sensing• Yeast

– Pheromone signaling for haploid yeast mating• Multicellular Organisms

Many signaling pathways (G proteins, channels, kinases)

Modes of Cell Communication

Lodish, 20-1

• Intracellular ReceptorsLigands need to be

lipophilic– Steroids– Thyroid hormone– Retinoids

• Cell surface receptorsLigands can be either

water soluble orlipophilic--but bind atthe surface

Lodish, 20-2

Four classes of cell-surface receptors

Lodish, 20-3

Transmitting 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 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

Signaling speed matches with function

• 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

Detecting Receptors by Ligand Binding

Saturation Binding studiesCan be performed in intactcells, membranes, or purifiedreceptors1. Add various amounts oflabeled ligand (drug, hormone,growth factor)2. To determine specificbinding, add an excess ofunlabeled ligand to competefor specific binding sites.QU: Why is there non-specificbinding?3. Bind until at equilibrium4. Separate bound fromunbound 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

Ligand Binding Reversibility, Affinity & Kinetics

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)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

Kinetics & Affinity

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

Kinetics and Half-life

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

Half-lives differ greatly

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 subunitdecreases affinity of subsequent binding events.

Positive cooperativity:binding of ligand tofirst subunit increasesAffinity of subsequentbinding events.Example: hemoglobinbinding O2

Cooperative binding

The Hill equation accounts for the possibility that not all receptorsites 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 singleclass of binding sites

If slope > 1, then positivecooperativity

If slope < 1, then negativecooperativity

Competitive bindingHow many different types of ligands can a receptor bind? Are some ligandsmore avid for a receptor than others?You can use the ability of a compound (could be agonist or antagonist) tocompetitively 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]

α-adrenergic receptor β-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)

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-receptor complexes, during chemotaxis of living Dictyostelium cells

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

Experimental system: Dictyostelium discoideum, a soil amoeba

Seeing is believing, Total InternalReflection Fluorescence

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

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

Approach: Tag cAMP ligandwith a fluorescent dye

Bound cAMP stays in oneplace on cell surface;unbound tagged cAMPdiffuses rapidly away

Evanescent wave excites onlytagged cAMP near slide

Seeing is believing* . . .

*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-1Tail

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

Other methods of measuring binding

• Surface plasmon resonance (BiaCore)Can measure “on” rates and “off” rates to calculate binding affinities

• Isothermal calorimetryVery accurate, requires lots of protein and expensive equipment

• Equilibrium dialysisUseful 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 theprotein/complex, the slower the tumble rate and the greater theanisotropy

• Co-immunoprecipitation• Yeast two-hybrid

Many receptors regulate cell function byproducing second messengers

• Cyclic nucleotides: cAMP, cGMP• Inositol phosphate (IP)• Diacylglycerol (DAG)• Calcium• Nitric oxide (NO)• Reactive oxygen species (ROS)

Molecular mediators of signal transduction. Cellscarefully, and rapidly, regulate the intracellularconcentrations. Second messengers can be used bymultiple signaling networks (at the same time).

Earl Sutherland1971 Nobel laureate

Rall, et al. JBC 1956

Fischer & Krebs, Nobel 1992

Discovered thatphosphorylase activitywas regulated by thereversible step ofphosphorylation. IdentifiedPKA and some of the firstphosphatases.

cAMP regulates PKA activity

Alberts 15-31,32

Positive cooperativity--binding ofincreases affinity for second cAMP

PKA targets include Phosphorylase kinaseand the transcription regulator, cAMPresponse element binding (CREB) protein

Diacylglycerol and Inositol Phosphates assecond 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 bindto multiple targets, such as membrane transport proteins,calcium pumps, CaM-kinases

CaM-kinase II regulation

Alberts, 15-41

Frequency of calcium oscillationsinfluences 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 mechanismto decode frequency of calcium spikes.

Requires the ability of the kinase tostay active after calcium drops. This isaccomplished 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 thandoes 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 bybinding heme ring (allostericmechanism)

Gases can act as second messengers!

Discovery of NO signaling

Robert F Furchgott showed thatacetylcholine-induced relaxation ofblood vessels was dependent on theendothelium. His "sandwich"experiment set the stage for futurescientific development. He used twodifferent pieces of the aorta; one hadthe endothelial layer intact, in theother it had been removed.

Louis Ignarro reported that EDRF relaxed bloodvessels. He also identified EDRF as a molecule byusing spectral analysis of hemoglobin. Whenhemoglobin was exposed to EDRF, maximumabsorbance moved to a new wave-length; andexposed to NO, exactly the same shift in absorbanceoccurred! 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’sadaptation to stress

Many of longevitymutations map to ROSpathways

Mutations in SuperoxideDismutase (SOD)cause amyotrophiclateral sclerosis (ALS,Lou Gehrig’s Disease)

Unfortunately, no greatclinical data showingthat anti-oxidants willhelp us live longer!

ROS activates multiple pathways

Finkel & Holbrook, Nature (2000)

Activation mechanisms ????

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

Inactivation of phosphatasesHydrogen peroxide inactivates protein-Yphosphatase 1B

Redox sensorsThioredoxin (Trx) binds and inhibitsASK1, an upstream activator of JNK/p38pathways. ROS dissociates Trx-ASK1complex

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

G proteins:switches linking receptors & 2nd messengers

• Discovery and Structure of Heterotrimeric Gproteins

• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms• Alliance for Cell Signaling (AfCS)

Discovery of G proteinsMartin Rodbell first proposed the concept of “discriminator-transducer-amplifier” to address the problem: “How canmany hormones (epinephrine, ACTH, TSH, LH, secretin,and glucagon) activate lipolysis and cAMP production inadipocytes through presumably a single cyclase? He calledthis problem “too many angels on a pinhead.” His workidentified GTP as important for the “transducer”.

His work was notinitially receivedwell by thescientificcommunity:

Nobel prize, 1994

Discovery of G proteinsAl Gilman purified the first G proteins. His lab tookadvantage of S49 lymphoma cells that lacked Gsα(although at the time, the cells were thought to lackadenylate cyclase, thus the name cyc-).

Reconstitution experiment rationale: Isolate membranesfrom cyc- cells, then add back fractions from donor wtmembranes that restore adenylate cyclase activity. Nobel prize, 1994

Donor membranes were incubated forincreasing time at 30oC, whichinactivates the adenylate cyclaseactivity (- - - - -). Fortunately, G proteinsare relatively heat stable.

Addition of NaF, Gpp(NH)p, GTP, orGTP and isoproterenol restored activityin the cyc- membranes.

Ross, et al. JBC (1978)

Signal Transduction by G proteins



G protein signal transduction

Neves, Ram, Iyengar, Science 2002

Structure of G proteins

Iiri, et al. NEJM (1999)

Hydrolysis of GTP

• Arg & Gln stabilize the β and γphospates of GTP molecule incorrect orientation for hydrolysisby H2O

• Hydrolysis leads to majorconformation change in Gs α

• Mutations in the Gln or Arg (orADP ribosylation by cholera toxin)blocks the ability to stabilizetransition state, and thereforelocks G protein in the “on”position.

• Examples include adenomas ofpituitary and thyroid glands (GHsecreting tumors, acromegaly),and McCune-Albright syndrome.

Iiri, et al. NEJM (1999)

Canonical Gs Signaling PathwayFor interactive pathways atSTKE:

Gs pathwayhttp://stke.sciencemag.org/cgi/cm/CMP_6634

Gi pathwayhttp://stke.sciencemag.org/cgi/cm/CMP_7430

Gq pathwayhttp://stke.sciencemag.org/cgi/cm/CMP_6680

G12 pathwayhttp://stke.sciencemag.org/cgi/cm/CMP_8022

Neves, Ram, Iyengar, Science 2002




G protein signaling

• Many ligands• Robust switches• Multiple effectors• Conserved 7 TM

architecture• More than 50% of

drugs targetGPCRs

Bockaert & Pin, EMBO J (1999)

G protein-coupledreceptors

• 5 main families• Conserved 7 TM

architecture

GPCRs in the Human GenomeSteve Foord, GlaxoWelcome

Rhodopsin Secretin Metabotropic

Liganded 163 25 11Orphan 140 34 4Olfactory 350 6Taste 15 3

Identifying Ligands for Orphan GPCRS

Big Pharm approach: set upindividual stable cell linesexpressing each orphan GPCR.Fractionate peptides, tissuefactors, etc. and apply to eachcell line. Example: Orexinreceptors

Cottage industry approach:expression cloning strategy inXenopus oocytes. Use sibselection to identify cDNAs thatencode desired receptor.Example: Thrombin receptor

GPCR desensitization mechanisms

10 seconds is too long! αt-GTP must be inactivated in < 1 sec

Many variations: eg, effectors with RGS activity

eg, phospholipase C β acts on αqE

E*EPi

EFFECT

Regulators of G Signaling (= RGS1-~RGS16; RGS9 in ROS)

GTP

RGSRGS

RGSPi

GDPαt GTP

αt αt

Most RGSs act on αi or αq families

RGSSwi1 Swi2

GTPAccelerate GTPase from < 1/sec to >10 3/sec

GTP GDPαq GTP

αq αq

eg, γ subunit of cGMP PDE enhances effect of retinal RGS on αt

New concepts for GPCR signalingUsing mainly two-hybrid screeningapproaches, many proteins have beenfound to interact with portions of theGPCRs.Non-PDZ scaffolds: AKAPs (A-KinaseAnchoring Proteins, JAK2 (JanusActivated Kinase), homer, β-arrestinsPDZ scaffolds: InaD, PSD-95 (Post-Synaptic Density), NHERF (Na/HExchanger Regulatory Factor).

The arrestins have been found to bindto other signaling proteins and activatedownstream effectors:Examples: src, Raf & ERK, ASK1 &JUNK3

Lefkowitz reviews

Arrestins act as scaffolds for ERK andJNK signaling pathways

Lefkowitz reviews




Discovery of Small G proteinsRas genes first identified in‘60’s as transforming genes ofrat sarcoma viruses.

Weinberg, Varmus, Bishop andothers in the early ‘80’s showedthat many cancer cells havemutated versions of ras.

Activated form of ras found in90% of pancreatic carcinomas,50% of colonadenocarcinomas, and 20% ofmalignant melanomas.

Ras-GTP vs. Ras-GDP

Signaling GTPases are Allosteric Switches

γ-phosphate

Ras = classical “monomeric” GTPase

Binding γ-phosphate changes the conformations of two small surface elements, called “switch 1 and 2”

Swi1Swi2

Rho/Rac/Cdc42

In early ‘90’s, Alan Hall discovered that newly characterizedRas homologs (Rho, Rac, Cdc42) induced cytoskeletalchanges.

Hall, Science 1998

Actin Stress fibers Focal adhesions

Lamellipodia Filopodia

Ras superfamily of small G proteins

Takai, et al. Physiological Reviews, 2001

GTPases: How to use reverse genetics to identify their roles in cell regulation

Depends on understanding how the machines work

Epistasis question: Where in a pathway does a specific protein convey its particular message?

A B

C D E

Response

M N Q

Idea: 1. Inhibit activity of the protein of interest

2. Increase activity of the protein of interest

How to do this? Drugs, genetic diseases, mouse KOs, and . . .

Reverse genetics: express one or two mutant versions of the protein of interest

Depends on understanding how the machines work

1. Inhibit activity of the protein with a “dominant-negative” interfering mutant of that protein

2. Increase activity of the protein with a “dominant-positive” or “constitutively active” interfering mutant of the protein

The mutant titrates (binds up) a limiting component to block the normal protein’s signal

The mutant exerts the same effect as the normal protein would, if it were activated in the cell

Reverse genetics: small GTPases as examplesDepends on understanding how the machines work

“Dominant-negative” mutation “Dominant-positive”

mutation

The mutant titrates (binds up) a limiting component to block the normal protein’s signal

The mutant exerts the same effect as the normal protein would, if it were activated

GAP

GTP

Pi

GDPGEF

GEFGDP

Binds GEF but cannot replace GDP by GTP; so GEF not available for activating normal protein

Cannot hydrolyze GTP, so remains always active

Reverse genetics: advantages/pitfalls of using dominant-interfering mutants

Pro:Quick-and-dirty; no biochem

Many different families of signaling proteins amenable . . . once we understand how one of them works

Examples:

RTKs?Other kinases?Adaptors?

Con:Dominant-negatives

Dominant positives

Therefore . . .Still need biochemistry

Hard to apply to complex networks

Over-expression can titrate too many proteins (or the wrong proteins

Not always precise mimics of the normal protein (e.g., may be in the wrong place))

Can induce adaptation, turn-off mechanisms

Hierachy of small G protein activation

Ras

Use of constitutively active or dominant negative mutant small Gproteins revealed that ras and cdc42 can activate rac. Rac, in additionto inducing lamellipodia, also activates Rho.

y

Rho/Rac/Cdc42 signaling in actin assembly

Takai, et al. Physiological Reviews, 2001



• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms

Small G protein “turn on” mechanisms

First mammalian GEF,Dbl, isolated in 1985 asan oncogene in NIH 3T3focus forming assay. Ithad an 180 amino aciddomain with homology toyeast CDC24. Thisdomain, named DH (Dblhomology) is necessaryfor GEF activity.

In 1991, Dbl shown tocatalyze nucleotideexchange on Cdc42.

Schmidt & Hall, Genes & Dev. (2002)Dbl= Diffuse B-cell lymphoma

Small G proteins “turn off” mechanismsRhoGAPs outnumber the small Gproteins Rho/Rac/Cdc42 by nearly5-fold.Why so much redundancy?Luo group did RNAi against 17 ofthe 20 RhoGAPs in fly.

Six caused lethality whenexpressed ubiquitously. Tissuespecific expression of RNAirevealed unique phenotypes.

P190RhoGAP implicated in axonwithdrawal. Increasing amounts ofRNAi caused more axonwithdrawal (panels C-G).

Why so many RhoGAPs?Billuart, et al. Cell (2001)

Identification of RasGAP

McCormick injected Xenopus oocyteswith oncogenic ras (V12) versus wt ras(G12) and monitored germinal vesiclebreakdown (GVB) (top panel)

% G

VB

[ras] (ng)

V12

G12

Time (min)

% R

as-G

TP V12

G12

Then loaded ras with α-32P GTP, injectedinto oocytes, did immppt at increasingtimes and determined if GTP or GDP wasbound (bottom panel)

Purified the factor that promoted GTPaseactivity, cloned and named it GAP (or ras-GAP). Another ras-GAP later identified isNF1 (the gene mutated in neurofibromatosis,i.e., Elephant Man Syndrome).

Rate of GTP hydrolysis is 300-fold fasterin oocytes than in vitro!

Rho/Rac/CDC42 activation ofdownstream effectors

Rho

Effectors: PI 3-Kinase, PLD, Rho Kinase, Rhophilin, and others.

Rac-interacts via a CRIB domain in downstream effectors. CRIB(Cdc42/Rac interacting binding)

Effectors: NADPH oxidase, PAK, PI 3-Kinase, MLK2,3, POSH,DGK

Cdc42

Effectors: PI ε-Kinase, PAK, WASP, S6-Kinase, MLK2,3, Borg

The GTPase switch

Schmidt & Hall, Genes & Dev. (2002)

mcb cell signaling lectures 1 and 2 362-1668 lecture...

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