MCB Cell Signaling Lecture 1 Ken Blumer Dept. of Cell ... · 3’,5’-cyclic AMP Ca2+ Questions: Discovery (separate, re-combine) Specificity Amplification Complexity Signaling machines

MCB Cell Signaling Lecture 1

Ken Blumer

Dept. of Cell Biology & Physiology

506 McDonnell Sciences

[email protected]

362-1668

Lecture 1

General Concepts of Signal Transduction Cell Communication Types of Receptors Molecular Signaling

Receptor Binding

Scatchard Analysis Competitive Binding

Second Messengers G proteins

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)

“Cell Signaling”

Signals cross the plasma membrane

Cytoplasmic pathways & networks

Signaling to the nucleus

Responses

A B C

PQ

R

ST

Modes of Cell Communication

Lodish, 20-1

•  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, 20-2

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 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 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 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 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 constant Kd =

Bmax

Kd

Kinetics and Half-life

Kd = k1 k2 k1 = association rate constant

= dissociation rate constant k2

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, and practical 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 increases Affinity 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 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]

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 Internal Reflection 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 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 slide

Seeing is believing* . . .

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

0 5 10 20 15 25 0

400

Time (sec)

Pseudopod k2 = 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

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

Many receptors regulate cell function by producing 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).

The first established signaling pathway

cAMP mediates epinephrine-stimulated release of glucose from the liver

Phosphorylase kinase

cAMP- dependent protein kinase (PKA)

Glycogen

PhosphorylaseGlucose

Epinephrine

3’,5’-cyclic AMPCa2+

Questions:Discovery (separate, re- combine)SpecificityAmplificationComplexitySignaling machines

Earl Sutherland 1971 Nobel laureate

Rall, et al. JBC 1956

The first established signaling pathway

cAMP mediates epinephrine-stimulated release of glucose from the liver

Phosphorylase kinase

cAMP- dependent protein kinase (PKA)

Glycogen

PhosphorylaseGlucose

Epinephrine

3’,5’-cyclic AMPCa2+

Questions:Discovery (separate, re- combine)SpecificityAmplificationComplexitySignaling machines

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+ oscillations Low 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 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)

G proteins: switches linking receptors & 2nd messengers

•  Discovery and Structure of Heterotrimeric G proteins

•  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 proteins Martin Rodbell first proposed the concept of “discriminator-transducer-amplifier” to address the problem: “How can many hormones (epinephrine, ACTH, TSH, LH, secretin, and glucagon) activate lipolysis and cAMP production in adipocytes through presumably a single cyclase? He called this problem “too many angels on a pinhead.” His work identified GTP as important for the “transducer”.

His work was not initially received well by the scientific community:

Nobel prize, 1994

Discovery of G proteins Al Gilman purified the first G proteins. His lab took advantage of S49 lymphoma cells that lacked Gsα (although at the time, the cells were thought to lack adenylate cyclase, thus the name cyc-). Reconstitution experiment rationale: Isolate membranes from cyc- cells, then add back fractions from donor wt membranes that restore adenylate cyclase activity.

Nobel prize, 1994

Donor membranes were incubated for increasing time at 30oC, which inactivates the adenylate cyclase activity (- - - - -). Fortunately, G proteins are relatively heat stable. Addition of NaF, Gpp(NH)p, GTP, or GTP and isoproterenol restored activity in the cyc- membranes.

Ross, et al. JBC (1978)

Trimeric G Proteins: GTPase CycleAdded complexity

GTPGDP R* βγαe

αGTP

αGDP

R*

βγ

R*

βγ

Pi

E1

2E

RGS

GEF function requires cooperation between GPCR (R*) and βγGTPase is faster (2-6/min) than for small GTPasesBut RGS (Regulators of G Signaling) proteins accelerate GTPase even more (>1,000/sec)

TWO effectors, α-GTP and βγ

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 γ phospates of GTP molecule in correct orientation for hydrolysis by H2O

•  Hydrolysis leads to major conformation change in Gs α

•  Mutations in the Gln or Arg (or ADP ribosylation by cholera toxin) blocks the ability to stabilize transition state, and therefore locks G protein in the “on” position.

•  Examples include adenomas of pituitary and thyroid glands (GH secreting tumors, acromegaly), and McCune-Albright syndrome.

Iiri, et al. NEJM (1999)

Canonical Gs Signaling Pathway For interactive pathways at STKE: Gs pathway http://stke.sciencemag.org/cgi/cm/CMP_6634 Gi pathway http://stke.sciencemag.org/cgi/cm/CMP_7430 Gq pathway http://stke.sciencemag.org/cgi/cm/CMP_6680 G12 pathway http://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 target GPCRs

Bockaert & Pin, EMBO J (1999)

G protein-coupled receptors

•  5 main families •  Conserved 7 TM

architecture

GPCRs in the Human Genome Steve 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 up individual stable cell lines expressing each orphan GPCR. Fractionate peptides, tissue factors, etc. and apply to each cell line. Example: Orexin receptors

Cottage industry approach: expression cloning strategy in Xenopus oocytes. Use sib selection to identify cDNAs that encode 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 Cβ acts on αq

EE*

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

>103/sec

GTP GDPαq GTP

αq αq

eg, γ subunit of cGMP PDE enhances

effect of retinal RGS on αt

New concepts for GPCR signaling Using mainly two-hybrid screening approaches, many proteins have been found to interact with portions of the GPCRs. Non-PDZ scaffolds: AKAPs (A-Kinase Anchoring Proteins, JAK2 (Janus Activated Kinase), homer, β-arrestins PDZ scaffolds: InaD, PSD-95 (Post-Synaptic Density), NHERF (Na/H Exchanger Regulatory Factor).

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

Lefkowitz reviews

Arrestins act as scaffolds for ERK and JNK signaling pathways

Lefkowitz reviews




Discovery of Small G proteins Ras genes first identified in ‘60’s as transforming genes of rat sarcoma viruses. Weinberg, Varmus, Bishop and others in the early ‘80’s showed that many cancer cells have mutated versions of ras. Activated form of ras found in 90% of pancreatic carcinomas, 50% of colon adenocarcinomas, and 20% of malignant 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”

Swi1 Swi2

Ras

Gα

Gαt-GTP vs. Ras-GTP

Swi2

Swi3

Swi1

α-helical domain

Rho/Rac/Cdc42

In early ‘90’s, Alan Hall discovered that newly characterized Ras homologs (Rho, Rac, Cdc42) induced cytoskeletal changes.

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 examples Depends 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

GDP GEF

GEF GDP

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 G proteins revealed that ras and cdc42 can activate rac. Rac, in addition to inducing lamellipodia, also activates Rho.


Rho/Rac/Cdc42 signaling in actin assembly




•  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 as an oncogene in NIH 3T3 focus forming assay. It had an 180 amino acid domain with homology to yeast CDC24. This domain, named DH (Dbl homology) is necessary for GEF activity. In 1991, Dbl shown to catalyze nucleotide exchange on Cdc42.

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

Small G proteins “turn off” mechanisms RhoGAPs outnumber the small G proteins Rho/Rac/Cdc42 by nearly 5-fold. Why so much redundancy? Luo group did RNAi against 17 of the 20 RhoGAPs in fly. Six caused lethality when expressed ubiquitously. Tissue specific expression of RNAi revealed unique phenotypes. P190RhoGAP implicated in axon withdrawal. Increasing amounts of RNAi caused more axon withdrawal (panels C-G). Why so many RhoGAPs?

Billuart, et al. Cell (2001)

Identification of RasGAP

McCormick injected Xenopus oocytes with oncogenic ras (V12) versus wt ras (G12) and monitored germinal vesicle breakdown (GVB) (top panel)

% G

VB

[ras] (ng)

V12

G12

Time (min)

% R

as-G

TP V12

G12

Then loaded ras with α-32P GTP, injected into oocytes, did immppt at increasing times and determined if GTP or GDP was bound (bottom panel)

Purified the factor that promoted GTPase activity, cloned and named it GAP (or ras-GAP). Another ras-GAP later identified is NF1 (the gene mutated in neurofibromatosis, i.e., Elephant Man Syndrome).

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

Rho/Rac/CDC42 activation of downstream 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 Lecture 1 Ken Blumer Dept. of Cell ... · 3’,5’-cyclic AMP Ca2+ Questions: Discovery (separate, re-combine) Specificity Amplification Complexity Signaling machines

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