MCB- Signal Transduction Lecture 1

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MCB- Signal Transduction Lecture 1
General Concepts of Signal Transduction Cell
Communication Types of Receptors Molecular
Signaling Receptor Binding Scatchard
Analysis Competitive Binding Second Messengers
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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)

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The Integration of Biochemical Networks
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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
Begin genetic analysis by bombarding
functioning radio with small metal objects
misfunctioning radios will display phenotypes
Lazebnik, Cancer Cell 2002
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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)
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Cell Communication
Lodish, 20-1
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• 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
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Four classes of cell-surface receptors
Lodish, 20-3
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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

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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 5-34
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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
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Specific binding of insulin to cells
Receptor ligand binding must be specific,
saturable, and of high affinity

• 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
32267.
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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
k1
k1
association rate
(A) (B)
(AB)
dissociation rate
k2
k2
Forward reaction rate (A)(B)
k1
At equilibrium, the forward reaction goes at
exactly the same rate as the backward reaction
k2
Backward reaction rate (AB)
So . . .
(A)(B) (AB)
k2
k1
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Reversibility Timing
If . . .
(A)(B) (AB)
k2
k1
Define
So . . .
(A)(B)
dissociation constant

Kd
Kd

(AB)
Equilibrium binding is saturable
1.0
Bmax
Kd conc of A at which half of B binds
A
(AB)
0.5
Kd
(A)
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Reversibility Timing
Units
(M-1)(sec-1)
k1
association rate constant
Kd
dissociation rate constant
k2
(sec-1)
k1
usually 108M-1 sec-1 (diffusion-limited)
k2
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
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Reversibility Timing
Half-life of (AB)
k2
Kd
LIGAND
(sec)
(M)
(sec-1)
Acetylcholine
102
0.007
10-6
Norepinephrine
100
0.7
10-8
Insulin
10-2
70
10 -10
Half-life 0.69 k2
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Scatchard Analysis
(Bound Lig)
Slope - 1/Kd
(Free)
X intercept rec
(Bound Lig)
For an excellent discussion of principles of
receptor binding, and practical considerations,
see http//www.graphpad.com also posted on MCB
website.
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Scatchard Analysis
Positive cooperativity binding of ligand to
first subunit increases Affinity of subsequent
binding events. Example hemoglobin binding O2
(Bound Lig)
(Free)
(Bound Lig)
Negative cooperativity binding of ligand to
first subunit decreases affinity of subsequent
binding events.
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Cooperative binding
The Hill equation accounts for the possibility
that not all receptor sites are independent, and
states that
Fractional occupancy Lfn/ (Kd Lfn)
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
If slope 1, then single class of binding sites
log B/(Rt - B)
If slope gt 1, then positive cooperativity
Slope n
If slope lt 1, then negative cooperativity
log Lf
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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)
a-adrenergic receptor
b-adrenergic receptor
100
100
ISO
PHEN
ISO
PHEN
competitor
competitor
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So thats 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
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Seeing is believing . . .
Experimental system Dictyostelium
discoideum, a soil amoeba
Question Does GPCR signaling differ at front vs.
back of the cell?
Assess duration of ligand-GPCR complexes, during
chemotaxis of living Dictyostelium cells
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Seeing is believing, Total Internal Reflection
Fluorescence
Question Does GPCR signaling differ at front vs.
back of the cell?
Approach Tag cAMP ligand with a fluorescent dye
Evanescent wave excites only tagged cAMP near
slide
Bound cAMP stays in one place on cell surface
unbound tagged cAMP diffuses rapidly away
http//www.olympusmicro.com/primer/techniques/fluo
rescence/tirf/tirfintro.html
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Each point is a separate cAMP/R
complex
Seeing is believing . . .
Off On cAMP-R complexes (movie 7 sec
total)
Cell surface facing the slide
cAMP-R complexes dissociate 2.5 x faster at
the front than at the back!
Pseudopod
k2 1.1 and 0.39 s-1
400
Tail
Cy3-cAMP bound
k2 0.39 and 0.16 s-1
True for cells in a ligand gradient and also in
a uniform concentration of the ligand
0
0
5
10
20
15
25
Time (sec)
Ueda et al., Science 294864,2001
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Seeing is believing . . .
Each spot 1 cAMP/R complex
spots per m2 of surface area equal at
front and back of the cell (like
receptor density)
Spots move 1-2 m/sec
Ueda et al., Science 294864,2001
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Seeing is believing . . .
Inferences
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
The functional difference is not created by the
gradient, but instead reflects some difference
between the front and back of the cell
Questions
What biochemical mechanism underlies this
difference?
(Probably reflects residence of the GPCRs and
G proteins in different macromolecular complexes)
Ueda et al., Science 294864,2001
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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

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Second messengers
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).

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

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Earl Sutherland
1971 Nobel laureate
Rall, et al. JBC 1956
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Fischer Krebs, Nobel 1992
Discovered that phosphorylase activity was
regulated by the reversible step of
phosphorylation. Identified PKA and some of the
first phosphatases.
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cAMP regulates PKA activity
PKA targets include Phosphorylase kinase and the
transcription regulator, cAMP response element
binding (CREB) protein
Alberts 15-31,32
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Diacylglycerol and Inositol Phosphates as second
messengers
Alberts, 15-35
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Calcium acts as second (third?) messenger
Lodish, 20-39
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Calmodulin transduces cytosolic Ca2 signal
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
Alberts, 15-40
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CaM-kinase II regulation
Alberts, 15-41
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Frequency of calcium oscillations influences a
cells response
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
CaM-kinase II activity
CaM-kinase II activity
High frequency Ca2 oscillations
Low frequency Ca2 oscillations
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Calcium signaling also occurs in waves
Calcium effects are local, because it diffuses
much more slowly than does InsP3
InsP3 receptor is both stimulated and inhibited
calcium
Sperm binds
Sensitivity of InsP3 R to Ca 2
0 sec
10 sec
20 sec
40 sec
InsP3
Ca 2
Alberts, 15-37
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NO signaling
Gases can act as second messengers!
NO effects are local, since it has half-life of
5-10 seconds (paracrine). NO activates guanylate
cyclase by binding heme ring (allosteric
mechanism)
Lodish, 20-42
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Discovery of NO signaling
Furchgott, Ignarro, Murad, Nobel Prize 1998
Robert F Furchgott showed that acetylcholine-induc
ed 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.
http//www.nobel.se/medicine/laureates/1998/illpre
s/index.html
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Reactive Oxygen Species (ROS) Signaling
ROS important in cells adaptation to
stress Many of longevity mutations map to ROS
pathways Mutations in Superoxide Dismutase (SOD)
cause amyotrophic lateral sclerosis (ALS, Lou
Gehrigs Disease) Unfortunately, no great
clinical data showing that anti-oxidants will
help us live longer!
Finkel Holbrook, Nature (2000)
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ROS activates multiple pathways
Activation mechanisms ???? Mimic ligand effect
for GF receptors Oxidants enhance phosphorylation
of RTKs and augment ERK/Akt signaling Inactivatio
n 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)
Finkel Holbrook, Nature (2000)