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
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
Pathogenic virus
Growth factors
Cell cycle and
DNA repair
Cell suicide
(Apoptose)
Cytokines
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
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 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
Transmission of signals from
one molecule to another
3 basic modes (may be combined)
1. Allostery
Shape change, often induced by binding a protein
or small molecule
Switching can be very rapid
P
2. Covalent
modification
Modification itself changes molecule’s shape
Memory device; may be reversible (or not)
3. Proximity (= regulated recruitment)
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 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
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. Cocrystallization 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
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 32:267.]
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
(A) + (B)
(AB)
k2
At equilibrium, the forward reaction
goes at exactly the same rate as
the backward reaction
So . . .
k1 = association rate
k2 = dissociation rate
Forward reaction rate = (A)(B) k1
Backward reaction rate = (AB) k2
(A)(B) k1 = (AB) k2
Reversibility & Timing
If . . .
(A)(B) k1 = (AB) k2
Define
dissociation
Kd = constant =
So . . .
k2
(A)(B) k2
Kd =
=
(AB)
k1
k1
Equilibrium binding is saturable
Kd = conc of A at which
half of B binds A
(AB)
1.0
Bmax
0.5
Kd
(A)
Reversibility & Timing
Units
Kd =
k2
k1 = association rate constant
(M-1)(sec-1)
k1
k2 = dissociation rate constant
(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
Reversibility & Timing
Kd
LIGAND
k2
Half-life
of (AB)
(M)
(sec-1)
(sec)
Acetylcholine
10-6
102
0.007
Norepinephrine
10-8
100
0.7
Insulin
10 -10
10-2
70
*Half-life = 0.69 ÷ k2
Scatchard Analysis
(Bound Lig)
(Free)
Slope = - 1/Kd
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.
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.
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)]
Slope= n
log Lf
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)
a-adrenergic receptor
100%
b-adrenergic receptor
100%
ISO
PHEN
[competitor]
PHEN
ISO
[competitor]
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* . . .
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
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/fluorescence/tirf/tirfintro.html
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!
True for cells in a ligand gradient and also
in a uniform concentration of the ligand
Pseudopod
k2 = 1.1 and 0.39 s-1
400
Tail
k2 = 0.39 and 0.16 s-1
0
0
5
10
15
20
25
Time (sec)
*Ueda et al., Science 294:864,2001
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 294:864,2001
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 294:864,2001
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
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)
Earl Sutherland
1971 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
Positive cooperativity--binding of
increases affinity for second cAMP
PKA targets include Phosphorylase kinase
and the transcription regulator, cAMP
response element binding (CREB) protein
Alberts 15-31,32
Diacylglycerol and Inositol Phosphates as
second messengers
Alberts, 15-35
Calcium acts as second (third?) messenger
Lodish, 20-39
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
CaM-kinase II regulation
Alberts, 15-41
Frequency of calcium oscillations
influences a cell’s 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.
CaM-kinase II activity
CaM-kinase II activity
Alberts 15-39,42
Low frequency Ca2+ oscillations
High frequency Ca2+ oscillations
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
0 sec 10 sec
Alberts, 15-37
20 sec 40 sec
Sensitivity of
InsP3 R to Ca 2+
Sperm binds
InsP3
[Ca 2+]
NO signaling
Gases can act as second messengers!
NO effects are local, since it has halflife of 5-10 seconds (paracrine).
NO activates guanylate cyclase by
binding heme ring (allosteric
mechanism)
Lodish, 20-42
Discovery of NO signaling
Furchgott, Ignarro, Murad, Nobel Prize 1998
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.
http://www.nobel.se/medicine/laureates/1998/illpres/index.html
Reactive Oxygen Species (ROS) Signaling
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!
Finkel & Holbrook, Nature (2000)
ROS activates multiple pathways
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)
Finkel & Holbrook, Nature (2000)