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
Cell cycle and DNA 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 Signaling”
Signals cross the plasma membrane
Cytoplasmic pathways & networks
Signaling to the nucleus
Responses
A B C
PQ
R
ST
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
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
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. 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 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
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
(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 constant Kd =
Bmax
Kd
Reversibility & Timing
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
Reversibility & Timing
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 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 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
Seeing is believing* . . .
*Ueda et al., Science 294:864,2001
Each spot = 1 cAMP/R complex
Spots move ~1-2 µ/sec
# spots per m2 of surface area equal at front and back of the cell (like receptor density)
Seeing is believing* . . .
*Ueda et al., Science 294:864,2001
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).
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)