MCB Signal Transduction Lecture 1 General Concepts of

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 2. Covalent modification P 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 SH 2 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 Ig. G antibodies can bind to Staph A, Staph G, RF, and neonatal Fc. R. 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 (A) + (B) k 1 k 2 At equilibrium, the forward reaction goes at exactly the same rate as the backward reaction So. . . (AB) k 1 = association rate k 2 = dissociation rate Forward reaction rate = (A)(B) k 1 Backward reaction rate = (AB) k 2 (A)(B) k 1 = (AB) k 2

Reversibility & Timing If. . . (A)(B) k 1 = (AB) k 2 Define dissociation Kd = constant = So. . . k 2 (A)(B) k 2 Kd = = k 1 (AB) k 1 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 = k 2 k 1 = association rate constant (M-1)(sec-1) k 1 k 2 = dissociation rate constant (sec-1) k 1 usually ~ 108 M-1 sec-1 (diffusion-limited) k 2 just a time constant (sec-1) Thus, knowing the Kd and assuming a “usual” rate of association, you can calculate. . . k 2, and therefore the duration (or half-life*) of the (AB) complex *Half-life = 0. 69 ÷ k 2

Reversibility & Timing Kd k 2 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 LIGAND *Half-life = 0. 69 ÷ k 2

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 O 2 (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 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 k 1 = “on-rate” Assess k 2 = “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 c. AMP ligand with a fluorescent dye Evanescent wave excites only tagged c. AMP near slide Bound c. AMP stays in one place on cell surface; unbound tagged c. AMP diffuses rapidly away http: //www. olympusmicro. com/primer/techniques/fluorescence/tirfintro. html

Each point is a separate c. AMP/R complex Seeing is believing*. . . Off & On: c. AMP-R complexes (movie: 7 sec total) Cell surface facing the slide True for cells in a ligand gradient and also in a uniform concentration of the ligand Pseudopod k 2 = 1. 1 and 0. 39 s-1 Cy 3 -c. AMP bound c. AMP-R complexes dissociate ~2. 5 x faster at the front than at the back! 400 Tail k 2 = 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 c. AMP/R complex # spots per m 2 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 (k 2) at the front must be matched by faster association (k 1) 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 (Bia. Core) 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: c. AMP, c. GMP 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.

c. AMP regulates PKA activity Positive cooperativity--binding of increases affinity for second c. AMP PKA targets include Phosphorylase kinase and the transcription regulator, c. AMP 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 Ca 2+ 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, Ca. M-kinases Alberts, 15 -40

Ca. M-kinase II regulation Alberts, 15 -41

Frequency of calcium oscillations influences a cell’s response Ca. M-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. Ca. M-kinase II activity Alberts 15 -39, 42 Low frequency Ca 2+ oscillations High frequency Ca 2+ oscillations

Calcium signaling also occurs in waves Calcium effects are local, because it diffuses much more slowly than does Ins. P 3 receptor is both stimulated and inhibited calcium 0 sec 10 sec Alberts, 15 -37 20 sec 40 sec Sensitivity of Ins. P 3 R to Ca 2+ Sperm binds Ins. P 3 [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 1 B Redox sensors Thioredoxin (Trx) binds and inhibits ASK 1, an upstream activator of JNK/p 38 pathways. ROS dissociates Trx-ASK 1 complex HSF 1, NF-k. B, and ERK activities change with age (Pink boxes) Finkel & Holbrook, Nature (2000)