Cell Signaling I:Sex, Drugs and

Violence

A Metaphor for Cell Signaling: The milkman is the signal.

JoeW.Ramos,PhDjoeramos@hawaii.edu

http://www2.hawaii.edu/~joeramos/

From Genes and the Biology of Cancer, Varmus and Weinberg, 1993

Organization of lectures:

l General Principles of signaling cascadesl Relevance to Diseasel Hormone Signalingl Signaling via G-protein coupled receptors: cAMPl Signaling via G-protein coupled receptors: IP3 &DAG

l Signaling via enzyme-linked receptorsl Signal integration

l Apoptosis and Autophagy

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What is signaling?

Courtesy Peter Devreotes

www.hopkinsmedicine.org/cellbio/devreotes

A Neutrophil Chasing a Bacterium

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One event-Multiple Signals

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Cell signaling touches all aspects of cell biology:

Cell Signaling pathways regulate: l the cell cycle- proliferationl the cytoskeleton- migrationl transcription- differentiationl membrane traffic- exocytosisl cell survival and death- apoptosisl Development- body plan

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Cell signaling is central to modern medicine

Viagra

SteroidsCancer Diabetes

Alzheimer�s

AIDS

Angina

Cholera

Whooping cough….among others

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Forms of Cell Signaling

Adrenaline, Estradiol

Acetylcholine

Epidermal Growth Factor, NO

Delta, FasL

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Cell matrix proteins may also serve as signals

(e) Extracellular matrix-dependent

Target Cells

ECM protein (fibronectin)

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Same signaling molecule elicits different responses in different cells

Different Receptor-ligand complexes can also activate the SAMEresponse:

epinephrin or glucagon can activate glycogen breakdown and release of glucose into the blood.

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Cells depend on multiple signals

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Cell-to-cell communication by extracellular signaling usually involves six steps

l SYNTHESIS of the signaling molecule by the signaling cell

l RELEASE of the signaling molecule by the signaling cell

l TRANSPORT of the signal to the target cell

l DETECTION of the signal by a specific receptor protein

l RESPONSE: a change in cellular metabolism, function, or development triggered by the receptor-signal complex

l REMOVAL of the signal, which usually terminates the cellular response

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An intracellular signaling cascade

15-7 12

Phosphorylation

Recall: Produced by cellular respiration

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Intracellular signaling proteins that act as molecular switches

15-1714

Other signaling modifications

l Acetylation, methylation

l Ubiquitination, Sumoylation

l Proteolysis (caspases)

l Second messenger Binding (cAMP, cGMP, DAG, IP3, Ca2+)

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Fast and slow responses to signals

15-2116

Figure from Molecular Biology of the Cell, 4th edn.

Turning off the signal

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Signaling Molecules:

1. Steroids (Estradiol, Testosterone)

2. Amino acid derivatives (Histamine)

3. Gases (NO, CO)

4. Peptides and Proteins (Insulin,PDGF)

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Hormones

15-9

Small hydrophobic molecules:-can diffuse across membrane

(Adrenal gland)(Ovary) (Testis)

(Thyroid gland)

=cholesterol derivatives(steroids)

=tyrosine derivative(as is adrenaline)

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Steroid hormone cortisol

l Cortisol acts on metabolism of proteins, carbohydrates and lipids

l Made in Adrenal Glandl Diffuses across PM

l In blood causes increased blood pressure and glucose concentrations.

l Gives a quick burst of energy.l �the Stress Hormone�

secreted during fight or flight responses.

15-1020

The nuclear receptor superfamily

All hormone receptors bind as either heterodimers or homodimers-to simplify things it is shown only as a monomer here.

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Androgen Insensitivity Syndrome (AIS)

l Some very rare individuals are born as genetic males but lack the testosterone (androgen) receptor.

l They make the hormone, but their cells cannot respond to it.

l They therefore develop outwardly as femalesl Female genitalial However, have non-descended testisl No secondary female characteristics (no

estrogen!)l Hence, the importance of receptors! 22

NO is smooth muscle relaxation

15-11

-NO diffuses locally, half life is 2-30 seconds-Nerve cell releases acetylcholine which binds endothelial cell.-Relaxation of smooth muscle cells around blood vessels causes the vessels to expand increasing blood flow.

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cGMP mediates local signaling by NO: Viagra and nitroglycerin

Viagra inhibits phosphodiesterase that normally lowers cyclic GMP levels

Nitroglycerin relieves Angina

Acetylcholine is released by the autonomic nerves of the penis.

Local blood vessel dilation, Penile erection.

cGMP can regulate kinases, ion channels, and phosphodiesterases

Angina is pain resulting from poor blood flow to heart-nitroglycerine used for 100 years-is converted to NO

Viagra maintains erection by -keeping cGMP levels high-Keeping smooth muscle relaxed24

Viagra and NO?

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Something’s Gotta Give

Cell surface receptor-mediated signaling

l G-Protein Coupled Receptors

l Enzyme-linked receptors

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Three classes of cell-surface receptor

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A Neutrophil Chasing a chemoattractant

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The neutrophil is guided by GPCR signaling

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G protein-coupled receptors and their effectors

l Many different mammalian cell-surface receptors are coupled to a trimeric signal-transducing G protein.

l Function in:l Light detection l odorant detection l detection of certain hormones and neurotransmitters

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One event-Multiple Signals

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Clinical relevance

l Half of all known drugs work through G-protein coupled receptors

l The genome project has revealed vast numbers of new GPCRs (new drug targets?)

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G-protein-linked receptor

15-14

7 Pass Transmembrane proteins

Orange region binds G-protein

Light green region binds ligand

1 2 3 4 5 6 7

G-protein

Ligand

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Demonstration of functional domains in G protein-coupled receptors: chimeric proteins

Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G. & Lefkowitz, R. J. Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science 240, 1310-1316 (1988)

Expressed in Xenopus oocytes

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GPCR Structure changes upon Ligand Binding

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Ligated GPCR Structure

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The G-Protein

G-proteins are composed of three subunits: a, b, g

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G-proteins disassemble into two signaling proteins when activated

l Tethered to membrane by hydrophobic lipid anchor

l In response to ligand the G-protein is released from the GPCR into two signaling proteins: asubunit and bg complex

l 20 known G-proteins in mammals (different targets)

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The a subunit switches itself off by hydrolyzing its bound GTP

After a few seconds the GTP on the a subunit is hydrolyzed by the intrinsic GTPase activity.

This inactivates the subunit, which dissociates from the target and re-associates with the bgcomplex.

Both the activated a subunitand the bg complex can regulate target proteins.

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A Go protein opens a K+ channel in the heart muscle

15-17

The Heart�s contraction is controlled by two sets of nerve fibers: one speeds and one slows contraction.

To slow, nerves release acetylcholine.

Acetylcholine binds a GPCR on heart releasing the G-proteins.

Here the Gobg complex is the active signaling component-binding to the K+ channel and thus opening it: less frequent contractions.

Gobg

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G-proteins activate enzymes that catalyze the synthesis of messenger molecules

15-18

The intracellular message molecules are called Second Messengers

An example enzyme is Adenylate cyclase

The signal is thus greatly amplified.

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G-protein coupled receptors signal by several mechanisms

l We will examine four GPCR second messengers:l cAMPl Inositol triphosphate (IP3)l Diacylglycerol (DAG)l Ca+

l cGMP is also a second messenger

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cAMP increase activates gene transcription

15-22Earl Sutherland, discovered cAMP

This signaling pathway controls many processes in cells including hormone synthesis.

cAMP activity mediated by cAMP-dependent protein kinase (A kinase or PKA).

Gs

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Synthesis and degradation of cyclic AMP

15-31

Adenylate cyclase forms cAMP from ATP by a cyclization reaction that removes two phosphates and joins the remaining phosphate to the sugar.

cAMP phosphodiesterase is continually active and breaks the bond to form AMP.

ATP is always present in cells.

cGMP is an analogous second messenger.

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Effect of Cholera toxin on Gsα

Cholera toxin is a hexameric protein produced by bacteria:Vibrio cholerae which causes Cholera.

cAMP levels 100 fold higher:In epithelial cells this permits massive flow of water from blood into intestines

Important tool in early studies of Gs proteins.NAD+=nicotinamide adenine dinucleotide

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PKA Structure and regulation

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CREB links cAMP signals to transcription

cAPK=PKA48

The two branches of the inositol phospholipid pathway

PKC

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Modification of a common phospholipid precursor generates several second messengers:

synthesis of DAG and IP3

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Phospholipase C causes an increase in intracellular calcium

25 of the known GPCRs activate PLC

2 messengers:-DAG=PKC-IP3=Calcium release

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Cytosolic Ca2+ levels are tightly controlled

Ca2+ is actively pumped to the cell exterior and into the ER and mitochondrion.

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Calcium signaling in Rat brain glial cells

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Ca2+ calmodulin structure

Dumbell structure when inactive.Alpha helix �jack-knifes� when Ca2+ bound to surround target protein (CaM Kinase II).

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The Activation of Cam Kinase II

CaM Kinase II phosphorylates a wide range of cellular proteins 55

PLC-mediated responses

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Two major pathways by which G-protein-linked cell-surface receptors generate small intracellular

mediators

Figure from Molecular Biology of the Cell, 3rd edn.

PKA (cAPK) calmodulin

PLCAdenylate cyclase

GqGs

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Turning off GPCRs: arrestin

Figure from Molecular Biology of the Cell, 4th edn.58

Multiple G proteins transduce signals to different effector proteins

Figure from Molecular Biology of the Cell, 4th edn.59

cAMP-mediated responses

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