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Cell Signaling I: Organization of lectures: Sex, and Violence z General Principles of signaling cascades z Signaling z Signaling via G- coupled receptors: cAMP z Signaling via G-protein coupled receptors: IP3 &DAG Joe W. Ramos [email protected] z Signaling via -linked receptors www.crch.org/Profiles/jramos z integration

z

A Metaphor for Signaling: The milkman is the signal. From and the of , Varmus and Weinberg, 1993

Cell signaling touches all aspects of cell What is signaling? biology:

Cell Signaling pathways regulate: A Neutrophil Chasing a Bacterium z the - proliferation z the -migration- z transcription- differentiation z membrane traffic- z cell survival and death- apoptosis Courtesy Peter Devreotes z Development- body plan

www.hopkinsmedicine.org/cellbio/devreotes

Cell signaling is central to modern Forms of Cell Signaling medicine

Cancer , Epidermal Estradiol Growth AIDS Factor, NO Cholera Viagra Angina Delta, FasL Alzheimer’s Whooping cough ….among others

1 Cell matrix may also serve as Same signaling elicits different responses in different cells

(e) -dependent Different - Target Cells complexes can also activate the SAME response:

epinephrin or glucagon can ECM protein () activate glycogen breakdown and release of glucose into the blood.

Cell-to-cell by extracellular Cells depend on multiple signals signaling usually involves six steps

z (1) synthesis of the signaling molecule by the signaling cell

z (2) release of the signaling molecule by the signaling cell

z (3) transport of the signal to the target cell

z (4) detection of the signal by a specific receptor protein

z (5) a change in cellular , , or development triggered by the receptor-signal complex

z (6) removal of the signal, which usually terminates the cellular response

An intracellular signaling cascade

Recall: Produced by

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2 Intracellular signaling proteins that Other signaling modifications act as molecular switches z Acetylation,

z Ubiquitination, Sumoylation

z Proteolysis (caspases)

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

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Fast and slow responses to signals Turning off the signal

15-21 Figure from of the Cell, 4th edn.

Signaling :

1. Steroids (Estradiol, Testosterone)

= derivatives 2. derivatives (Histamine) (steroids)

() (Testis) 3. Gases (NO, CO) (Adrenal ) =tyrosine derivative 4. and Proteins (,PDGF) (as is adrenaline) ( gland) Small hydrophobic molecules: -can diffuse across membrane

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3 Androgen Insensitivity Syndrome hormone cortisol (AIS)

z Some very rare individuals are born as z Cortisol acts on metabolism of proteins, carbohydrates and genetic males but lack the testosterone (androgen) receptor. z Made in z They make the hormone, but their cells z Diffuses across PM cannot respond to it. z In blood causes increased z They therefore develop outwardly as females blood and glucose concentrations. z Female genitalia z Gives a quick burst of energy. z However, have non-descended testis However, have non-descended testis z “the Stress Hormone” secreted z No secondary female characteristics (no during fight or flight responses. !) z Hence, the importance of receptors! 15-10

The superfamily NO is relaxation

-NO diffuses locally, half is 2-30 seconds -Nerve cell releases acetylcholine which binds endothelial cell. -Relaxation of smooth muscle cells around blood vessels causes the All hormone receptors bind as either heterodimers or homodimers-to simplify things it is vessels to expand increasing blood flow. shown only as a monomer here. 15-11

cGMP mediates local signaling by NO: -mediated Viagra and Viagra and nitroglycerin signaling

Acetylcholine is released by the autonomic nerves of the penis.

Local blood vessel dilation, Penile erection. z G-Protein Coupled Receptors

cGMP can regulate , channels, and phosphodiesterases z Enzyme-linked receptors Viagra maintains erection by Nitroglycerin -keeping cGMP levels high relieves Angina -Keeping smooth muscle relaxed

Angina is pain resulting from poor Viagra inhibits phosphodiesterase that blood flow to heart normally lowers cyclic GMP levels -nitroglycerine used for 100 years -is converted to NO

4 Three classes of cell-surface A Neutrophil Chasing a chemoattractant receptor

The neutrophil is guided by GPCR -coupled receptors and their signaling effectors

z Many different mammalian cell-surface receptors are coupled to a trimeric signal-transducing G protein. z Function in: z Light detection z odorant detection z detection of certain hormones and

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

Ligand 7 Pass Transmembrane proteins

1234567 Light green region binds ligand

Orange region binds G-protein G-protein Expressed in Xenopus oocytes

See B. Kobilka et al., 1988, Science 240:1310 15-14

5 Clinical relevance 10 minute Break

z Half of all known drugs work through G- protein coupled receptors z The genome project has revealed vast numbers of new GPCRs (new targets?)

G-proteins disassemble into two The G-Protein signaling proteins when activated

z Tethered to membrane by •G-proteins are composed of three subunits: α, β, γ Tethered to membrane by hydrophobic anchor

z In response to ligand the G-protein is released from the GPCR into two signaling proteins: α subunit and βγ complex

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

The α subunit switches itself off by A Go protein opens a K+ channel in hydrolyzing its bound GTP the heart muscle

The Heart’s contraction is controlled After a few seconds the GTP on by two sets of nerve fibers: one the α subunit is hydrolyzed by speeds and one slows contraction. the intrinsic GTPase activity. To slow, nerves release This inactivates the subunit, acetylcholine. which dissociates from the target Goβγ and re-associates with the βγ Acetylcholine binds a GPCR on heart releasing the G-proteins. complex.

Here the Goβγ complex is the active Both the activated α subunit signaling component-binding to the and the βγ complex can K+ channel and thus opening it: less regulate target proteins. frequent contractions.

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6 G-proteins activate that catalyze G-protein coupled receptors signal the synthesis of messenger molecules by several mechanisms

An example enzyme is z We will examine four GPCR second Adenylate cyclase messengers: z cAMP The intracellular message molecules are called z Inositol triphosphate (IP3) Second Messengers z Diacylglycerol (DAG) z Ca+ The signal is thus greatly amplified. z cGMP is also a second messenger

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cAMP increase activates Synthesis and degradation of cyclic transcription AMP

cAMP activity mediated by G ATP is always present in cells. s cAMP-dependent protein (A kinase or PKA). Adenylate cyclase forms cAMP from ATP by a cyclization reaction that This signaling pathway controls removes two and joins the many processes in cells remaining to the sugar. including hormone synthesis. cAMP phosphodiesterase is continually active and breaks the bond to form AMP.

cGMP is an analogous second messenger.

Earl Sutherland, discovered cAMP 15-22 15-31

Effect of Cholera toxin on Gsα PKA Structure and regulation

Cholera toxin is a hexameric protein produced by : 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

7 The two branches of the inositol CREB links cAMP signals to transcription pathway

PKC

cAPK=PKA

Modification of a common phospholipid precursor C causes an generates several second messengers: increase in intracellular calcium synthesis of DAG and IP3

25 of the known GPCRs activate PLC

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

Cytosolic Ca2+ levels are tightly in Rat brain glial controlled cells

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

8 Ca2+ structure The Activation of Cam Kinase II

Dumbell structure when inactive. Alpha helix “jack-knifes” when Ca2+ bound to surround target protein (CaM Kinase II). CaM Kinase II phosphorylates a wide range of cellular proteins

Two major pathways by which G-protein-linked cell- PLC-mediated responses surface receptors generate small intracellular mediators

Gq Gs

Adenylate cyclase PLC

PKA (cAPK) calmodulin

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

Multiple G proteins transduce signals to Turning off GPCRs: arrestin different proteins

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

9 cAMP-mediated responses

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