BIOL 350 Cell Biology Sec3on 1

Natalia Caporale. PhD. ncapo@sfsu.edu

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GENERAL PRINCIPLES

CELL SIGNALING

Why communicate? Cells need to sense their environment and respond to it.

It senses for: Nutrients Foreign substances Tropic signals Hormones Etc

Mating Factor causes yeast to extend and

mate

Lonely Yeast

Why communicate? In mul3cellular organisms this has to be done in a coordinated manner, so cells need to talk to each other.

Cell Signaling

Signal transduction begins when the receptor protein on the target cell receives an incoming extracellular signal and converts it to the intracellular signal that alters cell behavior.

Cells can communicate in various ways

a.k.a. contact-dependent signaling

Cells have fast and slow (usually involving the nucleus) responses to signals

Signaling molecules can bind a receptor in the cell membrane, or if lipophilic, one in the cytoplasm.

Steroid Hormone Cor3sol acts by ac3va3ng a transcrip3on regulator

Gases can also be signaling molecules

Nitric oxide is a local signaling molecule that can

travel through membranes. It is quickly converted to

nitrates and nitrites (5-10 sec half life).

Nitric oxide accounts for the effects of nitroglicerine being taken for angina (chest pains). Nitroglicerine becomes NO and dilates the blood vessels, reducing the effort of the heart.

How does NO cause smooth muscle relaxa3on?

Nitric oxide binds guanylyl cyclase and activates it, leading to PKG activation, which leads to a reduction in Ca concentration and thus muscle relaxation

Viagra In the Penis, the cavernous nerve releases NO to cause vasodilation, leading to penile erection. Viagra (Sildenafil) is an inhibitor of the phosphodiasterase that transforms cGMP to GMP. The increase in cGMP concentration enhances muscle relaxation, facilitating and prolonging erections.

Intracellular proteins can act as molecular switches

Switching by Phosphorilation Dephosphorilation.

Adding or removing phosphate groups.

Intracellular proteins can act as molecular switches

Switching by Phosphorilation Dephosphorilation.

Kinases: Enzymes that add phosphate groups.

Serine/Threonine kinases Phosphorilate proteins on serines and threonines

Tyrosine Kinases Phosphorilate proteins on tyrosines.

Phosphatases: Enzymes that remove phosphate groups.

Intracellular proteins can act as molecular switches

Switching by GTP-binding.

Binding of GDP or GTP results in activation or

inactivation of the protein.

Most of these proteins have GTPase activity.

Intracellular proteins can act as molecular switches

Switching by GTP-binding.

Cell Surface Receptors fall in three basic classes.

The three classes dier in the transduc3on mechanisms. Ion channel-coupled receptors G-protein coupled receptors Enzyme coupled receptors

Common Second Messengers

Common Second Messengers Calcium.

3,5- Cyclic AMP (cAMP) 3,5- Cyclic GMP (cGMP) 1,2 Diacylglycerol (DAG)

Inositol 1,4,5-triphosphate (IP3)

Signal Amplica3on

G-PROTEIN COUPLED RECEPTORS

CELL SIGNALING

G-protein coupled receptors There are over 700 GPCRs in humans. Involved in a wide variety of signaling. They all have similar structure:

Single polypeptide chain. Crosses the membrane 7 times (serpentine receptors)

Similar proteins can be found in bacteria (so evolutionary ancient), though they do not act through G-proteins.

General Organiza3on: A GPCR and a G protein

General Organiza3on: A GPCR and a G protein

Biological Func3ons Mediated by 7TM Receptors

Embryogenesis Taste Carcinogenesis Exocytosis Neurotransmission Smell Hormone secre3on Vision Chemotaxis Development Control of blood pressure Cell growth and dieren3a3on Viral infec3on

G Protein-Coupled Receptors and Their Second Messengers

Signal Transduc3on by G Protein-Coupled Receptors Ligand binding on the extracellular domain changes the intracellular domain.

Anity for G proteins increases, and the receptor binds a G protein intracellularly.

GDP is exchanged for GTP on the G protein, ac3va3ng the G protein.

One ligand-bound receptor can ac3vate many G proteins.

The G-Protein Cycle

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G Protein-Coupled Receptors and Their Second Messengers

Termina3on of the Response Desensi7za7on by blocking ac3ve receptors from turning on addi3onal G proteins.

G protein-coupled receptor kinase (GRK) ac3vates a GPCR via phosphoryla3on.

Proteins called arres7ns compete with G proteins to bind GPCRs.

Termina3on of the response is accelerated by regulators of G protein signaling (RGSs).

Fluorescence Resonance Energy Transfer (FRET)

It is a technique to study the interac3on between two proteins. This can be done to, for example, test if 2 known proteins interact in

vitro and in vivo. Find out WHEN two proteins interact (as this is marked by the

genera3on of a specic uorescence).

It relies on 2 uorescent proteins of dierent absorp3on and emission spectra, with the special design characteris3c that the emission wavelength of the rst one matching the absorp3on wavelength of the second one.

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How to Interpret FRET ndings

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STIMULATE WITH EMISSION MEANING

405/440nm 475nm A and B are NOT interac3ng.

405/440nm 530 nm A and B are likely to interact

It should be noted that if A and B are expressed in high enough amounts, then it is more likely to have false positives (see 530nm by chance) than false negatives (do not see 530nm even though the proteins are interacting).

Measuring Ac3va3on of G-Proteins Through FRET

CFP is added to G. YFP is added to G. When the G-protein is inac3ve, all G-protein subunits are

together in the membrane and interact, which allows for FRET to happen and for the emission of yellow uorescence.

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Inactive G-protein

Measuring Ac3va3on of G-Proteins Through FRET

CFP is added to G. YFP is added to G. When the G-protein is ac3ve, the G-protein subunits separate

and now G will be too far away to be excited and FRET wont occur. We will see emission from CFP (cyan).

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Active G-protein

Measuring Ac3va3on of G-Proteins Through FRET

CFP has been added to G. YFP is added to G. Thus, if we monitor the emission of yellow uorescence, we

have a readout of the 3me at which the G-protein is ac3vated.

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Major Classes of G Proteins And Their Eectors

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G-proteins can ac3vate dierent messenger pathways

G CAMP PATHWAY

G-PROTEIN COUPLED RECEPTORS

cAMP rises rapidly in response to an extracellular signal

Binding of serotonin to neurons results in an increase in cAMP. This can be visualize using a fluorescent dye whose fluorescence increases as the concentration of cAMP increases.

cAMP signaling regulates the ac3va3on of Protein Kinase A (PKA)

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Protein Kinase A is activated when cAMP binds to its regulatory (inhibitory) subunit

Adenylyl cyclase can be ac3vated and inhibited by G-proteins

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ADP - Ribosyla3on

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Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD+ to acceptor proteins.

Cholera Cholera is an infec3on of the small intes3ne that is caused by the bacterium Vibrio cholerae. Symptoms:

watery diarrhea vomi3ng.

Transmission occurs primarily when drinking water or food is contaminated by the diarrhea from an infected person or by the feces of an infected but asymptoma3c person.

Primary treatment is with oral rehydra3on solu3on (ORS) and if this is not tolerated, intravenous uids.

Worldwide it aects 35 million people and causes 100,000130,000 deaths a year as of 2010.

Cholera: Mechanism

Cholera Toxin is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD+ to the subunit of Gs.

This ADP ribosylaEon alters the subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an ac3ve state that s3mulates adenylyl cyclase indenitely.

The resul3ng prolonged eleva3on in cyclic AMP levels within intes3nal epithelial cells causes a large eux of Cl- and water into the gut, thereby causing the severe diarrhea that characterizes cholera.

Cholera: Mechanism

Pertussis (Whooping Cough)

Pertussis toxin (PT) is a protein-based toxin produced by the bacterium Bordetella pertussis, which causes whooping cough.

PT is involved in the coloniza3on of the respiratory tract and the establishment of infec3on

Symptoms are: a paroxysmal cough (sudden attack) inspiratory whoop vomiting after coughing

Pertussis is re-appearing, and an epidemic was being debated in California in 2009

Pertussis: Mechanism

Pertussis toxin made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylaEon of the subunit of Gi.

This prevents the subunit from interac3ng with receptors; as a result, this subunit retains its bound GDP and is unable to regulate its target proteins.

Pertussis Toxin

Ac3va3on of PKA results in several eects:

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Activation of other enzymes through phosphorilation

Activation of transcription factors through phosphorilation

The Subunit can also act on Targets

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GQ PLC PATHWAY

G-PROTEIN COUPLED RECEPTORS

Ac3va3on of GPCR can also lead to ac3va3on of phospholipase C (PLC)

Note: If we run out of calciumin the ER, a protein associated with the IP3 gated calcium channels binds to and opens store-operated Ca2+ channels in the membrane. [8]

Another Target of Calcium: Calmodulin

Binding of Ca2+ to calmodulin induces a conformational change that enables it

to wrap around a target protein

Ca2+/Calmodulin-dependent protein kinases (CaM-Kinases)

simplified

Ca2+ Signaling

Is key for a large number of physiological events: Fer3liza3on Learning and Memory Synchroniza3on of ac3vity Secre3on of hormones and neurotransmioers Muscle contrac3on Etc

GENERAL PRINCIPLES

SIGNALING PATHWAYS CONTROLLING GENE

ACTIVITY

Extracellular Signals can Induce Long Term Responses

Receptors can act be located in the membrane or in the cytosol.

Aect many aspects of cell func3on: Division Dieren3a3on Expression levels of receptors, etc

They act by modula3ng gene transcrip3on Alter chroma3n structure Turn transcrip3on factors on or o

Families of receptor signaling pathways covered in Chapter 16

RECEPTOR TRYOSINE KINASES

SIGNALING PATHWAYS CONTROLLING GENE

ACTIVITY

Enzyme Coupled Receptors

Transmembrane proteins Contain an extracellular ligand

binding domain Usually one transmembrane domain

(so poor chance of ligand-induced conforma3onal changes)

The intracellular domain: Either acts as an enzyme Forms a complex with another protein

that acts as an enzyme.

Can mediate fast or slow responses.

Receptor Protein Kinases

Binding of the ligand induces the dimerization of the receptor subunits. Contact between the intracellular tails result in their activation and cross-phosphorilation.

Receptor Protein Kinases

The phosphorylation of the tyrosine tail results in the assembly of an elaborate intracellular signaling complex. Termination of signal:

Phosphates are removed by protein tyrosine phosphatases in response to extracellular signaling or by endocytosis and degradation.

Receptor Tyrosine Kinases (RTKs) have diverse extracellular structures and ligands

RAS AND MAP KINASE PATHWAYS

SIGNALING PATHWAYS CONTROLLING GENE

ACTIVITY

No3ce: There are many dierent MAP kinase pathways

Ras Proteins

It is a family of small GTP-biding protein.

Has GTPase ac3vity. Bound by a lipid tail to the

cytoplasmic tail of the plasma membrane.

Almost all RTKs ac3vate a Ras protein.

Note: There are several Ras proteins (family)

30% of human cancers contain an activating mutation in Ras genes.

Ras Protein Ac3va3on-Inac3va3on Cycle

Ras ac3va3on by RTKs

In this example, we are looking at epidermal growth factor and its pathway.

Here, GRB2 works as an adapter between a phosphotyrosine and the cytosolic Sos protein.

Ras ac3va3on by RTKs

Now, GRB2-SOS can reach and interact with the inac3ve Ras protein. (Ras-GDP)

Ras ac3va3on by RTKs

The associa3on of SOS to Ras promotes the exchange of GDP for GTP.

Now, Ras-GTP is the ac3ve form of the protein.

Ras GTP has a low anity for SOS so it goes away and starts signaling.

Ras then goes ahead and ac3vates the MAP Kinase Pathway

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MAP Kinase

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MAP Kinase Ac3vates Early-Response Genes

Early response genes are around 100 genes that are called this because they are induced well before the cell enters the S phase.

One of the most important ones are: c-Fos c-Jun

They induce the ac3va3on of a large number of genes that are necessary for the cell to progress through the cell cycle.

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MAP Kinase ac3vates c-fos

In the cytosol, MAP kinase ac3vates kinase p90RSK, which trhen moves into the nucleus and phosphorilates SRF.

Aser transloca3ng to the nucleus, MAP kinase phosphorilates TCF.

SRF and TCF are transcrip3on factors.

SRF and TCF work together to ac3vate the transcrip3on of genes that have the SRE element (such as c-fos) 73

G-protein coupled receptors can also lead to the ac3va3on of MAP kinases

This is an example of a yeast ma3ng pathway that involves MAP Kinases.

You do not need to know this.

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STOPPED HERE

Told them not to focus on the details of MAPK in the nucleus just know it diminerizes and ac3vates genes, among them cfos and cjun

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JAK KINASE AND STATPATHWAYS

SIGNALING PATHWAYS CONTROLLING GENE

ACTIVITY

JAK associated receptors

Receptors do not have enzyma3c ac3vity.

JAK, a protein kinase, associates with the receptors.

Biding of the ligand causes the dimeriza3on of the receptors.

Dimeriza3on results in the ac3va3on and cross-phosphorila3on of JAK.

Usually associated with the transcription factor STATs. Receptors for: cytokines, hormones, etc.

Ac3va3on of STATS

Following ac3va3on of a cytokine receptor, STAT binds to a phosphotyrosine in the receptor.

The JAK then phosphorilates the C-terminal tyrosine in STAT,

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Ac3va3on of JAK associated receptors

Ac3va3on of STATS

Phosphorilated STAT separates from the receptor and spontaneously dimerize.

The STAT dimer moves into the nucleus where it can bind to promoter sequences and ac3vate gene expression.

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PUTTING THINGS TOGETHER

SIGNALING PATHWAYS CONTROLLING GENE

ACTIVITY

There is Ample Convergence, Divergence and Crosstalk Among Dierent Signaling Pathways

Signaling pathways can converge , diverge, and crosstalk as follows: Signals form unrelated receptors can converge to ac3vate a common eector.

Iden3cal signals can diverge to ac3vate a variety of eectors.

Signals can be passed back and forth between pathways as a result of crosstalk.

Signaling pathways are highly interconnected

Convergence, Divergence and Crosstalk Among Dierent Signaling Pathways

Convergence GPCRs, receptor tyrosine kinases, and integrins bind to dierent ligands but they all can lead to a docking site for Gbr2.

Convergence, Divergence and Crosstalk Among Dierent Signaling Pathways

Crosstalk more and more crosstalk is found between signaling pathways: cAMP can block signals transmioed through the MAP kinase cascade.

Ca2+ and cAMP can inuence each others pathways.

Convergence, Divergence and Crosstalk Among Dierent Signaling Pathways

Divergence all of the examples of signal transduc3on so far are evidence of divergence of how a single s3mulus sends signals along a variety of dierent pathways.

Receptors undergo several types of down-regulation to limit the extent of their signaling

This enables a cell to adapt to an ongoing stimulus