Lecture 3 1 CELL SIGNALING - PowerPoint Presentation

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Lecture 3

1

CELL SIGNALING

AND

MOTILITY (BIOL 3373)

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WHAT IS The CELL SIGNALING?

2

How cells receive and respond to signals from their surroundings

On one hand, cell signaling regulates gene expression and controls the cell fate (proliferation, motility, differentiation and programmed cell death, or apoptosis).

On the other hand, cell signaling allows for the organization of cells into tissues, which, in turn, generate organs. In addition, cell signaling is essential for the maintenance of cells, tissues and organs

.

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Communication among cells is referred as

intercellular signaling.

Cells communicate with each other through

signaling molecules

.

Signaling molecules could be:

proteins, small peptides, amino acids,

nucleotides,

steroids, retinoids, fatty acid derivatives, nitric oxide, carbon monoxide

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CELL SIGNALING

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The 

androgens are the male sex hormones, responsible for the development and maintenance of reproductive function in the male. The principal androgen

, testosterone, is produced by the testes. Estrogens are female sex hormones. They are secreted mainly by the ovaries.Estradiol

is the most potent of the estrogens. the estrogens promote the development of the primary and secondary female sex characteristics.

Progestins

, the most important of which is 

progesterone

, are the other type of female sex hormone and are named for their role in maintaining pregnancy (pro-gestation).Forms of signaling molecules : Steroid hormonesSteroid hormones are a group of hormones that belong to the class of chemical compounds known as steroids. All steroid hormones are derived from cholesterol. They are transported through the bloodstream to the cells of various target organs where they carry out the regulation of a wide range of physiological functions. These hormones often are classified according to the organs that synthesize them.The adrenal cortex produces the adrenocortical hormones, which consist of the glucocorticoids and the mineralocorticoids. Glucocorticoids such as cortisol control many metabolic processes, including the formation of glucose and the deposition of glycogen in the liver. Mineralocorticoids such as aldosterone help maintain the balance between water and salts in the body, predominantly exerting their effects within the kidney.

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Forms of signaling molecules- Gas - NO

Nitric oxide (NO)

is a gas molecule produced and released by endothelial cells (

signaling cells

)

and rapidly diffuses across the membranes.

NO binds to an enzyme inside the smooth muscle cells (target cell), inducing cell relaxation

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Neurotransmitters are chemicals produced and released by neurons.Neurotransmitters travel across the synapse and allow communication between neurons.

Forms of signaling molecules

Neurotransmitters

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Forms of signaling moleculesNeurotransmitters

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Forms of signaling molecules

Peptide Hormones and Growth Factors

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Cells that produce and release the signaling molecules are

signaling cells.

Cells that receive the signal are target cells

Targets cells posses specific

receptors

that recognize signaling molecules.

signaling cell

target cell

signaling molecules

receptor

CELL SIGNALING

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Depending on the distance that the signaling molecule has to travel, we can talk about four types of signaling:

Contact- dependent signaling

requires cells to be in direct membrane-membrane contact.

The signaling molecule remains bound to the surface of the signaling cells. It influences only cells (target cells) contact it via a specific protein receptor.

Contact-dependent signaling is especially important during development and immune response

from Alberts et al.,

Molecular Biology of the Cell.

6

th

edition.

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CELL SIGNALING

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In paracrine signaling

the signaling molecules are local mediators and affects only target cells in the proximity of the signaling cell. An example is the conduction of an

electric signal from one nerve cell to another or to a muscle cell. In this case the signaling molecule is a neurotransmitter.

from Alberts et al.,

Molecular Biology of the Cell.

6

th

edition.

CELL SIGNALING

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autocrine signaling

signaling cell = target cell

In

autocrine signaling

cells respond to molecules they produce themselves. So signaling cells are also target cells.

Examples include cancer cells as they can produce signals that stimulate their own survival and proliferation.

CELL SIGNALING

from Alberts et al.,

Molecular Biology of the Cell.

6

th

edition.

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Large multicellular organisms use long- range signaling that coordinate the behavior of the cells in remote part of the body.

Synaptic signaling

is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which can be located far away from the neuron cell body.

from Alberts et al.,

Molecular Biology of the Cell.

6

th

edition.

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CELL SIGNALING

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In endocrine signaling

signaling molecules

(hormones)

are produce by endocrine cells and sent through the blood stream to distant cells

CELL SIGNALING

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The Three Stages of Cell Signaling

Earl W. Sutherland discovered how the hormone epinephrine acts on cells

Sutherland suggested that cells receiving signals went through three processes:

Reception

Transduction

Response

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Overview of cell signaling

EXTRACELLULAR

FLUID

Receptor

Signal

molecule

Relay molecules in a signal transduction pathway

Plasma membrane

CYTOPLASM

Activation

of cellular

response

Reception

1

Transduction

2

Response

3

Cell signaling consists of 3 stages

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Reception

1

EXTRACELLULAR

FLUID

Receptor

Signaling

molecule

Plasma membrane

CYTOPLASM

1

Three Stages of Cell Signaling:

1 Reception

Signaling molecule binds to the receptor protein at the cell surface

The receptor and signaling molecules fit together (lock and key model)

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1- Reception:

A signal molecule binds to a receptor protein, causing it to change shape

The binding between a signal molecule (

ligand

) and receptor is highly specific

A conformational change in a receptor is often the initial transduction of the signal

Most signal receptors are plasma membrane proteins

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Receptors in the Plasma Membrane

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Intracellular Receptors

Some

receptor

proteins are

intracellular

, found in the cytosol or nucleus of target cells

Small or hydrophobic chemical messengers can readily cross the membrane and activate receptors

Examples of hydrophobic messengers are the steroid and thyroid hormones of animals

An activated hormone-receptor complex can act as a transcription factor, turning on specific genes

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Hydrophobic signaling molecules can cross the plasma membrane and bind to nuclear receptors

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EXTRACELLULAR

FLUID

Plasma

membrane

The steroid

hormone testosterone

passes through the

plasma membrane.

Testosterone binds

to a receptor protein

in the cytoplasm,

activating it.

The hormone-

receptor complex

enters the nucleus

and binds to specific

genes.

The bound protein

stimulates the

transcription of

the gene into mRNA.

The mRNA is

translated into a

specific protein.

CYTOPLASM

NUCLEUS

DNA

Hormone

(testosterone)

Receptor

protein

Hormone-

receptor

complex

mRNA

New protein

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The nuclear receptor superfamily

Nuclear receptors are inactive without signaling molecules (

ligands

)

When the nuclear receptor interacts with the specific ligand, it becomes

activate

and induces the

transcription of target genes

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Reception

1

EXTRACELLULAR

FLUID

Receptor

Signaling

molecule

Plasma membrane

CYTOPLASM

1

Relay molecules in a signal transduction pathway

Transduction

2

The signal from the receptor is converted into a intracellular message that produces a cellular response.

2

nd

Messenger!

Three Stages of Cell Signaling:

2 Transduction

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SIGNAL TRANSDUCTION:

The study of the molecular circuits responsible for the generation of a cellular response after the delivery of a signal.

Includes a

NETWORK

of molecular and cellular events

conveying a

SIGNAL

from the outside to the inside of the cell.

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SIGNAL TRANSDUCTION

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Signaling molecules (

ligands

) bind to

receptors

on target cells.

After the binding with the ligand, the receptor activates one or more intracellular signaling within the target cells, that involves several proteins ( transducer Protein).The intracellular signaling modulates the activity of target proteins

(also known as effector proteins

) thereby the behavior of the cells.

from Alberts et al.,

Molecular Biology of the Cell.

6

th

edition.

Slide27

EXTRACELLULAR

FLUID

Plasma membrane

CYTOPLASM

Receptor

Signaling

molecule

Relay molecules in a signal transduction pathway

Activation

of cellular

response

Reception

Transduction

Response

1

2

3

Cellular responses can be different and complex (i.e. change in gene expression, cell motility, cell growth, cell differentiation, cell death), depending on cell types.

Three Stages of Cell Signaling:

3 Response

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Cytoplasmic and Nuclear Responses

The response may occur in the cytoplasm or in the nucleus

Many signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus

The final activated molecule in the signaling pathway may function as a transcription factor

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Many other signaling pathways regulate the

synthesis

of enzymes or other proteins, usually by turning genes on or off in the nucleus

The final activated molecule may function as a transcription factor

Cytoplasmic and Nuclear Responses

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Fine-Tuning of the Response

There are four aspects of fine-tuning to consider

signal Amplification

(and thus the response)

Specificity of the response,

Efficiency of response,

Initiation of the signal,

Termination of the signal,

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Signal Amplification

At each step of many signal transduction pathways, the number of activated participants in the pathway increases. This is referred as

signal amplification

,

For example, one epinephrine-activated GPCR activates 100s of G

as

-GTP complexes, which in turn activate 100s of adenylyl cyclase molecules, that each produce hundreds of cAMP molecules, and so on. The overall amplification associated with epinephrine signaling is estimated to be ~10

8

-fold (figure 1)31figure 1

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Specificity of the response

Different kinds of cells have different collections of proteins

These differences in proteins give each kind of cell specificity in detecting and responding to signals

The response of a cell to a signal depends on the cell’s particular collection of proteins

Pathway branching and “cross-talk” further help the cell coordinate incoming signals

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Signal

molecule

Receptor

Relay

molecules

Response 1

Response 2

Response 3

Cell B. Pathway branches,

leading to two responses

Cell A. Pathway leads

to a single response

Cell C. Cross-talk occurs

between two pathways

Response 4

Response 5

Activation

or inhibition

Cell D. Different receptor

leads to a different response

Specificity of the response

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The

same signal can induce different responses

in distinct cell types

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Efficiency of response depends on:

The expression of specific

receptors;

The bioavailability of transducer

molecules:

Expression levels/half-life

Localization within the cell

Modality of activation/inactivation

The bioavailability of effector molecules: cytoskeletal elements (morphological changes) transcription factors (changes in gene expression) proteolytic enzymes (cell death)35

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INITIATION of Signal: RECEPTOR ACTIVATION/

InACTIVATION

For EACH

signaling pathway there is a common feature that defines a sequence of events:The receptor is in an INACTIVE STATE

in the absence if the signal

When the signal arrives, it

BINDS

to the receptor, which

MODIFIES its conformation This change in the receptor ACTIVATES other molecules (i.e. a downstream signaling cascade)36

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Termination of the Signal:

Inactivation mechanisms are an essential aspect of cell signaling

When

signal molecules leave the receptor

, the receptor reverts to its

inactive state.

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THE SIGNALING PATHWAY

VIA G-PROTEIN38

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G-protein coupled receptors

G- protein coupled receptors consists of 3 components:

trans-membrane receptors (GPCRs)

known also as serpentine receptors.

2. G proteins:

Guanine nucleotide-binding proteins, trimeric G protein.

3. Effectors:

Effectors can be different :

adenylyl cyclase or Phosphodiesterase (PDE) or phospholipase C-β (PLC- β) or ion Channel.39Receptor

G protein

Effector

adenylyl cyclase

PLC-

β

PDE

ion Channel

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These

effectors in turn regulate the intracellular concentrations of

secondary messengers, such as cAMP, cGMP

,

diacylglycerol (DG), IP3

,

sodium (Na

+), potassium (K+) or calcium cations (Ca2+), which ultimately lead to a physiological response, usually via the downstream regulation of gene transcription.adenylyl cyclase

PLC- β

PDE

ion Channel

cAMP

cGMP

DG

IP3

Na

+

,

Ca2+

K+

Second messengers

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Several types of receptors are associated with G proteins, such as hormones, neurotransmitters, growth factors, glycoproteins, cytokines, odorants and photons

G-protein coupled receptors

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G-protein-coupled receptors

Transmembrane

helix

C

-Terminal chain

G-Protein

binding region

Variable

intracellular loop

Extracellular

loops

Intracellular loops

N

-Terminal chain

HO

2

C

NH

2

VII

VI

V

IV

III

II

I

Membrane

Single protein composed of

7

transmembrane

domains

with an extracellular amino terminus and an intracellular carboxyl terminus.

Intracellular C terminus

changes conformation in response to a stimulus and this results in changes in the ability to recruit

G proteins.

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G proteins are composed by 3 subunits, α,β and γ

.

G-protein

The α subunit contains the GDP binding site as well as the GTP hydrolysis domain.

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cAMP

ATP

Second

messenger

First messenger

(signal molecule

such as epinephrine)

G-protein-linked

receptor

G protein

Adenylyl

cyclase

Protein

kinase A

Cellular responses

GTP

Adenylyl cyclase

is a

multipass

transmembrane protein that converts ATP to form cyclic AMP (cAMP)

.

ADENYLYL CYCLASE : EFFECTOR OF GPCR

44

Slide45

ATP

Cyclic AMP

AMP

Adenylyl cyclase

Pyrophosphate

P

P

i

Phosphodiesterase

H

2

O

Cyclic AMP (cAMP)

The

Adenylyl Cyclase

catalyzes the reaction that leads to the production of cAMP.

cAMP is synthesized from ATP through a cyclization reaction that removes two phosphate group as pyrophosphate.

cAMP is an unstable molecule because it is soon hydrolyzed to AMP by specific Phosphodiesterases.

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ß

a

g

GDP

GTP

ß

a

g

Ligand

Receptor

G

Protein

Cell membrane

ß

a

g

Binding site for G-protein opens

Activation of G-protein

The binding of the

ligand

to the

receptor

changes the receptor conformation.

The

new receptor conformation

allows the binding of the

G protein

.

When the

G protein assembles to the receptor

it alters its own conformation, therefore the GDP binding site within the α subunit is distorted and GDP is released.

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1

2

3

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ß

a

g

GTP binds

Fragmentation

and release

ß

a

g

ß

a

g

GTP

4. The binding of GTP

to the

α subunit

promotes the closure of the nucleotide binding site within the α subunit.

5.

Therefore the

α subunit changes its conformation

and

6.

separates from both

the β-γ dimer

and

the receptor

.

This process is active while the ligand is bound to the receptor

One ligand can activate several G protein:

Signal amplification

Activation of G-protein

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α

subunit

changes conformation

4

6

5

Slide48

a

Subunit recombines with

b-g

dimerto reform inactive G protein.

Active site

(closed)

Binding site

for

a

subunit

cyclic AMP

ATP

Binding

Active site

(open)

P

cyclic AMP

ATP

GTP hydrolysed

to GDP catalysed

by

a

subunit

a

s

-subunit

Adenylyl

cyclase

GTP

GDP

Active site

(closed)

Signal

transduction

(con)

7.

The α-GTP subunit interacts with the

Adenylyl cyclase

8. Adenylyl cyclase

becomes active and

converts ATP in

cAMP

.

9.

The cycle is completed when GTP is hydrolyzed to GDP within the α subunits. This causes the re-association of α subunit with β-γ dimer and the binding of G-protein to the receptor, which terminates the cycle.

Activation of G-protein

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7

8

9

Slide49

The

target proteins of cAMP vary depending on cell types, however the best characterized cAMP target is the PROTEIN KINASE A (PKA). PKA phosphorylates serine or threonine residues on target proteins thereby regulating their activity. Among the target proteins of PKA the phosphodiesterases are responsible to lower cAMP concentration thus keeping PKA activation short and localized.

cyclic AMP

ATP

Adenylate

cyclase

Enzyme

(active)

P

Enzyme

(inactive)

PKA

Activation

Activation of protein Kinase A (PKA)

P

Target

Protein

49

Slide50

Examples of a signaling pathway mediated by

cAMP

-PKA include the activation of transcription regulator,

the CRE binding protein (CREB).

PKA phosphorylates CREB on a single serine.

Active phosphorylated CREB (

p-CREB

) recruits the coactivator CREB-binding protein (

CBP) (not known) to the regulatory sequence (CRE) present in many genes (i.e. somatostatin gene) that are activated by cAMP.CREB signaling plays an important role in the learning and memory process in the brain.Target Proteins of protein Kinase A (PKA)50

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Activation of protein Kinase A (PKA)

PKA is a complex protein consisting of 2 regulatory subunits and two catalytic domains.

The regulatory domains bind to the cytoskeleton or membrane organelle, thereby tethering PKA in specific subcellular compartment.

Four cAMP molecules bind to the PKA regulatory domains, causing their dissociation from the protein complex and the consequent activation of the catalytic subunits.

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https://youtu.be/0nA2xhNiAow

Double Click on box below for video

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Smell depends on GPCRs that regulate cyclic-nucleotide-gated ion channels

Olfactory receptor neurons

Olfactory receptor neurons line on the nose.

These cells use GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the modified cilia that extend from each cell.

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Sensory transduction in olfaction

Odorant molecules bind to olfactory receptors activating a G protein, which in turn activates adenylyl cyclase . Second-messenger systems are activated, Na+ (sodium) and Ca2+ (calcium) or Ca2+ channels are opened, and the cilia are depolarized. This depolarization

iniziates

a nerve

implulse

thart

travels alomg its axon to the brain

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Vision depends on GPCRs that regulate cyclic-nucleotide-gated ion channels

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GMP

Light

Na+

cGMP gated ion channel

Rod cells

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Photo- transduction in photoreceptor cells

In the retina the rod cells contains a pigment protein,

rhodopsin

,

which is a GPCRs

associated with the G protein,

Transducin

.

Signal =

light

G-protein coupled receptor=

Rhodopsin

(Rods)

G protein =

transducin

enzymatic activity =

phosphodiesterase (PDE)

second messenger =

DECREASE in GMP

Na+ channel

closure

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In dark condition

the rhodopsin are inactive cGMP-gated ion channels are opened the rod cell are depolarized high release of neurotransmitters

In light condition

rhodopsin absorbs light and activates transducing

cGMP decrease cGMP-gated ion channels become closed reduced the release of neurotransmitters.

Photo- transduction in photoreceptor cells

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Amplification of Photo- transduction

one rhodopsin

activates

500s of G

-GTP

complexes, which in turn activate

500s of GMP phosphodiesterase , that each produce 105 of cAMP molecules, and so on. The overall amplification associated with rhodopsin signaling result in the alteration of membrane potential of 1 mV

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Stimulatory G-protein and Inhibitory G protein

Stimulatory G proteins (

Gs

) increase the cAMP concentration and activate PKA.

However several extracellular signals activate a different class of G proteins known as

Inhibitory G proteins (

G

i

). Inhibitors G protein blocks the Adenylyl Cyclase ( AC) activity thereby reducing cAMP levels.59

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Binding of epinephrine to G-protein-linked receptor (1 molecule)

Reception

Transduction

Inactive G protein

Active G protein (10

2

molecules)

Inactive adenylyl cyclase

Active adenylyl cyclase (10

2

)

ATP

Cyclic AMP (10

4

)

Inactive protein kinase A

Inactive phosphorylase kinase

Active protein kinase A (10

4

)

Active phosphorylase kinase (10

5

)

Active glycogen phosphorylase (10

6

)

Inactive glycogen phosphorylase

Glycogen

Response

Glucose-1-phosphate

(10

8

molecules)

Epinephrine-induced breakdown of glycogen

.

Breakdown of glycogen

to glucose monomers by

phosphorolysis in liver

and muscle cells

Best characterized example of

G

s

protein coupled receptor is the epinephrine receptor.

The binding of epinephrine to G-protein-linked receptor triggers a signaling pathway leading to the release of glucose from glycogen, which takes places in hepatocytes (liver cells).

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Both Stimulatory G proteins (

Gs) and Inhibitory G proteins (Gi) are targets for bacterial toxins:Cholera toxin stimulates

Gs: catalyzes

ADP ribosylation of the α subunit of

G

s

and blocks

its GTPase activity thereby compromising the GTP hydrolysis.  As a result, α subunit of Gs becomes permanently “active” causing a persistent stimulation of adenylate cyclase and elevated cAMP  large efflux of water and Cl- in the gut that causes diarrhea. Stimulatory G-protein and Inhibitory G protein61

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Stimulatory G-protein and Inhibitory G protein

Pertussin

toxin stimulates

G

i

:

catalyzes

ADP ribosylation of the α subunit of Gi keeping it in the inactive GDP bound state.

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In class presentation on FeBRUARY

11, 2019Establish 4 team groups (12 max people per group ): Each student should join a group of choice. (Each of you is free to join a group of interest but, if you prefer, I can make up the group)

Each team needs to:Choose 1 out 4 scientific articles I have sent for today class;

Read the chosen article and make a 20 min presentation to be delivered for in class presentation on February 11;The presentation should be shared among team members;

Be prepared to answer in class questions from the professor and your peers.

Please send to me (

antonio.giordano

@ temple.edu)

the following: Name of the students for each group and the paper chosen by February 7 PPT presentation along with information for each group by 12 AM on February 11.