Chapter 14

Signal-Transduction Pathway

Signal-transduction circuits in biological systems have molecular on-off switches that, like those in a computer chip, transmit information when ”on”.

Chemical signals are crucial to coordinating physiological responses.

When organism is threatened, the adrenal glands release the hormone epinephrine, which stimulates the mobilization of energy stores and leads to improved cardiac function.

After full meal, the cells in the pancreas release insulin, which stimulates the uptake of glucose from the bloodstream and leads to other physiological changes.

The release of epidermal growth factor in response to a wound stimulates specific cells to grow and divide.

Signal transduction depends on molecular circuits

1. Release of the primary messenger.

- ligand

2. Reception of the primary messenger.

3. Delivery of the message inside the cell by the second messenger(cAMP, cGMP, Ca2+, IP3, DAG…).

4. Activation of effectors that directly alter the physiological response.

5. Termination of the signal.

※ The use of second messenger :

-> the signal may be amplified.

-> free to diffuse to other cellular compartments.

-> creates both opportunities and potential problem

by cross talk.

14.1 Heterotrimeric G proteins transmit signals and reset themselves

-Epinephrine ↔ β–adrenergic

receptor(β-AR)

-β–adrenergic receptor : member

of 7 transmembrane helix(7TM)

receptors.

-7TM receptor : initiated by

signals as diverse as hormones,

neurotransmitters, odorants,

tastants, and protons.

- 7TM (= serpentine

receptor)

The single polypeptide chain

“snake” through the

membrane 7 times.

- Rhodopsin : essential

role in vision. Member of

7TM family.

- 7TM family members are similar in structure to rhodopsin.

- The binding of epinephrine

to β–adrenergic receptor

triggers conformational

changes in the cytoplasmic

loops and the C-terminal

region.

Ligand binding to 7TM receptors leads to the activation of heterotrimeric G proteins

Epinephrine binding

β–AR conformational

change

Activate G protein

Stimulate adenylate cyclase

Increase cAMP

concentration

※Inactivated state of G protein

-GDP bound at α subunit.-Exist as heterotrimeric(αβγ).-α subunit is p-loop NTPase(nucleotide binding) family member. -α and γ subunits are anchored to the membrane by lipid modification.

※Activated state of G protein

-GTP bound at α subunit-α subunit dissociates from β and γ subunits.

Hormone binding → receptor stimulate nucleotide exchange as GEF (GDP → GTP)

Activated G proteins transmit signals by binding to other proteins

-When G protein activated, α subunit dissociated from βγ subunits. And α subunit find new binding partner(Adenylate cyclase).

- Adenylate cyclase : convert ATP into cAMP. 12 membrane spanning helices. 2 large cytoplasmic domains.- Result : binding of epinephrine to the receptor on the cell surface increases the rate of cAMP production inside the cell.

3-6. Protein Switches Based on Nucleotide Hydrolysis

Figure3-12. Structure of the core domains of a typical GTPase and an ATPase

Most protein switches are enzymes that catalyze the hydrolysis of a nucleoside triphosphate to the diphosphate

- GTPase : major class of switch protein (G protein)- ATPase : usually associated with motor protein complexes or transporters

GTPase ATPase

-two-component response regulator: histidine kinase, response regulator proteins

Why ATP or GTP are used for trigger of switch?

3-6. Protein Switches Based on Nucleotide Hydrolysis

Figure3-13. Schematic diagram of the universal switch mechanism of GTPases

- Triphosphate-bound state = “on”, spring-loaded

- Loss of gamma phosphate group → conformational change.

- Two hydrogen bonds in the each switch (Ⅰ and Ⅱ).

- and - phosphates are bound to P-loop (GXXXXGKS/T)

-phospate is bound to both switch I and II (DXnT and DXXG respectively)

Although common structural and functional features in switch proteins, many insertions of other domains in individual GTPases present various functions.

3-7. GTPase Switches : Small Signaling G Proteins

Figure3-14. The switching cycle of the GTPase involves interactions with proteins that facilitate binding of GTP and stimulation of GTPase activity

The switching cycle of nucleotide hydrolysis and exchange in G proteins is modulated by the binding of other proteins

GTP hydrolysis rate is very low → GAP(GTPase-activating protein) increase the rate by 105 fold

GDP release is conducted by GEF(guanidine-nucleotide exchange factors)Opening up the binding site

Small GTPase Ras family: H-, N-,and K-ras, 21kDa, lipid attachment

Signal transduction by Ras is dependent on the GTP-bound state. A prolonged on state are found in up to 30% of human tumors. Reduction of GTP hydrolysis is caused by point mutations at 12, 13 or 61 resulting in uncontrolled cell growth and proliferation. Good target for anti-tumor therapy.

How the GAP facilitate GTP hydrolysis? - GAP insert an arginine side chain into the nucleotide-

binding site of the GTPase. The positive charge on the side chain helps to stabilize the negative charge in the transition state for hydrolysis of the -phosphate group of GTP

Heterotrimeric GTPase

- α, β and γ subunit.- α subunit consist of the canonical G domain and an extra helical

domain.- β and γ subunit are tightly associated with each other by coiled-

coil interaction.- G protein associated with G protein coupled receptor(GPCR).- GDP-bound G protein bind to GPCR = “off” state.- When activated by ligand, these receptors act as GEF for their

partner G protein. - When GDP is released and GTP binds, G protein dissociates from

the GPCR.- In the absence of β and γ, α does not bind to GPCR.

3-8. GTPase Switches : Signal Relay by Heterotrimeric GTPases

Regulator of G-protein signaling proteins (RGS proteins) are responsible for the GTPase catalytic rate. How it increase the rate?

subunit of G-protein has a “built-in” arginine residue in the extra helical domain that projects into the catalytic site. RGS proteins bind to the switch regions, reducing the flexibility and stabilization the transition state for hydrolysis.

Particular RGS proteins regulate particular GPCRs; specificity

GPCRs are the most numerous receptors in all eukaryotic genome (1-5% of the total number of genes)

various ligands such as light, orants, lipids, peptide hormones.

8 families

3-8. GTPase Switches : Signal Relay by Heterotrimeric GTPases

Figure3-15. Hypothetical model of a heterotrimeric G protein in a complex with its G-protein-coupled receptor

“Off” state

GPCR =

WD40

Coiled-coil interaction

Cyclic AMP stimulates the phosphorylation of many target proteins by activating protein kinase A

-The increased concentration of cAMP → affect a wide range of cellular processes(ATP production stimulation in muscle cell, enhances the degradation of storage fuels in other cell and so on…).

-The key enzyme of effects of cAMP is protein kinase A(PKA).

-PKA : stimulates the expression of specific genes by phosphorylating a transcriptional activator.

G proteins spontaneously reset themselves through GTP hydrolysis

-How is the signal switched off?

-Gα subunits have intrinsic GTPase activity(GTP → GDP+Pi).

(The hydrolysis is slow)

- GDP bound α subunit re-associates with βγ subunits to re-form

the inactive heterotrimeric protein.

The hormone bound activated receptor must be reset.

1. Hormone dissociates.2. Remaining receptor-hormone complex is

deactivated by the phosphorylation of Ser and Thr in the C’ terminal.

β-Arrestin bind to phosphorylated C-terminal tail and further diminishes its ability to activate G protein.

Some 7TM receptors activate the phosphoinositide cascade

-Angiotensin II receptor: control of blood pressure

-Angiotensin binds to Angiotensin II receptor and activate Gq

-Activated G-protein activates “phospholipase C”

-Phsopholipase C catalyzes the cleavage of PIP2 → IP3+DAG.

Free IP3 binds to Ca2+ channel

Ca2+ flow out from ER to cytoplasm

Ca2+ bind to protein kinase C

Ca2+ bind to calmodulin

Smooth muscle contraction

Glycogen break down

Vesicle release……

- DAG remains in the plasma membrane. It activates protein kinase C.

Calcium ion is a widely used second messenger

-Bind to negatively charged oxygen

atoms(Glu and Asp).

-Coordinated by 6~8 oxygen atoms.

Why Ca2+ ?1.Intracellular low concentration in 100nM 2.Ca2+ can bind tightly to proteins and induce conformational changes.

- Changes in Ca2+ concentrations can be monitored in real time.

-Fura-2 : specially designed dye. binds Ca2+ and change their fluorescent properties. binds Ca2+ through positioned oxygen atoms(red).

-When a dye is introduced into cells, changes in available Ca2+ concentration can be monitored with microscopes that detect changes in fluorescence.

- Red : high Ca2+ concentration - Blue : low Ca2+ concentration

Calcium ion often activates the regulatory protein calmodulin

※Calmodulin : 17kda, 4 Ca2+ binding

sites. Sensor of Ca2+. Activated by the

binding of Ca2+. EF-hand protein

family member.

-EF-hand protein : Ca2+ binding motif.

Two helices(E and F) are positioned

like the forefinger and thumb of the

right hand.

-The binding of to calmodulin induces conformational changes(=expose hydrophobic surface that can be used to bind other proteins)

14.2 Insulin signaling : phosphorylation cascades are central to many signal-transduction processes

The insulin receptor is a dimer that closes around a bound insulin molecule

-Insulin : peptide hormone, 2 chains, linked

by 3 disulfide bonds.

- Insulin receptor : dimer of 2 identical units,

Each unit consists of 1 α chain and 1 β chain.

(α chain = outside of the cell, ligand binding

site.

β chain = inside of the cell, membrane

spanning + protein kinase domain)

Insulin binding results in the cross-phosphorylation and activation of the insulin receptor

-Insulin binding → 2 α subunits close together → 2 β subunits close together → tyrosine kinase activation

When these tyrosine residues phosphorylated, a striking conformational change take place.

Insulin binding → tyrosine kinase activation

The activated insulin receptor kinase initiates a kinase cascade- Additional sites within the receptor also are phosphorylated → act as docking site for other substrate(ex>IRS-1).

- IRS(insulin receptor substrate) : signal transduction through a series of membrane anchored molecules to a protein kinase.

-N-terminal of IRS : pleckstrin homology

domain(binds phosphoinositide), phosphotyrosine

binding domain (SH2).

-Center~C-terminal of IRS : 4 YXXM seq.

(phosphorylated by insulin receptor tyrosine kinase)

- IRS = adaptor proteins ; they bind to the lipid

kinase and bring it to the membrane so that it can

act on its substrate.

- Phosphotyrosine in the IRS are recognized by SH2 domain.

※ SH2(Src homology)

-Present in many signal

transduction proteins.

-Bind to phosphotyrosine

containing peptide.

-Ex> phosphatidylinositide 3-

kinase

PIP3 activates PDK1(protein kinase)

PDK1 phosphorylates and activates other kinase Akt.

Akt phosphorylates targets

Contains SH2 domain

- The signal is amplified at several stages along this pathway.

Insulin signaling is terminated by the action of phosphatases

-Specific enzymes are required to hydrolyze these

phosphorylated proteins and convert them back into

the states that they were in before the initiation of

signaling.

-Lipid phosphatase; PIP3 PIP2.

-Tyrosine phosphatase; IR, IRS.

-Serine phosphatase ……

14.3 EGF signaling : signal-transduction pathways are poised to respond

EGF binding results in the dimerization of the RGF receptor

※ EGF

- Epidermal growth factor.

- 6kda polypeptide.(53 a.a)

- Stimulates the growth of epidermal and

epithelial cells.

- Three intrachain disulfide bonds stabilize

the structure.

※ EGF receptor

- Protein tyrosine kinase.

- Cross-phosphorylation reaction.

- Single transmembrane spanning helix.

- Dimer of two identical units. Exist as monomers

until EGF ligand binding them.

-The receptor dimer binds two ligand molecules.- EGF molecule lies far away from the dimer interface.- This interface includes a dimerization arm.

- Each monomer is in a conformation that is quite

different from that observed in the ligand-bound

dimer.

- The dimerization arm binds to a domain within the

same monomer. (= tethered)

Dimer

Monomer

- Her2(one of EGF receptor) exists in the extended

conformation even in the absence of bound ligand.

- 50% identical in amino acid seq. with the EGF

receptor and has the same domain structure.

- Does not bind any known ligand.

- Forms heterodimers with the EGF receptor and other

members of the EGF receptor family.

- Overexpressed in some cancers.

- Overexpression = tumor growth.

The EGF receptor undergoes phosphorylation of its carboxyl-terminal tail

- Dimerization → tyrosine kinase activation → C

terminal of kinase domain is phosphorylated.

- 5 tyrosine residues.

-The dimerization of the EGF receptor brings the C-

terminal region on one receptor into the active site of

its partner’s kinase.

EGF signaling leads to the activation of Ras, a small G protein-The phosphotyrosines on the receptors act as docking sites for SH2 domains on other partner.

- Grb-2 : adapter protein. Contains SH2 domain. recognizes phosphotyrosine residues of EGFR. then recruits Sos protein through SH3 domain.

- Sos binds to Ras(signal transduction component).

-Sos binds to Ras(signal transduction component, Small

G protein) and activates Ras.

- Sos opens up the nucleotide binding pocket of Ras.

(=Sos acts as a GEF)

Activated Ras initiates a protein kinase cascade

Ras activation(GTP bound)

Ras binds Raf(protein kinase)

Raf conformational change

Raf phosphorylates MEKs

MEK is activated and activates ERKERK(extracellular signal-regulated kinases) phosphorylates substrates

※ Ras = small G protein.

- Ras, Rho, Arf, Rab, and Ran…

- Plays a major role in a host of cell functions including

growth, differentiation, cell motility, cytokinesis, and

the transport of materials throughout the cell.

14.4 Many elements recur with variation in different signal-transduction pathways

1. Protein kinases are central to many signal-

transduction pathways. Protein kinases often

phosphorylate multiple substrates.

2. Second messengers participate in many signal-

transduction pathways(cAMP, Ca2+, IP3, lipid DAG).

3. Specialized domains that mediate specific

interactions are present in many signaling

proteins(pelckstrin homology domain, SH2 domain).

14.5 Defects in signal-transduction pathways can lead to cancer and other disease

※ Rous sarcoma virus(causes sarcoma in

chicken)

- v-src gene is an oncogene, tyrosine

kinase. SH2 and SH3 domains.

- c-src protein is a signal transduction

protein that regulates cell growth.

- In v-src, no C terminal tyrosine that is

phosphorylated to inactivate c-src v-

src is always active cancer.

Monoclonal antibodies can be used to inhibits signal-transduction pathways activated in tumors

-Mutated or overexpressed receptor tyrosine kinases are frequently observed in tumors.

-Ex> EGFR is overexpressed in some human cancers. Strategy : produce monoclonal antibodies to the extracellular domains of the offending receptors.

Cetuximab(Erbitux) : targeted the EGFR in colorectal cancer. inhibits EGF binding.Trastuzumab(Herceptin) : inhibits HER2. binds to domain 4 of HER2.

Protein kinase inhibitors can be effective anticancer drugs

-Translocation of genetic material between chromosome 9 and 12 produce c-abl and bcr fused protein.

-C-abl is tyrosine kinase.

- c-abl and bcr fused protein is not regulated expressed at higher level cell growth CML(chronic myelogenous leukemia)

-Gleevec : specific inhibitor of the Bcr-Abl kinase.

Cholera and whooping cough are due to altered G-protein activity

-Cholera and whooping cough are two pathologies of the

G protein dependent signal pathways.

-Cholera toxin : secreted by Vibrio cholerae.

subunit binds to Gm1 gangliosides of intestinal

epithelium.

A subunit catalyzes ADP-ribosylation to arginine of Gα

subunit always active G protein, PKA activation, Channel

open