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Signal Relay by Heterotrimeric GTPases

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									        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          After full meal, the cells   The release of
 threatened, the adrenal   in the pancreas release      epidermal growth
 glands release the        insulin, which stimulates    factor in response to a
 hormone epinephrine,      the uptake of glucose        wound stimulates
 which stimulates the      from the bloodstream         specific cells to grow
 mobilization of energy    and leads to other           and divide.
 stores and leads to       physiological changes.
 improved cardiac
 function.
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.
 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)
                                   - Rhodopsin : essential role
The single polypeptide chain
                                   in vision. Member of 7TM
“snake” through the membrane
                                   family.
7 times.

- 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
                                  Hormone binding → receptor
                                  stimulate nucleotide
                                  exchange(GDP → GTP)




※Inactivated state of G               ※Activated state of G protein
protein
                                      -GTP bound at α subunit
-GDP bound at α subunit.              -α subunit dissociates from β and γ
-Exist as heterotrimeric(αβγ).        subunits.
-α subunit is p-loop
NTPase(nucleotide binding) family
member.
-α and γ subunits are anchored to
the membrane by lipid modification.
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
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




            GTPa                                    ATPa
            se                                      se
-two-component response regulator: histidine kinase, response
  Why ATP or GTP are used for trigger of switch?
   Figure3-12. Structure of the core domains of a typical
3-6. Protein Switches Based on Nucleotide Hydrolysis

- Triphosphate-bound state = “on”, spring-loaded
- Loss of gamma phosphate group → conformational
change.
- Two hydrogen bonds in the a- and b- phosphates are
                             each switch (Ⅰ and Ⅱ).
                            bound to P-loop
                            (GXXXXGKS/T)

                            g-phospate is bound to both
                            switch I and II (DXnT and
                         Although common structural and
                            DXXG respectively)
                         functional features in switch
                         proteins, many insertions of other
                         domains in individual GTPases
 Figure3-13. Schematic diagram of the universal switch
                         present various functions.
  3-7. GTPase Switches
       : Small Signaling G Proteins
   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)
   Figure3-14. binding site
Opening up theThe switching cycle of the GTPase involves
• 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.
       3-8. GTPase Switches
            : Signal Relay by
      Heterotrimeric GTPases
• 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).
• Regulator of G-protein signaling proteins
  (RGS proteins) are responsible for the
  GTPase catalytic rate. How it increase the
  rate?
• a 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
3-8. GTPase Switches
     : Signal Relay by Heterotrimeric GTPases
               GPCR
               =




                                         “Off” state
      WD40




               Coiled-coil interaction
 Figure3-15. Hypothetical model of a heterotrimeric G
  3-6. Protein Switches Based on Nucleotide Hydrolysis
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?
    Figure3-12. Structure of the core domains of a typical GTPase and an ATPase
3-6. Protein Switches Based on Nucleotide Hydrolysis

- Triphosphate-bound state = “on”, spring-loaded
- Loss of gamma phosphate group → conformational change.
- Two hydrogen bonds in the each switch (Ⅰ and Ⅱ).


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

                                     g-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.

  Figure3-13. Schematic diagram of the universal switch mechanism of GTPases
   3-7. GTPase Switches
        : Small Signaling G Proteins
   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

    Figure3-14.The switching cycle of the GTPase involves interactions with
    proteins that facilitate binding of GTP and stimulation of GTPase activity
   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 g-phosphate group of GTP
3-8. GTPase Switches
     : Signal Relay by Heterotrimeric GTPases
    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.
   Regulator of G-protein signaling proteins (RGS proteins) are
    responsible for the GTPase catalytic rate. How it increase the
    rate?
   a 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
                    GPCR =




        WD40
                                                       “Off” state




                   Coiled-coil interaction

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


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



-Bind to negatively charged oxygen
atoms(Glu and Asp).
-Coordinated by 6~8 oxygen atoms.
- 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.
-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
                        Contains SH2
                        domain




           PIP3 activates PDK1(protein kinase)


   PDK1 phosphorylates and activates other kinase Akt.


Akt phosphorylates targets(ex> GLUT1-glucose transporter)
- 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.
 -Tyrosine phosphatase.
 -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.
Dimer                                                Monomer




 - 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)
- 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 ERK
                       ERK(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.
                binds to Gm1 gangliosides.
                modify Gα subunit  always active G protein

								
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