Chapter 14 Signal-transduction pathways by linzhengnd

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									Chapter 14 Signal-transduction pathways


 Hormones,
 Aromas (volatiles) sense
 1 messenger
 [threshold conc.]
 [ligand]
                                                      [proteins in cell membrane]
  Delivery
  [2 messenger]




             changes in enzyme activity, gene expression, or ion-channel activity
              metabolic pathways
             Branch / feedback / terminate
Common second messengers
 (1) Free to diffuse to other compartments of the
      cell
 (2) Significantly amplified signals
 (3) Common 2º messengers utilized
 — cross talk: opportunities and potential
    problems


                                                    [NO]
                                                    H2O2
                                                    cADP ribose
Hormones:
   Signal
transduction

                          Signal molecules

                                                                 ligand,
 steroids      nonpolar                      large and polar
                                                                 primary messenger
                                                 
            through membrane                 bind to receptor
                                                 
             bind to protein                 alter receptor structure intracellular

                                                 
            interact with DNA                [2° messengers] 

                                                
        modulate gene expression              protein P / de-P

                                                 
                                             biochemical reaction
Type 1: Seven-transmembrane-helix (7TM)
                 receptor
          –– serpentine receptor




                                                    C-terminal and
                                                    cytoplsmic loop
                                                    conformation
                           rhodopsin                changes




                       ca. 50 % therapeutic drugs
Guanyl nucleotide-binding protein (G protein)
  an intermediary in signal transduction from 7TM
  a heterotrimeric G proteins




                             7TM

                                 G-protein-coupled receptors
                                     (GPCRs)


                                   ATP  cAMP
 G protein: a heterotrimer
: bind the nucleotide (activated and unactivated state)
: a seven-bladed propeller
: a pair of -helices
p. 381
(02)   G-protein-coupled receptors (GPCRs)
             Activated G proteins
transmit signals by binding adenylate cyclase
                                            ATP cAMP
The epinephrine receptor signal-transduction

                pathway
            [cAMP]
                 stimulate ATP production for muscle
                contraction
               enhance the degradation of storage fuels
               increase the secretion of acid by gastric
               mucosa
               lead to the dispersion of melanin pigment
               granules
               diminish the aggregation of blood platelets
               induce the opening chloride channel
               mediated by protein kinase A(PKA)


            target protein—ser/thr-P
            cAMP- response element binding (CREB) protein, in nucleus
             - a transcriptional activator
Serotoni
   n




            cAMP- response element binding protein

           Close K+ channel
How to terminate the signal transduction?
   1. Resetting G subunit: a intrinsic GTPase activity,
                      spontaneous




      seconds ~minutes
      A build-in clock
      Mechanism?
          Signal termination

                       dep. hormone conc.




cytosol     Specific
            (for C-terminal
            and occupied)
                                            diminishes its ability to
                                            activate G proteins
           Phosphatidyl inositol bisphosphate
               hydrolysis two messengers


Vasopressin
    
  7TM
   
   Gq                          A1
   
                           A2
 Phospholipase C
                      D
 Cleavage PIP2
                           C
   
IP3 (soluble form) 1,4,5
DAG (insoluble form)
DAG and IP3
work in tandem
                                                       (02)
        IP3 open channels to release   Ca2+


  3IP3 + IP3 -gate channel
(nM) 

  Ca2+ release (from ER, SR)
                                              a short-lived
                                             messenger

  Smooth muscle contraction
  Glycogen breakdown
  Vesicle release
  Early fertilization
                   Diacylglycerol metabolism     (02)




                                phosphorylated


Arachidonate
Prostaglandin H2
                                hydrolyzed
  Ca2+ — a ubiquitous cytosolic
           messenger
The reasons for Ca2+ mediate many signaling processes
1. Fleeting changes in [Ca2+] are readily detected
  The low level of [Ca2+]cyto can be easily and abruptly   Plagiarism
   raised for signaling purposes        100 nM
   avoid insoluble compound formation
   (an apparent drawback is in fact an advantage)
   is via Ca2+-ATPase and Na+- Ca2+ exchanger (02)
2. Ca2+ can bind tightly to proteins and result in
    significant conformational changes
   coordinated with six to eight oxygen atoms
   from proteins or water

               6 O from aa/protein
               1 O from water
How to detect or monitor the variation of [Ca2+] in real
                        time
       - The fluorescent calcium-binding dye




                Red: high
                Blue: low
                 Calcium-specific reagents (02)

ionophores




  raise the cytosolic Ca2+ level      reduce the unbound Ca2+ level

                                   EDTA: ethylenediamine tetraacetic acid
Calmodium – a calcium sensor
  ¤ a 17-kd with 4 calcium-binding sites
  ¤ is activated when [Ca2+]cyto 500 nm
  ¤ a member of EF-hand protein family, a calcium binding motif
     - a helix-loop-helix unit
  ¤ Parvalbumin: vitamin D3-dependent Ca2+ binding proteins

                                         forefinger




                                           thumb
 Calcium bind                         Calmoduline-dependent protein kinase (CaM
                                      kinase)
  calmodium conformational changes   – recognize positively charged,
     expose hydrophobic surfaces       amphipatic  helix
     that can be used to bind other
     proteins




A pair of EF-
hand motifs

 a flexible
 helix
(02)
                  calcium + calmodulin


       CaM kinase                 Ca2+-ATPase pump
                                          
       active target protein      [Ca2+]cyto level decrease
                                          
       signal propagation           signal termination
       (the memory of a
       previous calcium pulse)
 Type 2: Insulin signaling
   receptors that include protein kinase as part of their structures
  receptor is a dimer of two identical units
  each unit: - and -chain linked by a disulfide bond


-chain        -chain

                                                       One insulin binding on
                                                       the outside of the cell
                                                          
                                                       A membrane-
                                                       associated kinase
                                                       within the cell is
                                                       activated;
                                                       cross-phosphorylation

           2 inter- and 1-intra-
           chain disulfide bond
                               Protein kinase A: Ser/Thr P




in  subunit




          3 tyr residues in
          activation loop of
           subunit
                          a series of membrane-
                          anchored molecules




IRS : insulin-receptor substrate
IRS : insulin-receptor substrate
IRS1/IRS2, act as adaptor proteins
N-terminal: Pleckstrin homology domain, binds phosphoinositide lipids
           phosphotyrosine-binding domain:
Tyr-X-X-M sequence: are phosphorylated by the receptor tyrosine kinase




                                                           Met
IRS   phosphoinositide 3-kinase:
 a lipid kinase, 110 kd catalytic subunit and 85 kd regulatory subunit

 containing a SH2 domain: Src homology 2, recognize the
 phosphotyrosine residues in the IRS, via two Arg residues that are
 conserved in all SH2 domain
phosphoinositide 3-kinase  PIP3
   PIP3-dependent protein kinase
    Akt: a kind of protein kinase, is not membrane anchored
Membrane-anchor molecules




 Glucose transporters (GLUT4)
 Stimulate glycogen synthesis
Amplicification/ termination




                    phosphatase    phosphatase




                                     phosphatase
Type 3: EGF (epidermal growth factor) signaling
 stimulate the growth of epidermal and epithelial cells
 a receptor tyrosine kinase, a 6 kd polypeptide




                               3 intrachain disulfide bonds




EGF receptor structure
EGF receptor:
 is a dimer of two identical units, but exist as monomers until EGF
   ligands bind to them
 each monomer binds a EGF molecule in its extracellular domain
 each EGF molecule lies far away from the dimer interface
 a dimerization arm from each monomer that reaches out and inserts
   into a binding pocket on the other monomer
If EGF is absent?



                          binds to a part of within the same monomer




Once EGF                   Change into a active conformation
present,


                    A constitutive active form ?
                     Her 2 receptor, 50 % identical in aa sequence
                     with the EGF receptor and has the same domain
                     structure
                     Her 2 is overexpressed in some cancers
EGF phosphorylation:
also like insulin receptor, cross-phosphorylation of one unit by another unit
  within a dimer, but
 its carboxyl - terminal tail containing tyrosine rich (5 residues)
 the kinase itself is an active conformation without phosphorylation




Dimerization  C-terminal region on one receptor into the active site
                  of its partner’s kinase
Grb-2: an adaptor protein
     SH2 domain  phosphotyrosine residues of receptor
     SH3 domain  proline-rich region of Sos
Sos: a guanine-nucleotide-exchange factor (GEF)
Ras: small G proteins, small GTPase
    localized to the inner surface of plasmamembrane
              two
                    GTPase-activating
                    proteins (GAPs)
phosphatase
          G proteins vs. small G proteins
                (divergent evolution)

      G proteins               small G proteins
      30-35 kd                  20-25 kd
      heterotrimer              monomer (similar to G)
      7TM                      dimerization
      GTPase act.               GTPase act. (low)
                               GTPase-activating proteins
(GAPs):
                                 facilitate GTP hydrolysis
                               Sos + GAPs adjust small G cycle
                               ras mutation  cancer
14.4 Many elements recur with variation
     in different signal transduction pathways
 Protein kinases are central
 Second messengers
 Specialized domains
  pleckstrin homology domains: interact with lipids PIP3
  SH2 domains: interact with the phosphorylated tyrosine residues
              Some virus induced cancer
   – to understand the signal-transduction proteins and
                           pathways
 Rous sarcoma virus: a retrovirus, a oncogenic RNA virus
 viral sarcoma (v-src): oncogene [A cancer-causing gene; any of several
     mutant genes that cause cells to exhibit rapid, uncontrol proliferation.]
 cellular sarcoma (c-src): proto-oncogene, does not induce cell
     transformation

                                           v-Src: 11 aa of C-terminal, lack Y residue
c-Src
                                             always active
                                   19 aa


                                             a. SH2 bind to tyr-P of C-terminal
                                             b. The linker between SH2 and
                                                protein kinase is bounded by SH3
                                              c-Src inactive



                                              Biology/chemical/physical factors
                       Ras: a small G protein or GTPase
             – localized to the inner surface of plasmamembrane
The small G proteins




      Three 21-kd Ras proteins in mammalian cells
        H-Ras: Harvey rat sarcoma
        K-Ras: Kirsten rat sarcoma         A loss of the ability to hydrolyze GTP
        N-Ras: Neuroblastoma rat sarcoma           continue on
      Tumor-suppressor genes (contribute to cancer development):
        to develop cancer only when both copies of the genes normally present in a cell
         are deleted or otherwise damaged.
        e.g., genes for some of the phosphatase
Monoclonal antibodies utilization:
  inhibit the signal transduction in activated tumor formation
In some human epithelial cancers, such as breast, ovarian, and
colorectal cancers, overexpressed the epidermal-growth-factor receptor (EGFR)
  Monoclonal Ab offend receptor
e.g., Cetuximab, target a receptor tyrosine kinase
    Trastuzumab (Herceptin): inhibit Her2 overexpressed in breast cancers
Protein kinase inhibitor– a potential anticancer
                      drugs

Chronic myologenous leukemia (CML)
chromosome defect:
 the translocation between chromosome 9
  and 22
  (reciprocally)
Bcr-Abl fused protein:
 overexpress kinase activity and is not        encode
  regulated appropriately                      tyrosine
STI-571: a specific Bcr-Abl kinase inhibitor   kinase




 To understanding the signal-transduction
 pathways is leading to conceptually new
 disease treatment.
 Metabolism disease
Choleragen
 a cholera toxin from Vibrio cholera (G -)
 two functional units:
  subunit B: bind to GM1 gangliosides of intestinal epithelium (p. 738)
  subunit A: enters the cell, catalyze the covalent modification of Gs
   protein
  Gs + subunit A  Gs-Arg-ADP-ribose
                   stabilize Gs-GTP form (perpetually
    stimulation)
                   activate adenylate cyclase
                   [cAMP]
                   activate protein kinase A
                   open Cl- channel / inhibit Na+-H+ exchanger
                   NaCl and H2O loss

  Treatment consists of rehydration with a glucose-electrolyte
     solution.
Pertussis toxin
 is secreted by Bordetella pertussis
 Gi + pertussis  Gi-ADP-ribose
                  reduced Gi-GTP affinity
                  inhibit adenylate cyclase
                  [cAMP]
                  close Ca2+ channels and open K+ channels
(02)
   Altered G-protein activity caused
               diseases
96T
(191)
96T (192)
(02)                  STAT5
       (signal transducers and activators of
                   transcription)
          –– a regulator of gene expression
          –– is phosphorylated by JAK2


                                   recirpocal interaction
97T



                                                                     Nelson p. 474



Which of the event is important for epidermal growth factor signaling?
(A) ADP-ribosylation (B) Farnesylation (C) Mono-oxygenation (D) Peroxidation
(E) Glycosylation (94,台大)                                 p. 284
96C

								
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