Transcript Cell Communication

Cell communication 1:
cell signaling and signal transduction.
3-7-2016
Can single cell live alone without
any kind of communication with
others or environment?
For bacteria,
• Communicate with environment
– Chemotaxis: to detect nutrients and avoid
toxicic substance
• Communicate with other bacteria
– Quorum sensing (來偷聽細菌們的對話吧！)
For multicellular organism,
• Communicate with environment
– Sensory system: see, smell, hearing, taste, touch etc.
• Communicate with others
– Chemical , visual , language and words.
• Communicate within
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Development : from single fertilized egg to adult body.
Transport system: circulation, digestion, secretion.
Neural system: fast communication, learning and memory.
Endocrine system: homeostasis, growth and reproduction.
Immune system: defense against non-self.
Failure of communication: cancer and aging.
Elements of Communication
• Signal
– What it is? who send? how to produce? and when to send?
• Receiver
– Receptor: specificity and sensitivity
– Localization
– Control: amount and sensitivity
• Signal interpretation
– Signal transduction: amplify, diversify and integration
• Responses
– Cell contend dependent
Cell: integration of various signals and to make proper response
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Cell communication within our body
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Generation of body pattern
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Path of signal transduction discovery
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Biochemical study of hormonal action:
how hormone activates liver/muscle
phosphorylase to break down glycogen?
Glc-1-P → Glc-6-P → Glycolysis
Glycogen
P
Glycogen
phosphorylase a*
ATP
Phosphatase
n
P
Glycogen
phosphorylase b
(inactive)
n-1
How hormone (glucogon or epinephrine)
activate hepatic enzyme to degrade glycogen?
Liver
Glycogen
Pancreas
Low blood sugar
Glucagon or
Epinephrine
stress
Glycogen
synthase
Glucose
Glycogen
phosphorylase
Effect of epinephrine on phosphorylase activity in dog liver slices.
Sutherland EW et al J. Biol. Chem. (1956)
EFFECT OF EPINEPHRINE AND GLUCAGON ON THE
REACTIVATION OF PHOSPHORYLASE IN LIVER HOMOGENATES
J. Biol. Chem. 224: 463 – 475; 1957
Effect of boiled E/G treated membrane extract on activation of
phosphorylase in the test tube
cAMP is converted from ATP by membrane bound enzyme
Adenylate Cyclase (AC)
AC in different tissues can only be activated by specific
hormone (liver/glucagon; ACTH/ adrenal gland).
Hormone may need interact with its specific receptor first!
?
?
The second messengers concept
Sutherland, E. W. Pharmac. Rev., 18, 145 (1966).
How cAMP activates glycogen phosphorylase
through phosphorylaion?
Isolation of phosphorylase kinase;
Is phosphorylase kinase a direct targert of cAMP?
NO! its activation also need to be phosphorylated – another
kinase?
EG Krebs isolated phosphorylase kinase kinase and
demonstrated that is the direct target of cAMP!
EH Fisher isolated protein phosphatase which reverse the
phosphorylation reaction.
Krebs and Fisher establish that protein phosphorylation is a
reversible control mechanism of many enzymes and proteins!
p.428
O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
Pi
H2O
Protein Phosphatase
 A protein kinase transfers the terminal phosphate of
ATP to a hydroxyl group on a protein.
 A protein phosphatase catalyzes removal of the Pi by
hydrolysis.
Epinephrine cascade.
Epinephrine triggers a series of
reactions in hepatocytes in which
catalysts activate catalysts,
resulting in great amplification of
the signal. Binding of a small
number of molecules of
epinephrine to specific βadrenergic receptors on the cell
surface activates adenylyl cyclase.
The numbers of molecules shown
are simply to illustrate
amplification and are probably
gross underestimates.
?
?
How adenyl cyclase is activated?
By hormone receptor directly? Or
Need some other activators?
GTP dependent transmembrane signaling?!
GORDON M. TOMKINS found the cytocidal effect of cyclic
AMP on clonal S49 lymphoma cells.
Proc. Natl. Acad. Sci. U.S.A. 70, 76-79 (1973)
Add Epinephrine
Beta adrenergic
receptor
Activation of AC
Increase cAMP
Activate PKA
Cell die!
What’s wrong about this cAMP
resistant cells?
Defect in regulatory subunit?
Defect in catalytic subunit?
Defect in down stream targets of kinase?
What else?
Gillman et al were able to select another S49 cell variant (cyc-) that appeared to have normal receptors and adenylyl
cyclase but that failed to generate a cyclic AMP signal in
response to appropriate hormones (b-adrenergic agonists or
prostaglandins).
Proc. Natl. Acad. Sci. U.S.A. 74, 2016 – 2020 (1977)
Solubilize membrane
from wild type cells
hormone
adenylyl
cyclase
unc
unc
hormone
Hormone
No cAMP
cAMP
G protein coupled receptor: 7 trans-membrane protein
G proteins dissociate into two signaling proteins when activated
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Adenylyl cyclase
ATP
Cyclic AMP
If cAMP can play as an important intracellular
second messager, how about other nucleotide?
In 1975, FERID MURAD identified
guanylate cyclase catalyzes the formation
of guanosine 3':5'-cyclic monophosphate
(cyclic GMP) from GTP.
Guanylate cyclase is activated by various
agents including sodium azide,
hydroxylamine, nitroglycerin, sodium
nitrite etc.
None of them physiological
relevant!
The obligatory role of endothelial cells in the relaxation of arterial
smooth muscle by acetylcholine
Furchgott RF, Zawadzki JF Nature 288, 373 – 376; 1980
Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived
relaxing factor from pulmonary artery and vein possesses
pharmacologic and chemical properties identical to those of nitric
oxide. Circ Res. 1987;61:866–879.
How is the receptor mediated
signaling desensitized?
How the signal is turned off ?
Hormone association
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• Arachidonic acid present in membrane lipids are released for
eicosanoid synthesis in the cell interior by phospholipase A2
Discovery of growth factor and
its signal transduction pathway
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8-day chick embryo sensory ganglia cultured in the presence of
fragments of mouse control or sarcoma 180 cells for 24 hrs.
Rita Levi-Montalcini Cancer Research 14: 49-54 (1954)
48
A soluble factors secreted from mouse sarcoma
180 cells which can promote nerve growth from
sensory ganglia (Nerve Growth Factor, NGF)
What is biochemical nature of NGF?
Protein or Nucleic Acid?
To remove nucleic acid by digesting with
snake venom.
Snake venom itself contains NGF activity!
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Mouse salivary gland extract (NGF) were
injected into newborn mice. Some unexpected;
non-NGF related phenomenon were observed!
Cohen S. Proc. Nat. Acad. Sci. USA 46: 302-311 (1960)
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J. Biol. Chem. 1962
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Cohen S. J. Biol. Chem. (1973)
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Characterization of growth factor receptors
• Label ligand with radioactive isotope
• Binding assay
• Determining specificity of the ligand
binding
• Scatcard analysis
– Km verse total receptor number
• How signal of the growth factor receptor
transduced?
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Epidermal growth factor stimulates
phosphorylation in membrane
preparations in vitro
Nature 276, 409 - 410 (23 November 1978)
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EGF stimulates protein phosphorylation in the
membrane fraction of target cells (PNAS 1978)
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Extrinic signals
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How the signal is turned off ?
a-subunit of G protein has GTPase activity.
Cholera toxin blocks a-subunit’s GTPase activity through
ADP-ribosylation of a-subunit. Then ?
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