Cell communication 1: cell signaling and signal transduction. 3-19-2018

Quiz 3-19-2018 how many different mutants you can isolate by Tomkins screening method (treat S49 cells with epinephrine plus prostaglandin and isolate survival mutants).

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) Difficulty? Essential genes for viability Diploid Easy isolated vs hardly isolated mutants Haploinsulficentcy Hormone receptor Gs and Adenylate cyclase PKA Substrate of PKA which can lead to apoptosis.

Proc Natl Acad Sci U S A. 2015;112(41):12681-6. Mechanisms of cyclic AMP/protein kinase A- and glucocorticoid-mediated apoptosis using S49 lymphoma cells as a model system. Proc Natl Acad Sci U S A. 2015;112(41):12681-6.

Quiz 3-19-2018 Suthland 証明荷尔蒙在test tube中亦可活化肝醣分解酵素实验成功的關鍵步骤為何? Add ATP! Why? ATP hydrolysis enzyme and dilution!

Quiz 3-19-2018 決定细胞接受訊號的specificity 和 sensitivity 分別是由那些因素所決定？ KD (dissociation constant) How to measure? Association rate constant Dissociation rate constant

Sutherland EW et al J. Biol. Chem. (1956) Why activity drops spontaneously Effect of epinephrine on phosphorylase activity in dog liver slices. Sutherland EW et al J. Biol. Chem. (1956)

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.

Ignarro LJ, Byrns RE, Buga GM, Wood KS 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.

What are cAMP intracellular targets?   如果不同hormone最後都是活化adenylate cyclase，產生cAMP，那不同細胞如何產生不同hormonal response? What are cAMP intracellular targets?

Specificity of Receptor Tyrosine Kinase Signaling: Transient versus Sustained Extracellular Signal-Regulated Kinase Activation Cell 80, 179-185, 1995

Prediction and validation of the distinct dynamics of transient and sustained ERK activation. Nature Cell Biology 7: 365-373; 2005

如何判斷這些ATP or GTP是作為原料、activator還是提供能量呢?

The non-hydrolyzable AMPPNP is used as a substitute for ATP.

課堂上有提到NaF可以使AC活化，此時我便非常納悶:為什麼科學家們不會想由NaF為出發點，去探討receptor和AC的關係呢?

Cholera toxin and activation of AC Discovery of NAD and ADP-ribosylation

ADP-ribosylatio of protein

GTP dependent transmembrane signaling?!

What happen if we add GTP to the membrane of cyc- S49 mutant?

Forskolin is produced by the Indian Coleus plant and is a direct activator of adenylate cyclase

Discovery of Gi and pertussis toxin

Desensitization Receptor phosphorylation to reduce affinity of the receptor to the ligand. Degradation of hormone bound receptor through endocytosis. Reduce affinity of the receptor to the ligand through negative coopertivity among receptor.

Discovery of growth factor and its signal transduction pathway

Rita Levi-Montalcini Cancer Research 14: 49-54 (1954) 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)

What is biochemical nature of NGF? Protein or Nucleic Acid? 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!

Cohen S. Proc. Nat. Acad. Sci. USA 46: 302-311 (1960) 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)

J. Biol. Chem. 1962

Cohen S. J. Biol. Chem. (1973)

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?

Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro Nature 276, 409 - 410 (23 November 1978)

EGF stimulates protein phosphorylation in the membrane fraction of target cells (PNAS 1978)

Extrinic signals

Scatchard analysis

Scatchard plot