Cell communication 1 cell signaling and signal transduction

Cell communication 1: cell signaling and signal transduction. 3 -23 -2020

https: //www. youtube. com/channel /UCtj. Az. Nwh. V 49 RPNW 5 e. VUl. V Dw/featured

Quiz 3 -23 -2020 • how many different mutants you can isolate by Tomkins screening method (treat S 49 cells with epinephrine plus prostaglandin and isolate survival mutants).

GORDON M. TOMKINS found the cytocidal effect of cyclic AMP on clonal S 49 lymphoma cells. Proc. Natl. Acad. Sci. U. S. A. 70, 76 -79 (1973) • • • Limitation? No mutant of essential genes for viability Diploid Easy to isolat vs hardly isolated mutants Haploinsulficentcy Hormone receptor Gs and Adenylate cyclase PKA Substrate of PKA which can lead to apoptosis.

Mechanisms of cyclic AMP/protein kinase A- and glucocorticoidmediated apoptosis using S 49 lymphoma cells as a model system. Proc Natl Acad Sci U S A. 2015; 112(41): 12681 -6.

如果不同hormone都是活化adenylate cyclase，產生c. AMP，那不同細胞如 何產生不同hormonal response? How about on the same cells?

What are c. AMP intracellular targets?

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

Directing NGF’s actions: it’s a Rap. Nature Cell Biology 7: 338 -340; 2005

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

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

ADP-ribosylatio of protein

GTP dependent transmembrane signaling? !

What happen if we add GTP to the membrane of cyc- S 49 mutant?

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

Discovery of Gi and pertussis toxin

Discovery of growth factor and its signal transduction pathway 21

22

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) 23

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! 24

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) 25

J. Biol. Chem. 1962 26

Cohen S. J. Biol. Chem. (1973) 27

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 28

Quiz 3 -23 -2020 • 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

Scatchard analysis

Scatchard plot

Quiz 3 -23 -2020 • How two hormones employ same c. AMP as second messenger but elicit different responses in the same type of cells?

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? 34

Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro Nature 276, 409 - 410 (23 November 1978) 35

EGF stimulates protein phosphorylation in the membrane fraction of target cells (PNAS 1978) 36

37

38

Cell signaling by receptor-tyrosine kinases. Cell. 2010; 141: 1117– 1134. 39

• , Insulin Induces Rapid Accumulation of Insulin Receptors and Increases Tyrosine Kinase Activity in the Nucleus of Cultured Adipocytes. Journal of cell physiology 157, 217 -28: 1993 Insulin-induced Translocation of IR to the Nucleus in Insulin Responsive Cells Requires a Nuclear Translocation Sequence Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1865 (4), 551 -559; 2018

The insulin receptor goes nuclear. Cell Research (2019) 29: 509– 511 41

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.