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Chapter 3

Chapter 3. History and Techniques of Cell Signaling. History. History. Hormone – “to excite” or “to arouse” 1905 Proposed by Starling First factor found about 30 years later Ca2+ experiments started in 1947 1960s – Ca2+ storage 1970s – Calmodulin 1980s – [Ca2+] changes measured.

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Chapter 3

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  1. Chapter 3 History and Techniques of Cell Signaling

  2. History

  3. History • Hormone – “to excite” or “to arouse” 1905 • Proposed by Starling • First factor found about 30 years later • Ca2+ experiments started in 1947 • 1960s – Ca2+ storage • 1970s – Calmodulin • 1980s – [Ca2+] changes measured

  4. History • cAMP signaling 1950s • 1989 adenylyl cyclase (adenylate or adenyl) cloned • Inositol phosphate pathway signaling 1950s • Role in Ca2= signaling 1975 • Membrane inositol 1988 • Sphingomyelin cycle 1986 • Protein phosphorylation • Glycolysis & phosphorylation 1960s • 1970s PKC (protein kinase C) • 1980s serine / threonine kinases • 1988 tyrosine kinases • 2000 kinase genes / 1000 phosphatase genes?

  5. History • G proteins • 1970s G proteins as intermediates • 1980s Gs and Gt identified • 1987 Endothelium-derived relaxing factor • Identified as (NO, nitric oxide) • Current • RNAi • Integration of signals

  6. The Questions • Does ligand change [X] • What is X’s activity • Does ligand cause effect without X • Can one manipulate X to change effect • Do other [] change with change in [X] • Does transcription or translation change with [X] • Is [X] involved in all ligand signaling for that response • Can we design a drug that mimics [X]

  7. Techniques • Biochemistry and Labeling • Traditional enzyme analysis • Kinetics • Active sites • Antibodies • Identify receptors involved • Microscopy • Confocal (focused laser instead of white light) • Radioisotopes • Labeled substrates

  8. Probes • Fluorescence and confocal microscopy • Non-fluorescent probe that fluoresces upon binding event • Specificity? • Size of signal • Light detection • FRET – fluorescence resonance energy transfer • FLIM – fluorescence lifetime imaging microscopy

  9. FRET filter λ = 540 nm No signal signal laser Alexa 405 λ = 401 nm λ = 421 nm detector λ = 540 nm λex = 434 nm λem = 421 nm

  10. Pharmacological Agents • Drugs • Stop one event • Visualize the backlog • Specificity? • Integration?

  11. Protein-Protein Interactions • Protein purification • Expasy.org • X-Ray studies • Membrane bound? • NMR studies • In vitro reconstitution

  12. Molecular Genetics • Knock-out • Knock-in mutant • RNAi • Overexpression • Dominant Negative • Cellular Location • GFP

  13. Microarray & Proteomics • Microarray • See thousands of genes at the same time • Novel pathways? • Proteomics • RT-PCR • 2D electrophoresis

  14. Computer Analysis • Integrations is too complicated, need logic of a computer • One pathway at a time easy • All pathways – too many calculations

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