Chapter 3 History and Techniques of Cell Signaling

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

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?

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

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]

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

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

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

Pharmacological Agents Drugs – Stop one event Visualize the backlog – Specificity? – Integration?

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

Molecular Genetics Knock-out Knock-in mutant RNAi Overexpression – Dominant Negative Cellular Location – GFP

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

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