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
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
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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 Gs and Gt identified
• 1987 Endothelium-derived relaxing factor
– Identified as (NO, nitric oxide)
• Current
– RNAi
– Integration of signals
The Questions
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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)
– 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
filter
λ = 540 nm
FRET
No signal
signal
laser
Alexa 405
λ = 401 nm
λ = 421 nm
detector
λ = 540 nm
λem = 421 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
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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