1. Approaches to signal transduction:
- pathway
- ligand/receptor
- protein/protein interactions
- second messengers
- protein phosphorylation
- cellular responses
Shutting down a
signaling pathway
(second messengers)

2. Approaches for identifying a pathway:
- genetic: screening for mutants (loss, gain of function),
misexpression/overproduction screens
- pharmacological: identifying second messengers
- clonal analysis to identify signaling, receiving cells

3. Approaches for identifying ligand or receptor:
to find receptor:
- biochemical: affinity chromatography
- molecular: expression library screening
to find ligand:
- biochemical: membrane fraction vs. secreted molecule
contact-dependent vs. secreted ligand:
- tissue culture cell aggregation (cell adhesion)
- clonal analysis

4. Signaling in contact-dependent cells

5. Protein-protein interactions:
- biochemical: tagged fusion proteins, co-IP
- molecular: yeast 2-hybrid
- genetic: screening with mutant receptor background

6. Dominant-negative inhibition of signaling through receptor kinases

7. Second messengers:
- pharmacological: reporter dyes

8. cAMP dynamics:
- serotonin/G-protein-linked receptor signaling causes a rapid rise in intracellular cAMP.
- cAMP-dependent protein kinase (PKA) - labeled with a fluorescent dye that changes color on binding to cAMP - blue is low level, yellow is intermediate, red is high.

9. Ca++ dynamics:
- sperm entry causes release of Ca++ .
- Ca++ dynamics indicators (aequorin, fura-2).

10. Protein phosphorylation:
- microscopy: P-epitope antibodies
- molecular/genetic: Ala-scanning mutants
- biochemical: in vitro labeling with radioactive P
Cellular activities:
- changes in gene expression, behavior
- reversible vs. permanent changes (development)

11. Target cells become desensitized