Approaches to signal transduction:

- pathway

- ligand/receptor

- protein/protein interactions

- second messengers

- protein phosphorylation

- cellular responses

Shutting down a signaling pathway

(second messengers)

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

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

Signaling in contact-dependent cells

Protein-protein interactions:

- biochemical: tagged fusion proteins, co-IP

- molecular: yeast 2-hybrid

- genetic: screening with mutant receptor background

Dominant-negative inhibition of signaling through receptor kinases

Second messengers:

- pharmacological: reporter dyes

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.

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

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)

Target cells become desensitized