Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions
description
Transcript of Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions
![Page 1: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/1.jpg)
Approaches to signal transduction:
- pathway
- ligand/receptor
- protein/protein interactions
- second messengers
- protein phosphorylation
- cellular responses
Shutting down a signaling pathway
(second messengers)
![Page 2: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/2.jpg)
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
![Page 3: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/3.jpg)
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
![Page 4: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/4.jpg)
Signaling in contact-dependent cells
![Page 5: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/5.jpg)
Protein-protein interactions:
- biochemical: tagged fusion proteins, co-IP
- molecular: yeast 2-hybrid
- genetic: screening with mutant receptor background
![Page 6: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/6.jpg)
Dominant-negative inhibition of signaling through receptor kinases
![Page 7: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/7.jpg)
Second messengers:
- pharmacological: reporter dyes
![Page 8: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/8.jpg)
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.
![Page 9: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/9.jpg)
Ca++ dynamics:- sperm entry causes release of Ca++ .- Ca++ dynamics indicators (aequorin, fura-2).
![Page 10: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/10.jpg)
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)
![Page 11: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/11.jpg)
Target cells become desensitized
![Page 12: Approaches to signal transduction: - pathway - ligand/receptor - protein/protein interactions](https://reader035.fdocuments.us/reader035/viewer/2022062816/56814d6c550346895dbabc6a/html5/thumbnails/12.jpg)