Enzyme-linked receptors Flashcards
What are common characteristics of kinase-linked receptors?
- Linked with slow transcriptional responses
- One transmembrane domain
- Cytosolic domain is an enzyme
What is the mechanism of the activation of RTK?
- Two ligand binds to the extracellular domains of two RTKs
- The two RTKs joins and forms one activated dimeric receptor, bringing together two kinases that each phosphorylate the other on a tyrosine
- Phosphorylation increases the activity of the kinase, thus reducing the Kd and increasing the ability of ATP and/or protein substrate to bind
- The activated protein kinases phosphorylate several other tyrosine residues in the receptor’s cytosolic domain
- The resulting phospho tyrosines function as docking sites for downstream proteins
What is a unique characteristic of FGF receptor activation?
FGF ligand is bound to polysaccharide heparan sulfate, a component of the extracellular matrix. This enhances its binding to the receptor.
What are the steps to the Ras/MAP kinase signal transduction pathway?
- SH2 domain of the cytosolic adapter protein GRB2 binds to a phosphotyrosine on an activated, ligand-bound receptor
- SH3 domains of GRB2 bind to cytosolic Sos protein, bring it near the cytosolic surface of the plasma membrane, and close to its substrate, inactive Ras-GDP
- SoS is a GEF and converts Ras-GDP to active Ras-GTP
- Ras is bound to the cytosolic surface of the plasma membrane by an anchor
- Activated Ras promotes formation of signal transduction complex, containing acting protein kinases (Ras -> Raf -> MEK -> MAP kinase family (includes ERK)), which in tern activate members of the MAP kinase family, etc.
What is the goal of the study of Raf in Drosophila compound eye?
Test whether Raf protein kinase activity mediate R7 development
What were the results obtained by the study of RTKs in Drosophila compound eye?
Perform screens with different drosophila variants:
* WT results in normal eye
* Raf CA results in a rough eyed mutant; pathway is overactive, causing the presence of more than 7 photoreceptor cells
* Su(Raf) results in phenotype closer to the WT, as the suppressor compensates for overactive Raf activity. For instance, if MEK only works at 50%, normal phenotype is restored. However, this only works if lower activity is downstream of Raf
* E(Raf) (enhancer mutation) results in an enhanced rough eye phenotype. For instance, a null phosphatase leads to an over-phosphorylation of MEK, leading to an even more overactive pathway
Perform genetic interaction screen:
* Hypomorph => Removing 50% of a gene in the pathway results in the wild-type phenotype, though the system is very sensitive to further mutations
* Double mutant sev, ras => Leads to cone cell phenotype of the R7 cell. However, we cannot identify whether the mutation happens upstream of downstream, so an epistasis screen is needed
What is an epistatis screen? Give an example of its application.
An epistasis screen is an experiment to identify the order of a certain pathway or genes. It can be used to determine the elements upstream and downstream of Raf. For example, we have pathway A => B(CA) => C. We make A a dominant mutant, there will be no supression of the effects of B because A is upstream. However, a dominant mutant in C will result in suppression.
For Raf, you can make Ras dominant mutant or MEK dominant mutant, and observe the phenotype resulting in Drosophila flies.
As a scientist, I want to study show that Raf dimerizes. How can I do this?
Using PALM (photoactivation localization microscopy).
* Fluorescently tag Raf such as a “halo” can be seen surrounding the protein kinase
* To address the issue of overlapping halos when two Rafs dimerize, use blinking fluorescent tag to distinguish between the Rafs that are bound to each other
* RESULT: In mutants, dimerization of Rafs form clusters of size 2+, whereas wild-type rarely has clusters of 2+
How do cells avoid a signaling mess?
- Cell/tissue specific developmental history ensures that only a subset of proteins is expressed
- Scaffold proteins can separate common kinases
What are scaffold proteins and their function?
Scaffold proteins are molecules that play a role in the organization and regulation of cellular processes by:
1. Speeding up pathway by proximity and proper orientation
2. Separate pathways
3. Modulating pathways externally
4. Mediate feedback, shape response dynamics
Give an example where scaffold proteins help the organization of signaling.
In the mating and osmoregulatory pathways, the same protein kinase can be found, which is a potential source for confusion. So, in the mating pathway, scaffold protein Se5 stabilizes a large complex that includes all the components needed for the pathway, including Ste11.
Similarly, in the osmoregulatory pathway, Pbs2 scaffold protein keeps Ste11 bounded, such that it cannot be activated by components from the mating pathway.
What are drawbacks of scaffold proteins?
- No amplification as scaffold proteins bind each kinase in a linear fashion
- Potential for component titration
How does cytokine receptors differ from tyrosine receptors?
Tyrosine receptors have a kinase domain within them, whereas cytokine receptors have a separate protein tyrosine kinase that binds it, JAK
What proteins do cytokine receptors activate?
STAT proteins
What is the result of the binding of STAT to the cytokine receptor?
STAT is phosphorylated by JAK, allowing for its dimerization. This exposes its NLS and so it is transported in the nucleus where it binds DNA and activates transcription.
