Huganir article Flashcards
Why did they use drugs? What drugs did they use?
They used isoproterenol, forbol ester (PMA) and forskalin (FR) to increase the phosphorylation of the GluA1 receptors.
What is the pathway of Forskalin?
Forskalin activates adenylyl cyclase, which converts ATP to cAMP. cAMP activates PKA. PKA phosphorylates its targets.
What is the pathway of Forbol Ester (PMA)?
Forbol ester acts like DAG and directly activates PKC without the use of calcium.
What is the pathway of isoproterenol? What is the result of this pathway
Isoproterenol binds to beta-adrenergic receptors, which is its GPCR. Which activates the same responses as forskalin does. PKA binds beta-adrenergic receptor kinases, which activate the beta-adrenergic receptors. They phosphorylate the beta-adrenergic receptors.
What is isoproterenol an analog of?
Epinephrine
What is an advantage of isoproterenol?
It is a natural way of stimulating Adenylyl Cyclase because it activates a G protein.
Why do the researchers use an Ab against VGLUT1?
VGLUT1 is a glutamate transporter and is everywhere in the spines, and it packages the glutamate inside the synaptic vesicles. You want its staining to overlap with the staining for the phosphoisoforms and the GluA1.
Even if you see basal level of phosphorylation, why can’t you definitely conclude that you are seeing lots of phosphorylation?
You can have one strong Ab against one strong AMPAR that might be giving rise to all this fluorescence. Therefore proportion of phosphorylation vs amount of phosphorylation might not correlate
How do the researchers know that the Ab 845 is specific?
Look at the figure in powerpoint.
How do the researches know that the Ab 831 is specific?
Look at the figure in powerpoint.
How do the researchers know that the CIP phosphatase works?
Look at slide 10
Why is it okay that CIP only slightly decreased the FR staining?
You want to show that the staining is related to the phosphorylation and not anything else. So even though you want less staining, you decreased it with the CIP, which is what matters.
How do you know the CIP doesn’t destroy proteins?
The other labeling procedures such as GluA2, VGlut1, were not altered by the CIP and they served as controls. So you know that the phosphatase isn’t destroying proteins.
What is the purpose of the immunoprecipitation?
You can measure the proportion of the phosphorylated AMPARs to the unphosphorylated ones.
Process of immunoprecipitation?
- Have an input
- centrifuge it
- add antibodies to pull the phosphorylated AMPARs down. Add igG to serve as control where nothing gets pulled down.
- Supernatant has unphosphorylated AMPARs. Pellet has phosphorylated AMPARs.
- Measure proportions.
What is the significance of the IgG control?
Gives you a baseline of how much total AMPARs you have.
Why can you not compare the different antibodies to each other?
They each have different affinities for their proteins.
What does it mean when the supernatant GluA1 decreases after FR and PMA treatment?
Figure E, slide, 12. It means that more GLUA1 subunits got phosphorylated and went into the pellet, since the supernatant only shows the unphosphorylated GluA1.
Why is using CIP on fixed tissue hard?
Fixed tissue crosslinks proteins and makes them denature. Since phosphoatases can only do their jobs when they recognize a specific motif, phosphatases are not as efficient at recognizing the denatured tissues and they can’t reduce phosphorylation.
What is the function of tubulin?
it is a loading control to ensure you have the same amount of protein in each condition. Therefore, any changes are related to the experiment and not other variables.
Why did the researchers look at the pellet during the CIP treatment?
They wanted to see if there was a reduction in phosphorylated GluA1, which would show you how efficient the CIP is
How does the CIP treated experiment raise the possibility that the AMPAR is phosphorylated on two sites?
When you pull down one phosphoisoform, the antibody against that phosphoisoform also recognizes the other one. Slide 15.
What was different about the second experiment using CIP to test for dual phosphorylation? What can you conclude from this experiment?
Greater exposure times. You can conclude that there is double phosphorylation.
Why did the antibody against p845 recognize the S831A in the pellet?
The mutation is only on the 831 site, not the 845. Therefore, the antibody against p845 knows that in that condition, everything besides 831 (including 845) is phosphorylated.
