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.
Why should both inputs when mutating serines into alanines show the presense of GluA1?
Mutation of S->A does not prevent the expression of GluA1 receptor.
Why did the researchers test double phosphorylation under denaturing conditions?
You can have 2 glua1 subunits that each have one phosphorylated site so that one glua1 never has two phosphorylated sites. Therefore, 1 glua1 is never doubly phosphorylated.
What did the researchers conclude from the denaturing test for double phosphorylation?
They found that the graph C (slide 17) was similar to the previous one where they were testing just phosphorylation. This shows that individual subunits are doubly phosphorylated.
How did the researchers induce LTD in the cultured neurons?
They added NMDA receptors without glycine, and kept the Mg2+ block in.
What induces LTD?
Calcium influx through NMDA receptor.
Why can calcium enter NMDA even if there is an Mg2+ block?
It is not glued into the receptor so calcium enters because there is a driving force on calcium.
How did the researchers induce LTP in the cultured neurons?
They added glycine and removed the Mg2+ block.
What did the researchers conclude after the LTP and LTD protocol?
LTD decreases phosphorylation and LTP increases phosphorylation.
How you know if LTP increases phosphorylation?
Less is retained in the supernatant when they tested the iso version of LTP. Slide 19.
How does EE affect serine phosphorylation in the PSD fraction?
It increases phosphorylation.
How does EE affect serine phosphorylation in P2? Why?
Not much of a difference, but still an increase because the P2 is less pure since it is a membrane fraction.
How should and how does EE affect other scaffolding proteins?
If you have LTP induced by EE, then you should increase scaffolding proteins. Which it does.
Does EE increase serine 845 pplation through the immunoprecipitation method in the P2? How do you know?
It does, but the decrease is not significant. You have slightly less supernatant, but not enough to say that there is a significant change.
What does the PSD fraction say about EE?
EE significantly decreases phosphorylated S845 in the supernatant, which means that EE generates more phosphoisoforms of GluA1, decreasing the amount of supernatant.
Do the EE pellet fractions show evidence of double phosphorylation?
Yes. Go through slide 23
How does Amphetamine increase LTP?
Amphetamine blocks the reuptake of norepinephrine, which activates beta-adrenergic receptors, which increases PKA, which increases LTP.
Does amphetamine treatment increase p845 levels in mice forebrain membrane fractions? How do you know this?
Yes. Slide 24.
- Amphetamine treated GluA1 decrease in the supernatant, which means more are phosphorylated in the Amph condition than the Veh condition. This difference is significant.
What can you conclude about the phos-tag SDS PAGE?
The Huganir group found phosphorylation in their Pho-tag SDS PAGE, like Hayashi group did. The only difference is that the present study found more phosphorylation in the control.
