c. Tutorial 3: Signal Transduction - Protein Modifications Flashcards
Describe signal transduction
A process in which extracellular signals lead to a cellular response
Describe a signaling pathway
The act of a signalling molecule (hormone/NTX) binding to a receptor cell and thus causing a cellular response
Many cellular signaling pathways are regulated by _______.
a) hormones
b) glutamate
c) G-proteins
d) cAMP
c
G-proteins are a family of proteins w/ intrinsic GTPase activity. What does this mean?
it means that these proteins are able to hydrolyse GTP producing GDP
Describe the following types of G-proteins
a) monomeric
b) heterotrimeric
a) consist of a single subunit
b) consist of three different subunits (alpha, beta, gamma)
Match the following terms
a) GTP
b) GDP
1. Active G-protein
2. inactive G-protein
a) 1
b) 2
Describe the following proteins wrt g-protein activation
a) GEF - 3
b) GAP - 3
c) RGS - 2
a) guanine nucleotide exchange factor = activator protein for G-proteins by converting GDP to GTP
b) GTPase-activating protein = uses GTPase to inactivate G-proteins by converting GTP to GDP
c) regulator of G-protein signaling = regulates G-protein signaling
Describe the 6 steps to the signal transduction pathway that involves G-protein activation
- hormone binds to GPCR (G-protein coupled receptor)
- conformational change in receptor allowing it to bind to the alpha subunit of G-protein
- conformational change in G-protein releasing GDP which is then replaced by a GTP
- alpha subunit of G-protein binds to the effector protein which activates it
- effector protein activates secondary effector molecules and activates GAP allowing for the hydrolysis of GTP to GDP
- alpha subunit of G-protein disassociates from the effector molecule and goes back to G-protein
During the signal transduction pathway, the GPCR is acting as a GEF. What does this mean?
guanine nucleotide exchange factor = molecules that facilitate the release of GDP from a G-protein
Through which processes are effector molecues activated during the signal transduction pathway? By which type of enzyme?
phosphorylation = addition of phosphate groups to another protein via a kinase enzyme
both kinases and phosphatases are enzymes involved in the phosphorylation and dephosphorylation of a protein. What is the difference b/w the two?
- kinase = phosphorylates (adds phosphate gr)
- phosphatase = dephosphorylates (removes phosphate gr)
name the three most phosphorylatable AAs
- serine
- threonine
- tyrosine
T or F - each kinase can only phosphorylate one different protein as long as it contains the target sequence
F - they can phosphorylate many as long as it has the target sequence
T or F - kinases can regulate themselves through phosphorylation
T
T or F - phosphorylation of a protein will always result in activation of that protein
F - phosphorylation will always cause a conformational change but sometimes that change inhibits the activity of that protein
Which signaling pathway utilizes both heterotrimeric G-protein activity and kinase activity?
the adenylyl cyclase pathways
what are the following wrt the adenylyl cyclase pathway?
a) ligand hormone that binds to the GPCR
b) the effector molecule that is activated by the a-subunit of the G-protein
c) the secondary messenger molecules generated by the activation of the effector molecule
d) the activated protein that phosphorylates the rest of the proteins down the cascade
a) epinephrine
b) adenylyl cyclase
c) cAMP
d) PKA = protein kinase A
What are the 3 methods used to study the steps in a signal transduction pathway?
- measuring the amounts of specific proteins that are involved
- by analyzing the modification of the proteins involved (phosphorylation)
- monitoring protein-protein interactions
What is the technique used to study the amount and the modifications of specific proteins?
western blotting = SDS-PAGE
Describe the following steps associated w/ western blotting
1. SDS step - 3
2. PAGE step - 2
3. immunoblotting step - 3
- SDS (sodium dodecyl sulfate) = denatures the protein and makes it negatively charged in order to allow for separation via mass only
- PAGE (Polyacrylamide gel electrophoresis) = separates proteins via mass by having them travel through a porous gel towards the positive end.
- immunoblotting = transfer the proteins from the polyacrylamide gel to a nitrocellulose membrane where a primary antibody is released and only binds to the target protein followed by a labeled secondary antibody binding to the primary antibodies in order to make the target protein visible
How does western blotting allow for the analysis of protein modification?
during the immunoblotting phase the primary antibodies can distinguish b/w phosphorylated and non-phosphorylated proteins allowing for the ability to distinguish b/w the two
What does co-immunoprecipitation do?
its used to confirm that certain proteins interact w/ each other (are part of the same complex)
distinguish co-IP (co-immunoprecipitation) and IP?
- IP isolates one protein of interest
- co-IP isolates protein complexes (consist of multiple proteins bound together) of interest
Breifly describe the 4 steps to immunoprecipitation
- use an antibody to targets a protein of interest w/in a sample
- add agarose beads which attaches to the antibody
- the agarose beads cause the target protein to be isolated from the rest of the sample
- use a western blot to confirm the presence of the target protein and its abundance