Flashcards in Lecture 8 - G Protein Signaling Deck (72):
What are 2 main intracellular signaling pathways? How can both be removed?
2. GTP-binding protein
Hydrolysis can remove P in both
What is the primary messenger of a GPCRs?
The ligand: hormone, NT, etc.
What are 3 characteristics of G proteins?
1. Need both the ligand and GTP for the trigger the production of a second messenger
2. Receptor agonists stimulate a low Km GTPase
3. GDP and GTP modulate the affinity of a receptor to agonists, but not to antagonists
How are G proteins classified?
By the identity of the alpha subunit
How many G protein alpha subunit genes do we have? how many proteins do they encore for?
9 genes encoding 12 proteins
What are the 2 parts of the G protein alpha subunit?
1. Guanine nucleotide binding site
2. GTPase activity
Describe the binding of GTP to the G protein alpha subunit?
Very high affinity
What do the kinetics of the GTPase activity of the G protein alpha subunit depend on? Explain how this works.
Presence or absence of the activated receptor: without the activated receptor kcat is 10 times faster than the dissociation rate of GDP = the rate of the GTPase is limited by the dissociation rate of GDP
In the absence of an activated receptor, what form is the G protein in? What is the purpose of this?
Alpha subunit bound to GDP
Purpose = default "OFF" state
Would GDP be able to dissociate from the alpha subunit of the G protein in the absence of an activated receptor?
Yes, but low probability event
What are the different types of G proteins that activate adenylyl cyclase?
Specific to different tissues: olfactory system and gustatory system
What are the 6 proteins that can be directly activated by G proteins?
2. K+ channels
4. cGMP phosphodiesterase
6. Rho family of GTPases
4 steps of G alpha s cycle of activation/deactivation?
1. Gs is bound to GDP = OFF
2. Gs comes in contact with an activated receptor which causes displacement of bound GDP by GTP
3. Alpha subunit bound to GTP dissociates from beta and gamma subunits and activates AC. This also USUALLY causes the ligand to dissociate from the receptor
4. Galpha s hydrolyzes GTP thereby turning itself off and reassociates with the beta-gamma dimer
How does the beta-gamma dimer affect the alpha subunit of the G protein?
WHEN IN THE HETEROTRIMER form, the dimer stabilizes the OFF state by increasing the affinity of the alpha subunit for GDP
How does the dissociation of the alpha subunit from the G protein heterotrimeric form affect the receptor? What is this called?
It inhibits its binding to the ligand by decreasing its affinity for it = heterotropic allosteric inhibition of the receptor
What is the built in OFF mechanism of the G alpha GPCRs?
They have low affinity for the ligand when the G alpha subunit dissociates
When is the enzyme targeted by the alpha subunit of the G protein active?
When bound by G alpha s bound to GTP
How is cAMP degraded after being activated by AC? Equation.
Cyclic nucleotide phosphodiesterase: cAMP => 5'-AMP
How is cAMP formed?
Adenylyl cyclase: ATP => cAMP + PPi (3' OH attacks alpha P)
What is the full name of cAMP?
Adenosine 3'-5'-cyclic monophosphate
Why is cAMP a good second messenger?
Very easily degradable
Will a GPCR agonist affect the affinity of the receptor to the ligand?
Yes! Because through activation of G alpha s, the affinity of the receptor to the ligand will decrease
Will a G alpha GCPR antagonist affect the affinity of the receptor to the ligand?
Usual # of transmembrane domains in GPCRs?
How does desensitization of GPCRs work? 2 ways (what do we call the first one?)
1. Removing the receptor off the cell surface = homologous desensitization
2. Phosphorylation of the receptor inhibiting its ability to interact with a G protein
Describe the extracellular domains of transmembrane proteins.
Glycosylated and have disulfide bonds
Are disulfide bonds and carb groups ever found in the cytosolic domains of transmembrane proteins? Why?
NOPE because of reducing environment
Where is the ligand binding site of transmembrane receptors?
Within the membrane
What parts of the GPCRs interact with the G protein alpha subunit?
2nd and 3rd cytosolic loops
What parts of the GPCRs interact with the G protein beta-gamma subunit?
Where is the N-terminal of GPCRs found?
What kind of kinases are activated by cAMP? What does this mean?
Multifunctional kinases that can phosphorylate many different kinds of molecules
What are 2 types of multifunctional kinases? Where do they phosphorylate?
1. Serine/Threonine kinases
2. Tyrosine kinases
Both on OH group
What does kinase phosphorylation add to a molecule?
How does phosphorylation of a protein affect it?
1. Changes its conformation, thereby its enzymatic activity
2. Provides new binding site
What are the 5 multifunctional serine/threonine kinases?
3. Calcium/Calmodulin-dependent protein kinase
5. MAP kinase
What is another name for PKA?
cAMP-dependent protein kinase
Describe the structure of PKA
1. 2 catalytic subunits that dimerize in the inactive state
2. 2 regulatory subunits = dimer
Describe the activation of PKA by cAMP. 2 steps
1. 2 cAMP bind to each regulatory subunits of PKA
2. 2 regulatory subunits release the catalytics subunits
What do a lot of tyrosine/serine kinases have in common? How do they differ (2 ways)?
Catalytic subunit features and AA sequences
Differ in what context they phosphorylate Ser/Thr and in the nature of their regulatory domain
Describe the 2 main parts of the catalytic subunit of protein kinases.
1. ATP binding region
2. Peptide binding and catalytic region
List the 5 domains of the PKA regulatory subunits.
1. Dimer interaction site
2. Antigenic sites
3. Peptide inhibitory site
4. cAMP binding domain A
5. cAMP binding domain B
What is the role of the peptide inhibitory domain of the PKA regulatory subunits?
They bind to the catalytic subunits of PKA
What is the role of the dimer interaction site of the PKA regulatory subunits?
It holds the regulatory subunits together
How are all multifunctional kinases regulated? Overall and 2 mechanisms?
Regulatory subunits have similar sequences to the substrates and fold over the binding sites of the catalytic domains
Type I: pseudo P site of regulatory subunit binds but does not get phosphorylated
Type II: auto P site of regulatory subunit binds and gets phosphorylated which creates a kink
Which regulation mechanism of multifunctional kinases allows them to be activated faster? Why?
Type II because of the kink in the binding of the regulatory subunits created by the phosphorylation
Which regulation mechanism of PKA is the most common?
Are there more kinases or phosphatases?
What are the 3 pathways of the phosphatidyl inositol 4,5-bisphosphate (PIP2) system? What determines which ones happens?
1. DAG-protein kinase C
2. Arachidonic acid/Prostaglandin
Regulation provided by which phospholipase acts on PIP2
What is PIP2?
Glycerophospholipid cleavage: phospholipase D: cleavage site? products?
Cleavage site = bond between polar head group and P
Products = phosphatidic acid + polar head group
Glycerophospholipid cleavage: phospholipase C: cleavage site? products?
Cleavage site = bond between P and O attached to C3
Products = head group-P-O2 + DAG
Glycerophospholipid cleavage: phospholipase A1: cleavage site? products?
Cleavage site = FA-O bond at C1
Products = FA + lysophospholipid
Glycerophospholipid cleavage: phospholipase A2: cleavage site? products?
Cleavage site = FA-O bond at C2
Products = FA + lysophospholipid
Which phospholipase does NOT have a phospholipid as a product?
What is phospholipase C beta? Products?
Subtype of PLPC that recognizes PIP2 as a substrate is is coupled to G alpha q
Products: DAG + IP3
What does IP3 stand for?
Very hydrophilic and charged
What are 3 isozymes of PLPC? What is each activated by? What is each specific to?
1. PLPC Beta: G alpha Q coupled specific for PIP2 (also requires Ca++ for activation)
2. PLPC Gamma: activated by RPTK and specific for PIP2
3. PLPC Delta: activated by Ca2+ and specific for PIP2
Very lipophilic membrane protein kinase
Other than DAG, what else is PKC dependent on?
On what 2 membranes can IP3 bind to release intracellular Ca++ stores?
How does intracellular Ca++ concentration fluctuate?
Quick spikes with low dips due to the combination of:
1. IP3 receptors: calcium channel with low Km activating site for Ca2+ (positive feedback) and high Km inhibitory site for Ca2+ (when Ca2+ is very high there is negative feedback)
2. Many mechanisms to keep [Ca++] low (uptakers, transporters, etc)
What about the fluctuation of intracellular [Ca++] is most important?
The frequency of the spikes, not their amplitude
Describe how calcium-calmodulin dependent protein kinases (CaM-kinase) are activated. 3 steps
1. 4 calcium molecules bind calmodulin
2. Complex binds the protein kinase, activating it
3. The protein kinase will INTRAmolecularly autophosphorylate to a minimum extent: FULL activation
Describe the binding of calcium to calmodulin?
Sigmoidal cooperative binding
Describe how calcium-calmodulin dependent protein kinases are deactivated. 2 steps
When does this happen
Clock mechanism: happens after a certain number of phosphorylations
1. Calcium is released from fully active protein kinase = Ca2+ independent protein kinase
2. Protein phosphatase hydrolyzes the P off = inactive protein kinase
How active is the Ca2+ independent protein kinase?
50-80% active for AUTO phosphorylation, NOT substrate phosphorylation
How does CaM-KII read [Ca++]? How does this activate it?
It exists as multimers and has a frequency decoder of Ca++ oscillations
It will be fully activated under conditions of high frequency calcium concentration spikes because the baseline [Ca++] will not be able to go back to normal
What kind of receptor does vasopressin bind to?
GPCR G q that activates PLPC beta
How does intracellular [Ca++] change as vasopressin concentrations increase?
As vasopressin concentration increases, the frequency of calcium spikes increase, but their amplitude does not