Desensitization, Phospholipase C and Calcium Flashcards
What is desensitization?
Reduced responsiveness of a tissue to an agonist that develops in response to the continuous or repeated stimulation
How is GPCR desensitization induced?
Receptor phosphorylation by PKA and B-arrestin binding to the receptor
How does PKA repress the B-adrenergic receptor?
When B-adregernic receptor binds epinephrine, PKA phosphorylates the cytosolic domain that normally interacts with Gs protein, diminishing its capacity to activate Gs.
The intensity of the signal by further stimulation will be diminished while its phosphorylated.
Affinity for epinephrine is not changed
Why does any signaling molecule that activated PKA have the ability to desensitize other GPCRs?
Because PKA will have its inhibitory affect regardless of what initial molecule activates the pathway.
What are G protein coupled receptor kinases (GRKs)?
Kinases that phosphorylate specific serine and threonine residues on the cytosolic domain of GPCRs only when the receptor isn in an active conformation by ligand or agonist binding
What is B-adrenergic receptor kinase (B-ARK)?
a GRK that phosphorylates specific serine and threonine residues on the B-adrenergic receptor.
These phosphorylated residues serve as binding sites for B-arrestin
What is B-arrestin?
A scaffolding protein that recruits other cytoplasmic proteins.
Binds to phosphorylated B-adrenergic receptor and blocks coupling to G proteins
After B-arrestin binds B-adrenergic receptor, what proteins does it recruit?
Clathrin and AP2 which promote endocytosis of the receptor.
Can also bind proteins that link the GPCR to other signal transduction pathways
E.g. Map kinase cascade/Jun kinase cascade
What are two fates of the internalized B-adrenergic receptors that are endocytized?
They can be degraded, resulting in a net decrease in receptor content
The can be dephosphorylated and translocated back to the plasma membrane
What are Gq proteins?
A family of heterotrimeric G proteins that activate the B isoform of phospholipase C
What is the function of Phospholipase C-B?
Catalyzes the hydrolisis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)
PIP2 –> IP2 + DAG
What is the function of diacylglycerol (DAG)?
A lipophilic molecule that remains in the plasma membrane where it binds to and activates certain isoforms of protein kinase C
What is the function of inositol 1,4,5-triphosphate (IP3)?
Diffuses into the cytosol and binds to the receptor domian of IP3-gated Ca2+ release channels on the ER
What happens as a result of the increase in Ca2+ from IP3 activation of ER calcium channels?
- Recruitment of PKC to the plasma membrane
- binding of Ca2+ to calmodulin
What are the three isoforms of PKC and what is required for each activaiton?
Conventional - DAG, Ca2+, phosphatidylserine
Novel - DAG phosphatidylserine
Atypical - no regulatory domain
Which PKC isoform is translocated to the plasma membrane as a result of intracellular calcium increase?
Conventional
Why is IP3-induced increase in cytosolic calcium transitory?
- IP3 is inactivated by rapid dephosphorylation
- Calcium pumps in the plasma and ER membranes remove calcium from the cytosol
How are depleted calcium stores replenished?
Via store-operated channels (Orai 1)
What is the mechanism of Orai 1 store operated channels?
When calcium in ER is high, calcium binds to luminal surface of transmembrane STIM proteins
When Ca is not bound, STIM form oligomeric complexes that translocate to regions of the ER that are in close proximity to the plasma membrane. STIM proteins bind to and open Orai 1 channels, promoting influx of extracellular calcium into the cell
The use of calcium for signaling relies on calcium concentration gradients between the cytosol and what two compartments?
Extracellular fluid
Endoplasmic Reticulum
What are the three principle mechanisms for maintaining calcium concentration gradients?
Relative impermeability of the plasma membrane to calcium
Maintenance of a pool of stored calcium in the ER
Transporters that remove calcium from the cytosol
What are two calcium ATPases and where do the transport calcium?
Plasma membrane Ca ATPase (PMCA) - Ca out of the cell
SERCA - Ca into the ER or SR
ATP dependent**
What is the function of the Na-Ca exchanger (NCX)?
An antiport carrier on the plasma membrane that utilizes sodium influx along its concentration gradient to move Ca out of the cell
3 Na in, 1 Ca out
What are voltage-gated calcium channels?
Calcium channels closed at resting membrane potential
Depolarization induces transient opening to allow Ca influx along its electochemical gradient
What three thing scan modulated the activity of voltage gated calcium channels?
Phosphorylation ( E.g. PKA)
G proteins
Drugs and toxings
How does calcium enteric cardiac muscle cells further increase local calcium concentration?
Calcium enters through voltage gated channels, the binds to and opens calcium release channels in the sarcoplasmic reticulum, which are mad up of ryanodine receptors
What is Calmodulin?
Principal Ca-binding protein in eukaryotic cells
Has four high affinity binding sights and exhibits positive cooperativity
Has no intrinsic enzymatic activity
What is the function of calmodulin?
When calcium binding sites are occupied, CaM binds to and modulates the activity of various enzymes or effector molecules
Typically transient, but can be a permanent regulatory subunit
Describe the mehcanism of Ca/CaM-dependent protein kinase II?
This kinase has an autoinhibitory domain that usually blocks the catalytic domain. The binding of CaM disrupts the interaction and initiates autophosphorylation of the regulatory domain, which allows the enzyme to remain active after the calcium concentration in the cytosol falls
What are four junctures that the Ca2+ and cAMP pathways intersect?
- PDE activity is inhanced by Ca/CaM (antagonistic)
- PKA can phosphorylate some VGCC, increasing the probability that they will open
- Ca2+ and cAMP can influence the same target protein
- Both PKA and Ca2+/CaM dependent kinase phosphorylate CREB
