Second Messengers 2 Flashcards
IP3 structure
Small sugar, triple phosphorylated (out of 6 available hydroxyl groups)
- The different hydroxyl groups phosphorylated influence the inositols binding ability/role
- IP3 = Inositol 1,4,5-trisphosphate (tris = non-linear, e.g. 1,2,3)
IP3 is made from the cleavage of the head group of a lipid (done by phospholipase C)
IP3 mediates various responses
Smooth muscle contraction, platelet aggregation (clotting) and hormonal secretion
Important in fertilization:
- when sperm fuses with egg, IP3 is generated in the egg’s barrier to prevent second egg-sperm fusion
Routes of activating the Phospholipase C (PLC) family
1) GPCR
- Wider range of receptor agonsts: light, odour, peptides…
- Both G-alpha and G-beta-gamma subunits activate PLC (Ga - PLC-B1; Gby - PLC-B2)
2) Growth factors
- Growth factor’s double-feature ligand binds 2 receptors causing receptor dimerisation
- Intracellular TK domains activated by autophosphorylation (in close proximity)
- SH2 of PLC-g docks onto active TK domain to become active
IP3’s role in calcium distribution
Ca2+ ligand-gated ion channels on ER have an IP3 receptor (aka IP receptors)
The ER is a Ca2+ store
- SERCA actively pumps Ca2+ in
- Contains a Ca2+ buffer protein (e.g. calsequestrin)
When IP3 binds to IP receptors, Ca2+ efflux can occur from ER into cytoplasm (important in muscle cells!)
IP3 receptor (Ca2+ ion channel) structure
Enormous protein (2701-2745 residues) but only 10% involved in pore channel
- Highly damaging to cell if leaky
- 3 isoforms, assembled as a tetramer
Highly specific inositol binding site at N-terminus (far from pore)
- Ins (1,4,5) P3 binds with high affinity
- Ins (2,4,5) P3 or Ins (1,3,4,5) P4 cannot bind
Ligand-gated Ca2+ channel regulation
Tight coupling between ligand concentration and time of pore opening:
- Ca2+ release is QUANTAL (in individual amounts)
Allosteric channel modification
- Cell’s ATP status can determine kinase levels and different allosteric pathways
Measuring intracellular Ca2+ levels
2 reporter groups used:
1) Photoprotein (aequorin)
2) Fluorescent dyes based on EGTA (Fura-2, Fluo-3)
Cytosolic Ca2+ concentration is extremely low and stable (0.1uM) to increase sensitivity
- Blood levels = high (1-2mM) to ensure immediate response in cells
Ca2+ signals for release and influx
Low cellular levels of Ca2+ are detected by the ER (the ER has a finite Ca2+ capacity) which trigger the STIM1 protein:
- STIM1 facilitates ‘Capacitative Ca2+ entry’ where Ca2+ enters the cell through Ca2+ channels on the plasma membrane
- STIM1 activation results for various protein-protein signals between different organelles
Method of inhibiting IP3 activation of Ca2+ signalling
Adding another phosphate to IP3 to become IP4
- IP4 can’t bind to receptor
Ca2+ regulation of activity
inactive in ER, active when bound to Calmodulin
Negative regulation: Ca2+ hidden in ER (unable to interact with other cellular proteins/molecules)
Positive action: exerts it’s effect through the Calmodulin (CaM) binding partner
- Abundant in cells, has 4 Ca2+ binding sites
- Affects cyclin nucleotide metabolism, glycogen metabolims and Ca2+ transport
Calmodulin (CaM) activation
- Ca2+ binding causes CaM’s a-helix to fold in half, bringing the 2 globular domains together (folding a dumbell in half)
- CaM wraps itself around target molecules (interacts with simple motif in target proteins of +ve charged segment followed by hydrophobic segment, targets pattern)
- Exposes Met-rich hydrophobic domain on surface (buried as it wraps on target)
CaM controlling neurotransmitter release
2 types of neurotransmitter vesicles in presynaptic knob:
1) Juxtamembrane vesicles adjacent to synapse (1st ones released)
2) Reserve vesicles ensnared inside the cytoskeletal meshwork, must be released to migrate to synapse:
- CaMKII phosphorylates Synapsin on vesicle to break its attachment to cytoskeleton
