Outline a1 activation by adrenaline
- Gq activates PLC
- PLC cleaves PIP2 which increases DAG + IP3
- IP3 interacts with Ca2+ channels on ER and SR
- effects: calcium added to proteins + PKC
- > prominent slow depolarising current
PKC characteristics
- cytosolic protein
- > increase cytosolic [Ca]2+ allows PKC to interact with membrane phospholipids
- > contact with DAG activates PKC, allowing it to phosphorylate its target membrane
Outline calcium calmodium binding
- Cytosolic [Ca++] ~ 0.1uM -> increase 100 fold
- Ca++ binds to calmodium calcium sensor
- Conformational change in calmodium -> binds proetins
- CaM kinases phosphorylate their target proteins
- Calmodium can also be a protein domain
Points of amplification of a1
- Adrenaline:receptor complex is able to catalyse GTP:GDP exchange on multiple G-protein
- Each PLC can catalyse many IP3 and DAG’s
- Each activated Ca channel releases many Ca2+
- Each activated PKC can phosphorylate many membrane proteins
How to shut off a1 adrenaline stimulation?
- DAG is either hydroxylated or phosphorylated with an alcohol
- IP3 is converted to Inositol by phosphatases
- Calcium taken into ER by Na/Ca exchanger, Ca/ ATPase pump
Receptor Tyrosine Kinases
Dimers with E.C. hormone-binding domain + I.C. protein tyrosine kinase domain
Tyrosin kinase mech of action
- Upon membrane binding
- RTK monomers cross phosphorylate each other
- Phosphorylation RTK makes it a site of attachment for proteins with SH2 domains / PTB
- Localising proteins at memb.
How does insulin become fully active?
Phosphorylation of kinase
EGFR activation mech
- Binding EGF to each EGFR monomer induces structural charge
- Monomers dimerise
- Proximity of cytosolic domains allows cross phosphorylation
- Tyrosine phosphates act as docking sites for Grb-2, attached to sos
- Sos catalyses GDP -> GTP on membrane-bound Ras, activating it
- GTP: Ras binds and activates Raf a membrane bound protein kinase
- Series of protein kinases are phosphorylated + activated => phosphorylation of sacral TF’s altering activity
Grb-2
‘Adaptor protein’ = bridge to link proteins
-> composed of SH2 domain sandwiched between 2 SH3 domains
What do the different domains on Grb-2 do?
SH2 - binds sequences containing phosphorylated-Tyr
SH3 - binds proline-rich sequences
Sos
Guanine nucleotide exchange factor
-> catalyses GDP -> GTP on Ras protein when recruited to membrane via Grb-2
Ras
Small G-protein -> monomeric
-> slower GTPase activity than heterotrimeric
What do GAPs do
Increase GTPase activity by ~10^5 fold
Outline insulin receptor signalling
- Binding of insulin to dimeric receptor forces PTK domains together
- > cross-phosphorylation - Fully activates kinase activity -> more cross-phosphorylation
- Phosphorylated tyrosine residues act as docking sites for IRS-1 which gets phosphorylated
- Phosphorylated IRS-1 can bind PI-2K which now, located at mem phosphorylated PIP2
- PIP3 made from PIP2
PIP3 effects
Allows PPK1 + PKB -> associate with membrane via pH domains
-> phosphorylated PKB dissociates from memb. + phosphorylated target proteins
IRS-1
Phosphorylated on several tyrosine residues
-> IRS-1 already associated with membrane due to pH domain which can bind PIP2
What happens when IRS-1 is phosphorylated
Dissociate from insulin receptor
Docking protein
Can bind many proteins including Grb-2 (MAPK pathway)
How can insulin response be short and long?
Other proteins e.g. IRS-2 can assemble at phosphorylated insulin receptor
-> simultaneous stimulation of neurons for short + long term
Which proteins bind PIP3 and what are the effects?
- PDKA 1 via PH domain -> localises it to memb+ activates it
- PKB via PH domain -> phosphorylated + activated by PDK1
- > phosphorylates several proteins in both cytosol + nucleus
Insulin points of amplification
- Insulin receptor can phosphorylate multiple IRS-1 proteins
- PI-3K can catalyse formation of multiple PIP3 molecules
- PDK1 catalyses formation of multiple PKB enzymes
- PKB can go on to phosphorylate many proteins
How are insulin receptors shut down?
- Lipid phosphatases, recruited by active insulin receptors, dephosphorylate PIP3
- Insulin activates protein phosphatase 1 which counteracts PKA actionm inhibiting PP1
- > stimulated by glycogen + AD
