W16 Flashcards
How does ligand binding activate a GPCR?
Ligand binding causes a conformational change that moves the transmembrane helix 6 (TM6) and opens the cytoplasmic binding pocket for the Galpha subunit to bind
How are heterotrimeric G proteins localised to the plasma membrane?
Alpha and gamma subunits are post-translationally modified with covalently bonded lipid molecules that comprise the plasma membrane
What happens when the GPCR cytoplasmic binding pocket is opened?
Galpha Ras domain alpha5 helix binds, causing a conformational change whereby the alpha5 helix rotates and the AH clamp domain releases GDP. GTP displaces the GDP bound to the alpha subunit, causing the alpha subunit to dissociate from the beta-gamma subunit
What is the function of the Galpha Ras domain?
GTPase activity hydrolyses GTP to GDP, inactivating the Galpha subunit in absence of receptor activation
What is the function of the Galpha AH domain?
Clamps GDP in place, keeping Galpha stable in the inactive state in the absence of receptor activation
What is the function of the Galpha s subunit in GPCRs?
Activates adenylyl cyclase, produces second messenger cAMP from ATP, cAMP activates protein kinase A, cascade reaction
Describe receptor desensitisation of GPCRs.
GRK phosphorylates the GPCR, allowing arrestin to bind, blocking Galpha binding, arrestin may couple the endocytic pathway, GPCR internalised into an endosome and either dephosphorylated and recycled back to membrane or degraded
Describe two ‘brakes’ within the GPCR signalling cascade.
Regulators of G-protein signalling (RGSs) or GTPase activating proteins (GAPs) activate Galpha GTPase to quench the signalling response.
Phosphodiesterases break down second messengers, minimising response
Describe the structure of phosphatidylinositol.
Two fatty acid chains derived from arachidonic acid, connected via a glycerol moiety to a hydrophilic sugar head group
Describe the extended members of the phosphoinositide family.
Three phosphoinositide monophosphates, three phosphoinositide diphosphates, and a single phosphoinositide triphosphate
What are the three functional roles of phosphoinositides?
Regulators of integral membrane proteins, membrane localised sites for the recruitment/activation of cytosolic proteins, substrates for phosphoinositide-specific phospholipase C
Describe the phosphoinositide-specific phospholipase C pathway.
Phosphatidylinositol is phosphorylated at the 4’-OH via phosphoinositide 4-kinase followed by phosphorylation at the 5’-OH via type I phosphoinositide kinase to form phosphoinositide 4,5-bisphosphate PI(4,5)P2. Both kinases are located at the plasma membrane. PI-PLC hydrolyses PIP2 into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3)
What is the function of DAG?
Activates protein kinase C
What is the function of IP3?
Regulates Ca2+ release in the cell
How is PLCbeta activated?
Effector molecule for Galpha q subunit, recruited to the plasma membrane as the Galpha q is tethered to the membrane and the PtdIns(4,5)P2 substrate is localised to the membrane
What are the intracellular and extracellular concentrations of Ca2+?
Intracellular = 100nM
IntraER = 0.2mM
Extracellular = 2mM
Why is intracellular [Ca2+] kept low?
At high [Ca2+], phosphate precipitates
How is [Ca2+] kept low in the cytosol?
Pumped out of cell via Ca2+ pumps, Ca2+/Na+ exchangers working alongside Na+/K+ pumps, and pumped into the ER via Ca2+ pumps, all pumps requiring ATP as they work against the concentration gradient
How does IP3 trigger an increase in cytosolic [Ca2+]?
Binds to IP3 ligand-gated Ca2+ ion channels on ER membrane, increasing cytosolic [Ca2+] by 10-fold (to ~1000nM), stimulating Ca2+ effectors to regulate Ca2+-sensitive processes on a large timescale from microseconds to hours
Describe the binding at IP3 receptors on the ER membrane.
IP3 binding in saturation (bound to at least 3/4 subunits of tetramer receptor) alone only triggers conformational change to an intermediate conformation. Cytosolic Ca2+ must also bind as a co-agonist to change conformation to the open state. Cytosolic [Ca2+] at rest favours channel opening (positive feed forward) until a concentration exceeding ~400nM by which the equilibrium shifts to favour the closed intermediate (negative feedback), producing Ca2+ oscillations
How do ER Ca2+ stores become depleted?
When cytosolic [Ca2+] reaches 10-fold resting state (~1000nM) Ca2+ is predominantly pumped out of the cell via plasma membrane channels and a small proportion is pumped back into the ER, eventually becoming depleted and inducing store-operated Ca2+ entry
Describe store-operated Ca2+ entry.
At resting [Ca2+] in the ER, Ca2+ binds the lumenal side of Stim1, but at low concentration it no longer binds, causing Stim1 dimers to form an oligomer on the cortical ER and stimulate Orai Ca2+ channels to open. When ER stores are replenished, Ca2+ binds Stim1 and dismantles the oligomer, closing Orai1
What is calmodulin?
Ca2+-binding protein binds Ca2+ at both N- and C-termini, acts on a target protein and locks said protein in an active conformation
Describe Ca2+/calmodulin-dependent protein kinase II (CaMKII).
Six kinase domains interspersed with hub domains, in the inactive state the kinase domains are free to move in and out of plane with the hexameric structure. When out of plane, kinase domain can bind Ca2+-bound calmodulin and become locked in the out-of-plane conformation. When two adjacent kinase domains are locked in the out-of-plane conformation, they autophosphorylate each other and become active. If Ca2+-bound calmodulin dissociates, non-phosphorylated kinase domains revert back to the in-plane conformation but autophosphorylated kinase domains remain partially active until phosphatase dephosphorylates them
