GPCRs 3 Flashcards
to use Ca2+ as an intracellular signal, the resting cytosolic Ca2+ levels are kept…
low
what is the concentration of free Ca2+ in the cytosol?
less than or equal to 10^-7 M
extremely low
what is the concentration in the extracellular fluid + ER?
10^-3 M
high
what does the difference In concentrations allow?
a large gradient which drives Ca2+ into cytosol across plasma + ER membrane
describe the events following a signal?
- calcium channels open
- calcium ions enter cytosol triggering calcium responsive proteins in cell
for the signalling mechanism to work, what must the resting calcium ion concentration in the cytosol be kept?
low
how can you keep the calcium ion conc low?
- keeping calcium ions out of cell
- keeping intracellular ca2+ low
keeping calcium ions out of cell
- cells have ca2+-ATPase in plasma membrane
- uses ATP to pump ca2+ out of cell
keeping intracellular ca2+ low
- ca2+ pump in ER helps keep cytosolic [ca2+] low
- if cell damaged, [ca2+] rises to high level (>10^-5 M) = dangerous
- low affinity, high-capacity ca2+ pump in inner mitochondrial membrane takes up ca2+
give an example of a specific ca2+ binding protein?
calmodulin
how many mechanisms are there altogether and how many are inside?
5 altogether
3 inside
ca2+ as an intracellular messenger
intracellular injection of ca2+ caused skeletal muscle to contract
ca2+ entry into cells
- depolarisation of PM -> influx of ca2+ via voltage-gated ca2+ channels
- binding of mol to cell-surface receptors -> ca2+ release from ER
- this = mediated via inositol trisphosphate (IP3)
what does the smooth muscle not need?
extracellular calcium to function
when was the role of the membrane first described?
1953
what are the most important inositol phospholipids?
- PIP (phosphatidylinositol-phosphate)
- PIP2 - PI- bisphosphate
hydrolysis of PIP2
PIP2 breakdown begins with binding of signalling mol to G protein linked receptor
- activated receptor stimulates trimeric G protein (Gq)
- activates enzyme phospholipase c-beta
what is the best example of hydrolysis of PIP2?
m3 rat ileum receptor
more than …. different cell-surface receptors use the hydrolysis of PIP2 pathway?
25
when PLC-beta cleaves PIP2
what 2 products does it generate?
- IP3
- diacylglycerol (DAG)
where does PLC cleave the bond?
cleaves between O of phosphate and CH2 of other mol
IP3 diffuses through…
cytosol and releases Ca2+ from ER
DAG remains in the membrane and …
activates protein kinase C
what are the 3 types of phospholipase C?
beta, gamma and delta
beta class activated by G protein linked receptors
what is the journey of IP3?
- leaves plasma membrane
- releases ca2+ from ER by binding to IP3-gated ca2+ release channels in ER memb
- channels regulated by POSITIVE feedback
- released ca2+ binds back to channel to inc ca2+ release (CICR) - sensitive to ryanodine
calcium + smooth muscle contraction
- smooth muscle cells store ca2+ in ER and SR
- released when IP3 receptor activated
- IP3 generated by Gq linked receptor activation
what is the contraction of smooth muscle activated by?
MLC phosphorylation
catalysed by myosin light chain kinases (MLCK)
activated when bound to ca2+ - calmodulin
what do myosin phosphatase reverse?
phosphorylation + cause relaxation
no regulatory proteins are involved in …
smooth muscle contraction
function of ca2+ calmodulin complex
- calmodulin binds to 4 ca2+ ions
- calmodulin changes conformation, resulting in active complex
- 2 globular hands of complex wrap around binding site on target protein
role of ca2+ in cells
- activate membrane ion channels
- regulate enzyme activity
- modulate protein function
- regulate gene expression
- kill cells! (necrotic + apoptotic death)
- life and death messenger
what is calcium the only link between?
electrical activity of neurone and end response of cell
what triggers muscle contraction?
ca2+ liberation from SR
what is different about the ca2+ unlike the other ions?
it is chemical signal carried that is important not the charge
what are the sources of ca2+ to cells?
- extracellular fluid (2mM)
- plasma membrane channels
- ligand gated channels (glutamate NMDA)
- store operated channels (open when less ER calcium stores)
what are the 2 mechanisms that operate to terminate the ca2+ response?
- IP3 dephosphorylated by enzymes
- ca2+ that enters cytosol rapidly pumped out
what happens to IP3 if it isn’t dephosphorylated?
phosphorylated to make IP4
the enzyme that catalyses the production of IP4 is activated by…
increase in cytosolic (ca2+) induced by IP3
why is the resynthesis of phospholipids important in regulation?
cell would run out of substrate
at least how many reactions are involved in the metabolism of inositol lipids?
13
the regulation reactions can be divided into 2 cycles
what are they?
- inositol lipid cycle - occurs in membrane
- inositol phosphate cycle - occurs in cytosol
what is PKC?
ca2+ dependent enzyme
what does DAG increase the affinity of?
PKC for ca2+ -> activating enzyme
what does activated PKC do?
phosphorylated specific target proteins that vary depending on cell type
what are the several physiological functions of PKC?
- secretion
- modulation of ion conductance
- control of growth + differentiation
where are the highest concentrations of PKC found?
in brain
phosphorylates ion channels there
G protein linked receptors can desensitise in 3 ways
what are they?
- become altered so longer interact with G proteins (inactivation)
- temp moved to interior of cell -> no longer have access to ligand (sequestration)
- destroyed in lysosomes after internalisation (receptor down regulation)
what does desensitisation depend on?
phosphorylation of receptor by PKA, PKC or G protein linked receptor kinases
what do GRKs phosphorylate?
serine + threonines
once receptor has been phosphorylated, what does it bind to?
member of arrestin family of proteins
what can receptor desensitisation be classified into?
- homologous
- heterologous
what are the enzymes involved in receptor phosphorylation?
- beta adrenergic receptor kinase (BARK)
- cAMP- dependent protein kinase
- protein kinase C (PKC)
homologous desensitisation
- BARK most imp
- capable of phosphorylating active agonist
desensitisation of GPCR?
- inhibitory structural alteration of receptor
- receptor internalisation
- receptor degration
arrestin takes to Cathrin coated pits + degradation
give examples of receptor up + down regulation?
- cholesterol UP by statins
- opiate UP by antagonist naloxone
- beta adrenoreceptors UP by antagonist
