lectures 11 and 12 Flashcards
g protein coupled receptors (GPCR)
present in all eukaryotic cells
involved in most biological responses
wide variety of different ligands - lipids, peptides, neurotransmitters, chemicals, light
low sequence conservation across the family makes identification of the genome more difficult
GPCR signalling
7 alpha helices span the membrane
on the plasma membrane side they interact with different proteins
GPCR second messengers
small molecules that relay signals from cell receptors to intracellular targets
3 main types - hydrophobic, hydrophilic and gases
levels increase in response to activation of appropriate receptors - increased synthesis or release from extracellular stores
bind to target proteins and change their activity
increase is transient and they are actively degraded or removed
activation of GPCR
have no catalytic domain so we need to know the structure to know how they work
it is not well understood
thought to exist in active or inactive forms
lipid binding affects the equilibrium between 2 states
no ligand = inactive state facvoured
agonist = equilibrium shifter toward active state
antagonist/inverse agonist = equilibrium shifted toward inactive state
neutral antagonists does not shift equilibrium but may block against binding
structure of GPCR
difficult to carry out due to membrane proteins being dificult to express and crystallise, the flexible loop regions ability to change conformation makes crystallisation difficult
to overcome these problems the use of detergents to extract gpcrs from lipid bilayers and the stabilisation of protein structures through: truncation of n or c terminals, mutations of intra or extracellular loops to decrease flexibility, fusion of gpcrs with stable protein domains - these may also stop proteins folding into the same conformation as found in the cell
agonist binding of GPCR affect on structure
thought to disrupt hydrogen binding in gpcrs
this allows the shift in orientation of 6 and 7 helical segments and the insertion part of the g subunit into the gpcr helical bundle
activating ligand binding affect on GPCR structure
conformational changes are transmitted to the intracellular aspects of the receptor
activated receptor then associates with a gdp bound heterotrimeric g protein and acts as a gef to promote release of gdp and binding of gtp to the g alpha subunit
leads to dissociation of the g-beta-gamma subunits
either g alpha or g beta gamma binds the down stream effectors to modulate their activity
g-beta/ g-gamma dimers
they are always found as a dimer
most combinations of different isoforms are possible - functional consequences of different dimers is not clear
c terminus of Ggamma is prenylates which results in localisation of GbetaGgamma dimer to plasma membrane
what is prenylation
involves the transfer of either a hydrophobic farnesyl or geranyl-geranyl group to a c terminal cystine on target protein
2 main functions of Gbeta/Ggamme dimers
- regulation of Galpha subunit
- promotes retention of gdp to galpha subunit
- helps localise g alpha subunit to the membrane - galpha independent signalling
- activation or girks
g alpha subunit
16 genes, at least 23 distinct g alpha protein subunits in humans
responsible for majority of signalling activated by heterotrimeric g proteins
g alpha subunits can be myristoylated to help localise them to the membrane
bind to various downstream effectors, the binding site for this is similar to that of the gbeta/ggamma dimer so they cannot both bind at once
poses intrinsic gtpase activity, hydrolysis of gtp to gdp inactivates galpha subunit and allows it to re-associates with gbeta/ggamma dimer
some effectors have gap activity, gtp hydrolysis can also be promoted by regulator of g protein signalling proteins - these also have gap activity and function to attenuate galpha signalling
what is myristoylation
invovles covalent attachment of a myristoyl group via an amide bond to the alpha amino group of an n terminal amino acid of a proteins as it is being made
cAMP signalling
a cyclic nucleotide found in organisms
is a good 2nd messenger molceuls
levels can be increased and decreased rapidly
small and diffusible
very close control of intracellular concentration
generated by the actions of adenylate cyclases and can be removed by phosphodiesterases
adenylate cyclases
found in all organisms
6 classes identified- 1,2,4,5,6 are bacterial enzymes
3 are in prokaryotes and eukaryotes
mammalian cells have 10 class 3
1-9 are transmembrane proteins and 10 is a soluble protein regulated by gpcr signalling
activation of adenylate cyclases
main activation through galpha subunit
Gialpha is inhibitory
different one are not all activated the same way
effects of cAMP
can activate 2 main cellular pathways by PKA or EPAC
PKA has protein kinase activity, EPAC has GEF activity - both bind and are activated by cAMP
synthetic analogues of camp have been described that activate PKA or EPAC but not both - this allows specific effects to be studied
guanine nucleotide exchange factors activates monomeric gtpases by stimulating the release of gdp allowing binding of gtp
EPAC signalling
2 different domains, one associates with the membrane the other with gef activity
n terminal - membrane association, camp binding
c terminal - gef activity, rap1 is a small gtpase, cell adhesion and gap formation
PKA signalling
member of acg family of protein kinses
inactive pka exists in a complex of 2 catalytic and 2 regulatory subunits
regulatory subunit contains a pseudosubstrate motif that binds the active site of the kinase substrate
PKA functions
it phosphorylates proteins at Arg-arg-xaa-ser
LEARN EXAMPLES FROM POWERPOINT
transcriptional regulation by pka
see notes
termination of cAMP signalling
camp is removed by phosphodiesterases which hydrolyse camp to amp
can be regulated at multiple levels - expression, subcellular localisation, protein-protein interaction, phosphorylation
targeting of PKA
PKA can interact with A-kinase anchoring proteins (AKAPs)
all akaps contain a amphipathic helix that binds to the regulatory subunit of PKA
different akaps show a wide variety of structural diversity and mechanism of action
AKAPs act by localising PKA to distinct subcellular compartments and recruiting pka to other signalling proteins
mAKAP anchors pka and pde to the perinuclear membrane in myocytes, controls both localisation and feedback inhibition of the cAMP signal
cellular calcium levels
resting cells cytoplasmic levels are approx 1200 lower than extracellular
there may be a requirement of phosphate used in biosynthesis and energy pathways, calcium phosphate is insoluble
low calcium conc provides ability to use transient changes in calcium levels for signalling
calcium only diffuses short distances before being bound by other factors in the sytosol
calcium sotres exist in the ER and mitochondria
maintaining resting calcuim
to allows the low levels of calcium against conc gradient across the plasma membrane, calcium must be exported by calcium exchange pumps in the plasma membrane which allows it to be removed
calcium is exported by plasma membrane ca2+ atpase (PMCA) or na+/ca2+ exchanger (NCX)
PMCA - high affintiy for calcium, low transport rate, v effective at maintaining low ca2+, uses atp to drive calcium out the cell
NCX - low affinity for calcium, high transport rate, effective at maintaining low calcium level, uses diffusion of sodium into the cell to move calcium out
advantages of calcium as a 2nd messenger
fast - fast increases and decreases of levels due to low intracellular conc and high extracelluar
revesible - can be pumps back into intracellular stores from the cytoplasm and is quickly reversible so repeated stimulation is possible
measurement of cytoplasmic calcium
alizarin sulphonate - binds to calcium from pruple crystals that can be seen under a microscope - quantification is poor as not real time
aequorin - phosphoprotein isolated from jellyfish, emits light when calcium binds - doesnt leak out of cells by has low light emission
quin2 - exhibits increased fluorescence when bound to calcium and allows quantification but has low fluorescence and require excitation wavelength that can give rise to autofluorescence in cells
ino1, fluo3/4 - same as quin 2 - added as an ester into cells so once it is added it cleaves and cannot leave
cameleons/GcAMP- fusion of GFP and calmodulin, protein fluoresces when it binds to calcium, can be stably expressed in cells of animals
GPCRs trigger calcium signalling
ligand binds to GPCR
g-alpha subunit is released and binds to phospholipase c
binds to the membrane and DAG+IP3 releases IP3 to bind t0 the calcium channel to activate it and allows for calcium to leave the cell
