GPCR Flashcards
What is the characteristic of GPCR
7 TM
EC amino
IC carboxyl terminal
Ga-s
cAMP increase
g A OLF
Regulates
> calcium channels
c-src TYROSINE KINASES
Ga -i
can regulate tyrosine kinase
reduce cAMP production
Ga-q
regulate the activate of PLC ( phospholipase c) of beta isoform
to generate IP3 and calcium signalling
G beta-gamma
***KIR3,1-3,4 inward rectifying potassium channel
- in CSN and PNS
»activation of this potassium channel»_space; HYPERpolarisation of the neuron, so INHIBITION of the neurotransmission
*** GRK ( g protein regulated kinases)
Receptor function
location of the receptor and its effector will have functional consequences
OPIOD receptor example for localisation
Location in the brain > tend to be in the dendrite and the soma >control potassium channel > inhibit initiation of action potential > dampen excitability
Interneurons in the pain pathway
>at the nerve terminal (not cell body)
>couple to the Cav to inhibit neurotransmitter release
same receptor can couple to different effector depending on where it is localised
Common tools for studying GPCR signalling
- Radioactive GTP-gamma-s
- Cholera toxin
- Pertussin toxin
- Measuring second messenger such as cAMP and Calcium using biosensors
- favourite tool, measure Ca2+ signalling via re-engineered GPCR that has the carboxyl terminus modified to allow it to couple to Gq
- radioactive GTP-gamma-s
* quantify activation of GPCR receptor
* when agonist bind to the receptor> exchange of GDP to GTP
* radioactive GTP-gamma-s , Alpha subunit will be loaded with this
* final phosphate group is not connected to oxygen, rather to a suphate –> conseqnce can NOT be hydrolized (alpha subunit now is permanently bound to the GTP-g-s)
* purify and quantify the radioactivity
* any effector will be penanently switched on - Cholera toxin
* bacteria that causes cholera
* irreversibly activates G-a-s
* add sugar group to the G-a-s subunit, causing the alpha subunit to be permanently active. cannot hydrolized
* massive cAMP increase
* increase upregulation of CFTR, increase Cl- in water into the gut, diaeorrhea - Pertussis toxin
* irreversibly activates Gai and Gao
* add sugar group to the alpha subunit
* stop the exchange of GDP to GTP
* G protein is irreversibly INACTIVATED - Measure 2nd mesenger using Fluorescent Biosensors
* Fluorescent engineered protein that binds to cAMP or lipid
* measure the fluorescence of the reporter protein - FAVOURITE TOOL - measure the ca2+ level
*small molecule that change fluorescence upon Ca2+ binding
*modify the interesting receptor by changing the carboxy terminus of the receptor to couple to the gq and lead to increase in Ca2+ . Does not have to change the ligand binding site
*re-engineer receptor to signal via calcium
*to find new agonist and antagonist
agonist if increase Ca2+, antagonist if otherwise
tools for studying GPCR signalling - radioactive GTP-gamma-s
- radioactive GTP-gamma-s
* quantify activation of GPCR receptor
* when agonist bind to the receptor> exchange of GDP to GTP
* radioactive GTP-gamma-s , Alpha subunit will be loaded with this
* final phosphate group is not connected to oxygen, rather to a suphate –> conseqnce can NOT be hydrolized (alpha subunit now is permanently bound to the GTP-g-s)
* purify and quantify the radioactivity
* any effector will be penanently switched on
tools for studying GPCR signalling - Cholera toxins
- bacteria that causes cholera
- irreversibly activates G-a-s
- add sugar group to the G-a-s subunit, causing the alpha subunit to be permanently active. cannot hydrolized
- massive cAMP increase
- increase upregulation of CFTR, increase Cl- in water into the gut, diaeorrhea
tools for studying GPCR signalling - Pertussis toxin
inactivating Gi and Go
- irreversibly activates Gai and Gao
- add sugar group to the alpha subunit
- stop the exchange of GDP to GTP
- G protein is irreversibly INACTIVATED
tools for studying GPCR signalling -Measure 2nd mesenger using Fluorescent Biosensors
- Fluorescent engineered protein that binds to cAMP or lipid
* measure the fluorescence of the reporter protein
tools for studying GPCR signalling - ca2+ level
- FAVOURITE TOOL - measure the ca2+ level using biosensors
*small molecule that change fluorescence upon Ca2+ binding
*modify the interesting receptor by changing the carboxy terminus of the receptor to couple to the gq and lead to increase in Ca2+ . Does not have to change the ligand binding site
*re-engineer receptor to signal via calcium
*to find new agonist and antagonist
agonist if increase Ca2+, antagonist if otherwise
Collision coupling theory vs precoupling theory
Precoupled theory
> GPCR and the g proteins are precoupled
Collison theory
> GPCR and the g protein are NOT precoupled
>Only come together when the receptor is in active configuration
> potential for more diversity in the signalling
>so single receptor can couple to multiple g protein
evidence? FRET analysis by hein et al 2005
FRET analysis by Hein et al 2005
Fluorescence resonance energy transfer
CFP - FRET donor put on the g protein (bg subunit)
YFP - FRET acceptor was put on the receptor GPCR
cyan and yellow fluorenscent protein
- Measure emission of yellow light by YFP
- Shine light that can make the CFP emit blue light
- Measure intensity of blue and yellow light
- Add nerepinephrine NE
- measure yellow light
- YFP can only emit yellow light by receiving blue light from the CFP. So, YFP can only emit yellow light when in close proximity/ coupled
result of the experiment
- without NE, they can only visualise blue light,
- add NE, they can now visualise the yellow light
- insert graph here (blue low, yellow high, yellow/blue high)
- without ligand, GPCR and the G protein are not precoupled ( missing yellow light)
- after ligand binding (NE) yellow to blue light ration drastically increase suggesting that the receptor and the g protein are now closer toether and coupled
Catecholamine receptor
[NE and Epinephrine (noradrenaline and adrenaline receptor)]
couple to Gas
increase cAMP
Lefkowitz 1997
- a cardiologist in New York
- research on GPCR (B-AR)
- treatment for heart disease
studying Beta Adreno Receptor (B-AR)
evidence that GPCR can couple to multiple G proteins and multiple signalling pathway
Beta Adreno Receptor (B-AR)
Can cause
1. increase in cAMP, PKA
and also
2. activate MAPK (erk) pathway
what is the evidence that GPCR can couple to multiple G proteins and multiple signalling pathway
Lefkowitz 1997 ( SWITCHING OF THE COUPLING OF THE B2-AR TO DIFFERENT G PREOTINS BY PROTEIN KINASE A PKA)
result of the experiment EARLIER 1. B-adrenoreceptor couple to Gs 2. increase cAMP -> increase PKA 3. PKA phosphorylate its B-AR receptor LATER 4. The phosphorylation of the receptor cause the B-AR to switch to couple to Gi 5. beta-gamma subunit then increase the activity of src,sos,ras 6 increase activity of MAPK
in a TIME-DEPENDENT MANNER
context specific of signalling pathway?
signalling pathway activated by receptors in a cell is CONTEXT SPECIFIC
different cell, locations , condition result in different signalling pathway
result in greater control
SPECIFIC SIGNALLING is controlled by
- scaffold protein
- lipid
- endocytosis
- splicing
- post translational regulation
RGS protein
Regulator of G protein signalling
small g protein like RAS and RAF ( they have activating and inhibiting protein)
works kinda lliddat
What molecule can regulates receptor function?
GRKs and B-arrestin
Desensitisation of GPCR
- KEY EVENT THAT restrict THE SIGNALLING
1) homologous desensitisation
> receptor activated by the agonist, act on their receptor to inhibiti the receptor function
2) heterologous desensitisation
- receptor being activated impacts on another type of receptor
insert diagram here
agonist + receptor -> activated receptor 1 + GRK ( GPCR kinase, disrupt coupling of GPCR and g protein by phosphorylating the serine residues on the carboxyl terminus of the receptor) -> arestin-receptor complex [arrestin+ligand+receptor comples] -> a) endocytosis b) loss of GPCR coupling [HOMOLOGOUS desensitisation]]
ACTIVATED RECEPTOR 1 –(another pathway)–> activation of PKA, PKC etc -> phosphorylate RECEPTOR 2,3,…etc to cause reduced in G protein coupling [[[ HETEROLOGOUS DESENSITISATION]]]
GRK?
PLECKSTRIN HOMOLOGY DOMAIN in GRK
G protein couple Receptor Kinase
is a serine-threonine kinase
phosphorylate the serine residue at the carboxyl terminal of the receptor and disrupt coupling with the g protein
when receptor is activated, g protein active, bg subunit will dissociate form the receptor, after a long time will bind to GRK due to PLECKSTRIN HOMOLOGY DOMAIN in GRK
next»_space; beta arrestin bind to the GPCR
Beta arrestin
ARRESTS SIGNALLING
attract machinery of endocytosis so that the receptor will be internalised
a)carboxyl terminal will be dephosphorylated
b)agonist come off from the receptor
NEXT
1) degradation
2) recycled
2 isoform , b-arr1, b-arr2 ( most famous)
Concern with opioid / morphine-like drug teatment
problem with tolerance
experiment : measure of pain-relief
- people with repeated drug, curve shift to the right
- more opioid / increase dose to get the same result for pain relief
WITHDRAWAL EFFECT
[[Mechanism underlying the tolerance with opioid]]
with increase use of the drug, we will be losing the receptor (internalisation of the receptor)
lower the receptor reserve, shift the curve to the right
Lefkowitz 2000, b-arr2 knockout mouse
a) gave the animals continuous low dose
b) repeated high concentration at high frequency
next, assess anaelgesia
WT - in 5 days, anaelgesia almost gone ( curve downward)
KO - no tolerance, maintai %MPE
KO- remove b-arr2, tolerance is removed
What caused tolerance with opioid and morphine like drugs?
Desensitisation at the level of beta arrestin 2 and GRK and trafficking
BARR2 - continuous admission of morphine and opioid drug caused desensitisation of the receptor by internalising the receptor after BARR-Receptor binding. the receptor will then be degraded or recycled to the plasma membrane
opioid tolerance and addiction
they are different pathway
because mice that does not develop tolerance towards opioid drug STILL develop addiction to the drug
Different agonist regulates desensitisation and internalisation
do experiment
Desensitisation occur at what point
Desensitisation occur at the point of
1) receptor is phosphorylated and can no longer signal to the g protein
What impact the internalisation of GPCR?
Depends on
1) which kinases
2) which amino acid being phosphorylated
next
which protein association and which signalling pathway
We can classify the receptor depends on what happen after internalisation
1) CLASS A
- rapid b-arrestin falling off the receptor and rapid dephosphorylation
- The receptor will be sent to degradation or sensitisation
- eg. mu-opioid receptor
2) CLASS B
- remain longer in the endosome
- such as the angiotensin receptor
- slow recycling and slow degradation
GRK
only phosphorylate agonist bound receptor
Beta arrestin
- act as adaptor proteins for endocytic machinery
- provide scaffold for endocytic machinery
- as scaffold and adaptor protein to extend the signalling capacity of the receptor
- important domains in the beta arrestin include SH3 and SH1 domain (domain for many sign. pathway such as RAF/mapkkk), clathrin ( endocytosis), JNK(other MAPk),
Beta arrestin
- act as adaptor proteins for endocytic machinery
- provide scaffold for endocytic machinery ( bringing signalling molecules together)
- as scaffold and adaptor protein to extend the signalling capacity of the receptor
- regulation of transcription to produce long term changes in cell fx
- important domains in the beta arrestin include SH3 and SH1 domain (domain for many sign. pathway such as RAF/mapkkk), clathrin ( endocytosis), JNK(other MAPk),
What other signalling molecule binds to beta arrestin 2?
- c-SRC
- ERK
- PP2A –| Akt
- Ikb –| NF-kB
What are the signalling molecue that binds to GRK
- | MEK1
- | Raf1
- | Akt
- -> PI3k –> RReceptor encocytosis
What are the signalling molecue that binds to GRK
- | MEK1
- | Raf1
- | Akt
- -> PI3k –> RReceptor encocytosis
From clinical point of view, what pathway is more favoured?
Since most agonist show biased ligand, (some biased for beta arrestin signalling pathway, some biased for the receptor -like G-ai), we want ligand that shows bias toward the receptor G-ai
because G-ai will increased anaelgesia ( pain relief)
we want to ideally have a ligand or agonist that shows no bias at all towards beta arrestin 2, which signalling pathway cause respiratory depression, nausea and constipation
