KIR3,1-3,4 inward rectifying potassium channel - in CSN and PNS >>activation of this potassium channel >> HYPERpolarisation of the neuron, so INHIBITION of the neurotransmission GRK ( g protein regulated kinases)

GPCR Flashcards by Dhyra Osem Science

What is the characteristic of GPCR

7 TM
EC amino
IC carboxyl terminal

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Ga-s

cAMP increase

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g A OLF

Regulates
> calcium channels
c-src TYROSINE KINASES

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Ga -i

can regulate tyrosine kinase

reduce cAMP production

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Ga-q

regulate the activate of PLC ( phospholipase c) of beta isoform

to generate IP3 and calcium signalling

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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)

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Receptor function

location of the receptor and its effector will have functional consequences

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Catecholamine receptor

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[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

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studying Beta Adreno Receptor (B-AR)

evidence that GPCR can couple to multiple G proteins and multiple signalling pathway

Beta Adreno Receptor (B-AR)

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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

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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?

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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

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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?

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GRKs and B-arrestin

Desensitisation of GPCR

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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 >> 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?

1. c-SRC 2. ERK 3. PP2A --| Akt 4. 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

GPCR Flashcards

(41 cards)