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Functional interaction between
mGlu1a and GABAB receptors
RIVES Marie-Laure
Institute of Functional GenomicsInstitute of Functional GenomicsDepartment of Molecular PharmacologyDepartment of Molecular Pharmacology
UNIVERSITE MONTPELLIER IIUNIVERSITE MONTPELLIER IISCIENCES ET TECHNIQUES DU LANGUEDOCSCIENCES ET TECHNIQUES DU LANGUEDOC
T H E S E:T H E S E:
Discipline : Discipline : Pharmacologie Moléculaire – NeurosciencesPharmacologie Moléculaire – NeurosciencesEcole Doctorale : Ecole Doctorale : Sciences Chimiques et Biologiques pour la Santé Sciences Chimiques et Biologiques pour la Santé
February 12th, 2009 February 12th, 2009
G protein-coupled receptors: Important targets in Drug DesignG protein-coupled receptors: Important targets in Drug Design
Source: University of Leiden, The NetherlandsSource: University of Leiden, The Netherlands
GPCRs = 3% of the human genomeGPCRs = 3% of the human genomeAround 800 human genesAround 800 human genes
ex: Isuprel® (Isoprenaline)-adrenergic agonist
Cardiac stimulant
Liorésal® (Baclofen)GABAB agonist
Muscle relaxant and AntispasticAlcool abstinence
Pin et al., 2004Pin et al., 2004
G protein-coupled receptors (GPCRs): Class CG protein-coupled receptors (GPCRs): Class C
22 33 44 55 6611 77
Domain VFTDomain VFTVenus FlytrapVenus Flytrap
Anxiety Anxiety PainPain
SchizophreniaSchizophreniaEpilepsy / Absence EpilepsyEpilepsy / Absence Epilepsy
SpasticitySpasticityNeurodegenerative dis.Neurodegenerative dis.
Drug addictionDrug addiction
Implicated in a lot of CNS diseases:Implicated in a lot of CNS diseases: Structure of the rhodopsin: Structure of the rhodopsin:
(Palczewski et al., 2000; 2006)(Palczewski et al., 2000; 2006)
7-transmembrane receptors7-transmembrane receptors
N termN term
C termC term
cytosolcytosol
extracellular
extracellular
Structure of 7-TM receptorsStructure of 7-TM receptors
Dimers …. And higher order oligomers
Rows of Dimers of rhodopsin Rows of Dimers of rhodopsin at the cell surface at the cell surface in vivo in vivo
(Fotiadis et al., 2003)(Fotiadis et al., 2003)
EXTRACELLULAR
INTRACELLULAR
CC CC
Romano et al., 1996Romano et al., 1996
mGluRsmGluRsconstitutive homodimersconstitutive homodimers
Within the class C, the dimer is the functional unit:
GABAB2GABAB2GABAB1GABAB1
Pagano et al., 2001; White et al., 1998Pagano et al., 2001; White et al., 1998
GABAGABABB obligatory heterodimerobligatory heterodimer
G proteinG protein
Diversity of the signaling pathways Diversity of the stimuli:
PhotonsIons
Pheromones
Amino acidsAmines
PeptidesProteins
NucleosidesLipids
1 2 3 4 5 6 7
Diversity of effectors: P P P
GRK -arrestin
c-Src ERKp38JNK
RhoA PP2A
Akt
MEK1 RKIP Akt PI3K
- GIRK- PLC
- AC- Calcium channels N/P/Q
- PI3K- ERK1/2, JNK…
EffectorsEffectors Effectors
Effectors
GGq:q:
- PLC; CaPLC; Ca2+2+; PKC; PKC-PI3KPI3K
- ERK1/2ERK1/2- JNK, p38MAPKJNK, p38MAPK
Gi/o:
- AC (-)- PKA (-)
Gs:
-AC (+)- PKA (+)
G12/13:
- Rho GTPases- ERK1/2, ERK5
- JNK- p38MAPK - ERK1/2, ERK5, p38MAPK
Effectors
Diversity of the signaling pathways Diversity of the stimuli:
PhotonsIons
Pheromones
Amino acidsAmines
PeptidesProteins
NucleosidesLipids
1 2 3 4 5 6 7
Diversity of effectors:
- GIRK- PLC
- AC- Calcium channels N/P/Q
- PI3K- ERK1/2, JNK…
GGq:q:
- PLC; CaPLC; Ca2+2+; PKC; PKC-PI3KPI3K
- ERK1/2ERK1/2- JNK, p38MAPKJNK, p38MAPK
Gi/o:
- AC (-)- PKA (-)
Gs:
-AC (+)- PKA (+)
G12/13:
- Rho GTPases- ERK1/2, ERK5
- JNK- p38MAPK - ERK1/2, ERK5, p38MAPK
Functional Interactions - Signal Integration
Signal Integration Signal Integration
Integration at the level Integration at the level of the signaling of the signaling
pathwayspathways
Effector 1Effector 1
Effector 2Effector 2+/-+/-
+/-+/-
a way to insure a way to insure specificityspecificity of the local integration? of the local integration?
Physical InteractionPhysical Interaction
+/-
+/-
Complex 5HT2a-mGlu2
HallucinogensHallucinogens
5HT2a5HT2a mGluR2mGluR2
Complex 5HT2a – mGluR2 Complex 5HT2a – mGluR2 co-expressed in cortical neuronsco-expressed in cortical neurons
(Gonzales-Maeso et al., 2008)(Gonzales-Maeso et al., 2008)::
Stoichiometry not clearStoichiometry not clear
ButBut
The physical interaction between both The physical interaction between both receptors seems to be responsible for the receptors seems to be responsible for the
functional interaction observedfunctional interaction observed
Modulation of the Biological responses associated to the Modulation of the Biological responses associated to the hallucinogens hallucinogens i/oi/o
GABAGABA
(Hirono et al., 2001, Nature Neurosciences)(Hirono et al., 2001, Nature Neurosciences)
Cross-talk mGlu1a–GABACross-talk mGlu1a–GABABB in Purkinje cells in Purkinje cells
Colocalization of mGlu1a and Colocalization of mGlu1a and GABAGABABB in Purkinje cells in Purkinje cells
in dendritic spines:in dendritic spines:
(From Kamikubo et al., 2007)(From Kamikubo et al., 2007)
PKCPKC
AMPA NMDA AMPA NMDA
mGluR1amGluR1a
GABAGABABB
GluGlu
+ + Mechanism ??Mechanism ??
Functional Functional Cross talkCross talk::Facilitation of the LTD Facilitation of the LTD
induction in Purkinje cellsinduction in Purkinje cellsLTDLTD
++PLCPLC
++
??
Signal IntegrationSignal Integration
i/oi/o qq
GqGq
Gi/oGi/o
No potentiation by another Gi/o protein-No potentiation by another Gi/o protein-coupled receptorcoupled receptor
(Hirono et al., 2001, Nature Neurosciences)(Hirono et al., 2001, Nature Neurosciences)
Some arguments in favor of a physical interaction
Co-immunoprecipitation of both receptors, Co-immunoprecipitation of both receptors, mGlu1a and GABAmGlu1a and GABAB B from cerebellar from cerebellar
membranesmembranes
From Kubo and Tateyama, 2006 From Kubo and Tateyama, 2006
Some functional experiments Some functional experiments independent of the Gi/o signalingindependent of the Gi/o signaling
(Tabata et al, 2004, PNAS)(Tabata et al, 2004, PNAS)
Physical interaction?Physical interaction?SpecificitySpecificity
1- Is there a real 1- Is there a real physical interaction physical interaction
between mGlu1a between mGlu1a and GABAand GABABB??
Aim of the study:
2- Mechanism of 2- Mechanism of the functional the functional
interactioninteractionGABAGABABBmGlu1amGlu1a
Functional cross talkFunctional cross talk
Physical interactionPhysical interaction
??
In In Cortical NeuronsCortical Neurons::
Cal
cium
rele
ase
(a.u
.)C
alci
um re
leas
e (a
.u.)
In In HEK293 cellsHEK293 cells::
Cal
cium
rele
ase
(a.u
.)C
alci
um re
leas
e (a
.u.)
Log [Glutamate] or [GABA]Log [Glutamate] or [GABA]
GluGlu
Glu + 50 Glu + 50 M GABAM GABA
GABAGABA
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
Functional cross talkFunctional cross talk
Physical interactionPhysical interaction
??
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
Functional cross talkFunctional cross talk
Physical interactionPhysical interaction
??
Technologies to study the physical interaction between receptors1/ Biochemical approaches: Co-immunoprecipitation
a/ Resonance energy transfer technologies: FRET (Förster Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer)
2/ Biophysical approaches: Living cells
Donor Acceptor
Excitation RETRET Emission
Ex EmEx Em
Inte
nsity
Wavelength (nm)exc. em.
YFPRlucYFPCFP
Technologies to study the physical interaction between receptors1/ Biochemical approaches: Co-immunoprecipitation
a/ Resonance energy transfer technologies: FRET (Förster Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer)
2/ Biophysical approaches: Living cells
b/ TR-FRET (Time-Resolved - Förster Resonance Energy Transfer)
Temporal selectivity: Spectral selectivity:
Fluorescence Intensity
Free Acceptor
Parasite Fluorescences
(cells, medium…)
Delay MeasureTime (s)
Free Donnor
Rel
ativ
e In
tens
ity
Wavelength nm
Technologies to study the physical interaction between receptors1/ Biochemical approaches: Co-immunoprecipitation
a/ Resonance energy transfer technologies: FRET (Förster Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer)
2/ Biophysical approaches: Living cells
b/ TR-FRET (Time-Resolved - Förster Resonance Energy Transfer)
STST
Extr.
Intr.
Antibodies
(Maurel and Comps-Agrar et al., 2008) (Maurel et al., 2004)
Extr.
Intr.
STST
Snap-Tags
Specific detection of protein-protein interactions at the Cell surface
STST
GABAGABABB
STSTSTST
mGlu1amGlu1a
Cell-surface Co-immunoprecipitation
++
++
++++
++
++++
++
HA-GB1HA-GB1
Flag-mGlu1aFlag-mGlu1a
Flag-GB1Flag-GB1
Flag-GB2 Flag-GB2
WB HAWB HA
HA-GB1HA-GB1Flag-GB1Flag-GB1
GB2GB2Flag-GB2Flag-GB2
WB FlagWB Flag
IP FlagIP Flag
Flag-mGlu1aFlag-mGlu1a
mGlu1a and GABAmGlu1a and GABABB seem not to be associated in complexes at the seem not to be associated in complexes at the cell surfacecell surface
BRET Technology BRET Technology B
RET
ratio
BR
ET ra
tio
YFP / RLucYFP / RLuc
YFP / RLucYFP / RLuc
BR
ET ra
tioB
RET
ratio
RLucRLuc RLucRLuc YFPYFP
RLucRLuc YFPYFP
VenusVenusRLucRLuc RLucRLucHomer3Homer3
YFPYFPRLucRLuc
YFPYFP RLucRLucYFPYFP
YFPYFP RLucRLuc
GABAGABABB
PAR1PAR1
mGlu1amGlu1a
TR-FRET technologiesde
lta 6
65de
lta 6
65 KK
HAHA FlagFlag
d2d2
GABAGABABBmGlu1amGlu1a
KK
HAHA FlagFlag
d2d2 KK
HAHA
KK
HAHA
FlagFlag
d2d2
FlagFlag
d2d2
mGlu2mGlu25-HT2a5-HT2a
delta
665
delta
665
Flag-GB2 surface expression (MCps)Flag-GB2 surface expression (MCps)
HA-mGlu1a + GB1 + Flag-GB2HA-mGlu1a + GB1 + Flag-GB2
GABAGABABBmGlu1amGlu1a
KK
HAHA FlagFlag
d2d2 KK
HAHA
No significant FRET signal between mGlu1a No significant FRET signal between mGlu1a and GABAand GABABB
even for high expression leveleven for high expression level
TR-FRET technologiesde
lta 6
65de
lta 6
65no
rmal
ized
on
HA
exp
ress
ion
norm
aliz
ed o
n H
A e
xpre
ssio
n
FlagFlag
d2d2
STST KK
FlagFlag
d2d2
STST KK
d2d2 d2d2
GABAGABABBmGlu1amGlu1a
FlagFlag FlagFlag
STST KK
No significant FRET signal between No significant FRET signal between mGlu1a and GABAmGlu1a and GABABB
even for high level of expressioneven for high level of expression
25002500
20002000
15001500
10001000
500500
00
delta
665
delta
665
ST-GB1 + GB2 + Flag-mGlu1aST-GB1 + GB2 + Flag-mGlu1a
Flag-GB2 or Flag-mGlu1a expression (MCps)Flag-GB2 or Flag-mGlu1a expression (MCps)
d2d2 d2d2
GABAGABABBmGlu1amGlu1a
FlagFlag FlagFlag
STST KK
TR-FRET technologies: Snap-Tags
22 11
STSTKK d2d2
STST
delta
665
delta
665
22 11
d2d2STST
22 11
STSTKK
22 11
d2d2STST STST
KK
No significant FRET signal between mGlu1a No significant FRET signal between mGlu1a and GB2and GB2
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
Signal IntegrationSignal Integration
Results:
2/ Mechanism of the functional interaction: Involvement of the 2/ Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
Signal IntegrationSignal Integration
Mechanism of the functional interaction
PTX PTX sensitivitysensitivity
Cal
cium
rel
ease
(a.u
.)
-8 -7 -6 -5 -4 -30
2000
4000
6000
8000
-2Log [Glutamate]
Cross Talk dependent on the Gi/o signalingCross Talk dependent on the Gi/o signaling
No potentiation with the GB2 mutant enable No potentiation with the GB2 mutant enable to couple Gi/o to couple Gi/o ((GB2-L686PGB2-L686P))
GABAGABAB1B1 GABAGABAB2B2
L686PL686P
Cal
cium
rel
ease
(a.u
.) -8 -7 -6 -5 -4 -3
0
1000
2000
3000
4000
-2Log [Glutamate]
5000
i/oi/o qq
ARK ARK = =
less less potentiationpotentiation
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2Log [Glutamate]Log [Glutamate]
44
33
22
00
11
CaCa2+2+
rele
ase
(AU
*100
0) re
leas
e (A
U*1
000)
Involvement of the subunits of the Go protein
rere ==
better better
potentiationpotentiation
Nor
mal
ized
Ca
Nor
mal
ized
Ca2+
2+
rele
ase
(%)
rele
ase
(%)
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2Log [Glutamate]Log [Glutamate]
300300
200200
00
100100
GABAGABABBmGlu1amGlu1a
i/oi/o qq
CaCa2+2+
Involvement of the subunits of the Go proteinC
aCa2+2+
rele
ase
(AU
*100
0) re
leas
e (A
U*1
000)
Log [Glutamate]Log [Glutamate]
FreeFree ==
occlusionocclusion
GABAGABABBmGlu1amGlu1a
i/oi/o qq
CaCa2+2+
Basal [CaBasal [Ca2+2+]i]i
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
CaCa2+2+
Only dependent on the Gi/o signaling Only dependent on the Gi/o signaling On the On the subunits subunits
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
CaCa2+2+
Only dependent on the Gi/o signaling Only dependent on the Gi/o signaling On the On the subunits subunits
Pre-stimulation of the GABAPre-stimulation of the GABABB receptor receptor
==mGlu1a - more potentiated responsesmGlu1a - more potentiated responses
Kinetics of activation1010
7,57,5
55
00
2,52,5
CaCa2+2+
rele
ase
(AU
*100
0) re
leas
e (A
U*1
000)
Log [Glutamate]Log [Glutamate]
ACAC PLCPLC
Decrease in the BRET signalDecrease in the BRET signal==
available for potentiating the available for potentiating the calcium signalingcalcium signaling
i/oi/o YFPYFP
RLucRLuc
YFPYFP i/oi/o
RLucRLuc
BR
ET ra
tioB
RET
ratio
Gales et al., 2006
Inhibition of the potentiation with Inhibition of the potentiation with the CGP54626the CGP54626
(GABA(GABAB B Antagoniste)Antagoniste)
Cross Talk only dependent on the Gi/o Cross Talk only dependent on the Gi/o protein signaling and the kinetics of protein signaling and the kinetics of
activation of the activation of the subunits subunits
Kinetics of activation
BR
ET ra
tioB
RET
ratio
TimeTimeCGP54626CGP54626GABAGABA
% o
f %
of
mG
lu1a
– m
axim
al
mG
lu1a
– m
axim
al
calc
ium
resp
onse
calc
ium
resp
onse
Re-association of the Re-association of the subunits after stimulation with subunits after stimulation with
the CGP54626the CGP54626
i/oi/o YFPYFP
RLucRLuc
YFPYFP i/oi/o
RLucRLuc
Results:
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
CaCa2+2+
Only dependent on the kinetics of Only dependent on the kinetics of activation of the activation of the subunits subunits
Time integration of the signal at the level of the PLCTime integration of the signal at the level of the PLC
Priming effectPriming effect
BAY367620 = Inverse agonist of BAY367620 = Inverse agonist of the mGlu1athe mGlu1a
GABAGABABB-mediated calcium responses -mediated calcium responses dependent on the activity of mGlu1adependent on the activity of mGlu1a
BAY367620BAY367620
Time (s)Time (s)
Fluo
resc
ence
(RFU
)Fl
uore
scen
ce (R
FU)
TimeTime
GABAGABAGlutamateGlutamate
Time (s)Time (s)
Fluo
resc
ence
(RFU
)Fl
uore
scen
ce (R
FU)
60006000
70007000
80008000
90009000
1000010000
1100011000
00 100100 200200Time (s)Time (s)
Fluo
resc
ence
(RFU
)Fl
uore
scen
ce (R
FU)
After After PTXPTX treatment treatment
TimeTime
mG
lu1a
m
Glu
1a
activ
ityac
tivity
Time IntegrationTime Integration
1010
7,57,5
55
00
2,52,5
CaCa2+2+
rele
ase
( AU
*100
0) re
leas
e ( A
U*1
000)
Log [Glutamate]Log [Glutamate]
GlutamateGlutamate
GABAGABAGABAGABA
No Calcium No Calcium responseresponse
Fluo
resc
ence
(RFU
)Fl
uore
scen
ce (R
FU)
Time (s)Time (s)
TimeTime
mG
lu1a
m
Glu
1a
activ
ityac
tivity
Time IntegrationTime Integration
1010
7,57,5
55
00
2,52,5
CaCa2+2+
rele
ase
( AU
*100
0) re
leas
e ( A
U*1
000)
Log [Glutamate]Log [Glutamate]
GlutamateGlutamate
No Calcium No Calcium responseresponse
Fluo
resc
ence
(RFU
)Fl
uore
scen
ce (R
FU)
Time (s)Time (s)
Fluo
resc
ence
(RFU
)Fl
uore
scen
ce (R
FU)
No calcium responseNo calcium response
Calcium responseCalcium response
Time (s)Time (s)Fl
uore
scen
ce (R
FU)
Fluo
resc
ence
(RFU
)
BAY367620BAY367620GABAGABA
Time (s)Time (s)
IP productionIP production
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
IP3IP3PLCPLC
CaCa2+2+
CaCa2+2+ CaCa2+2+
IP3IP3
cytosolcytosol
Endoplasmic Endoplasmic reticulumreticulum
extracellularextracellular
The constitutive activity of the mGlu1a receptor allows GABAThe constitutive activity of the mGlu1a receptor allows GABABB--mediated IP productionmediated IP production
But not Calcium signaling dependent on the activity of mGlu1aBut not Calcium signaling dependent on the activity of mGlu1a
Time Integration : Effect of the constitutive activity of mGlu1aTime Integration : Effect of the constitutive activity of mGlu1aIP productionIP production
0,100,10
0,080,08
0,060,06
0,040,04
0,020,02
IP P
rodu
ctio
nIP
Pro
duct
ion
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3-10-10Log [Glutamate]Log [Glutamate]
0,120,12
00
mGlu1a +/- GABAmGlu1a +/- GABABB activation activation
0,100,10
0,080,08
0,060,06
0,040,04
0,020,02
IP P
rodu
ctio
nIP
Pro
duct
ion
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3-10-10
Log [GABA]Log [GABA]
0,120,12
00
GABAGABABB +/- mGlu1a +/- mGlu1a
+ mGlu1a, + BAY367620
Constitutive activity of mGlu1aConstitutive activity of mGlu1a
GABAGABABBmGlu1amGlu1a
i/oi/o
CaCa2+2+
cytosolcytosol
Endoplasmic reticulumEndoplasmic reticulum
Homer3 dimersHomer3 dimers
extracellularextracellular
CaCa2+2+
GABAGABABBmGlu1amGlu1a
i/oi/o Monomeric Monomeric Homer1aHomer1a
IP3IP3
PLCPLC
ShankShank
Ango et al., 2001
Bettler et al., 2006 Bettler et al., 2006
Time integration of the signal Time integration of the signal
Cross-talk mGlu1a–GABAB: Conclusion
PLCPLC
AMPA NMDA AMPA NMDA
mGluR1amGluR1a
GABAGABABB
GluGlu
Functional Functional Cross talkCross talk::Facilitation of the LTD Facilitation of the LTD
induction in Purkinje cellsinduction in Purkinje cellsLTDLTD
GqGq
Gi/oGi/o
GABAGABA
Gi/oGi/o
++
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
Signal IntegrationSignal Integration
Results:
22/ Mechanism of the functional interaction: Involvement of the / Mechanism of the functional interaction: Involvement of the subunits subunits
3/ Kinetics of activation3/ Kinetics of activation
4/ Consequences in the GPCR field4/ Consequences in the GPCR field
1/ Do they oligomerize? Is the cross talk observed dependent of their potential 1/ Do they oligomerize? Is the cross talk observed dependent of their potential physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
i/oi/o qq
Signal IntegrationSignal Integration
Generalization to other pairs of GPCRs mGlu1a + mGlu1a + mGlu2mGlu2 + + GoGo
ControlControl+ LY354740+ LY354740
% C
alci
um in
crea
se (a
.u.)
% C
alci
um in
crea
se (a
.u.)
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2
5HT2c5HT2c + + GABAGABABB + + GoGoControlControl+ GABA+ GABA
% C
alci
um in
crea
se (a
.u.)
% C
alci
um in
crea
se (a
.u.)
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2Log [5-HT]Log [5-HT]Log [DHPG]Log [DHPG]
Functional cross talk Functional cross talk exists between other exists between other Gq- and Gi/o-protein Gq- and Gi/o-protein coupled receptors, coupled receptors,
even in native tissueeven in native tissue
mGlu3 and mGlu5 mGlu3 and mGlu5 co-expressed in co-expressed in
cortical astrocytescortical astrocytes
Cal
cium
rele
ase
(a.u
. * 1
000)
Cal
cium
rele
ase
(a.u
. * 1
000)
Log [Agonist]Log [Agonist]
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2-11-11 -10-10 -9-9 -8-8 -7-7 -6-6 -5-5 -4-4
75007500
50005000
25002500
00
1000010000
No physical interaction between these other pairs of GPCRs No physical interaction between these other pairs of GPCRs
22 11
FlagFlag
STST
KK
d2d2 d2d2STST
Flag-GB1 + GB2Flag-GB1 + GB2 ST-5-HT2cST-5-HT2c
delta
665
delta
665
Expression of the Snap-tagged Expression of the Snap-tagged subunit (MCps)subunit (MCps)
d2d2STST FlagFlag FlagFlag
KK KK
Flag-mGlu2Flag-mGlu2ST-mGlu1aST-mGlu1a
(C1 ou C2KKXX)(C1 ou C2KKXX)
Expression of the Snap-tagged Expression of the Snap-tagged subunit (MCps)subunit (MCps)
delta
665
delta
665
No physical No physical interaction interaction even for even for
these pairs of these pairs of receptorsreceptors
Functional cross talk between Gi/o and Gq receptorsFunctional cross talk between Gi/o and Gq receptors
No physical interaction requiredNo physical interaction required
Generalization to other pairs of GPCRs
L1L1
i/oi/o qq
CaCa2+2+
L2L2
i/oi/o qq
CaCa2+2+
L3L3
i/oi/o qq
CaCa2+2+
L4L4
What about a receptor both coupled to Gq and Gi/o?What about a receptor both coupled to Gq and Gi/o?
5HT4 known to generate 5HT4 known to generate Ca responseCa response
Auto-potentiation when a receptor couples to both Go and Gq
But 5HT4 also coupled to GoBut 5HT4 also coupled to Go
BR
ET ra
tioB
RET
ratio
150150
100100
00
5050
Auto-potentiation Auto-potentiation ==
can not be potentiated by the activation of can not be potentiated by the activation of another Gi/oPCRanother Gi/oPCR
i/oi/o qq
CaCa2+2+
L4L4
Log [5-HT]Log [5-HT]
CaCa2+2+
rele
ase
rele
ase
(flu
ores
cenc
e A
U)
(flu
ores
cenc
e A
U) 30003000
20002000
00
10001000
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3-10-10
PTXPTX
+ PTX
i/oi/o YFPYFP
RLucRLuc
Auto-potentiation vs Functional SelectivityAuto-potentiation vs Functional Selectivity
Functional cross talk between Gi/o and Gq receptorsFunctional cross talk between Gi/o and Gq receptorsNo physical interaction requiredNo physical interaction required
Auto-potentiation when a receptor is coupled to both Gq and Gi/oAuto-potentiation when a receptor is coupled to both Gq and Gi/o
Conclusion
L1L1
i/oi/o qq
CaCa2+2+
L2L2
i/oi/o qq
CaCa2+2+
L3L3
i/oi/o qq
CaCa2+2+
L4L4
Functional Interactions - Signal Integration
Signal Integration at Signal Integration at the membrane levelthe membrane level
+/-
+/-
Signal Integration Signal Integration
Formation of protein complexes:Formation of protein complexes:Scaffolding proteinsScaffolding proteins
PDZPDZ PDZPDZInteracting proteinInteracting protein
Signal Integration Signal Integration Integration at the Integration at the level of the signaling level of the signaling
pathwayspathways
Effector 1Effector 1
Effector 2Effector 2+/-+/-
Perspectives
Signal Integration at Signal Integration at the membrane levelthe membrane level
+/-
+/-
PDZPDZ PDZPDZInteracting proteinInteracting protein
Integration at the Integration at the level of the signaling level of the signaling
pathwayspathways
Effector 1Effector 1
Effector 2Effector 2+/-+/-
1/1/Are there scaffolding Are there scaffolding
proteins proteins in vivo in vivo to insure to insure the specificity of the cross the specificity of the cross
talk observedtalk observed between between mGlu1a and GABAmGlu1a and GABABB??
Scaffolding proteins between mGlu1a and GABAB in vivo?
?
Ango et al., 2001 Kitano et al., 2002; Balasubramanian et al., 2007
GABAGABABBmGlu1amGlu1a
No No Physical interactionPhysical interaction
Specificity of the functional crosstalkSpecificity of the functional crosstalk
Mupp1
Tamaline N P X X F
Homer3
Perspectives
+/-
+/-
2/2/Interacting proteins that Interacting proteins that
regulate the Gi/o coupling regulate the Gi/o coupling of the receptors and so, of the receptors and so,
calcium signaling? calcium signaling?
PDZPDZ PDZPDZInteracting proteinInteracting protein
PDZPDZ
Interacting proteinInteracting protein
i/oi/o qq
Effector 1Effector 1
Effector 2Effector 2+/-+/-
Regulation of the Gi/o coupling by Interacting proteins?
cytosolcytosol
extracellularextracellular
i/oi/o qq
i/oi/o qq
PDZPDZ
Interacting proteinInteracting protein
4.1G inhibits the Gs coupling of the mGlu1a receptor
(Tateyama and Kubo, 2006)
CaCa2+2+
rele
ase
rele
ase
Perspectives
+/-
+/-
3/3/Orphan GPCRs: Orphan GPCRs:
potential rolespotential roles
PDZPDZ PDZPDZInteracting proteinInteracting protein
PDZPDZ
Interacting proteinInteracting protein
i/oi/o qq
0,100,10
0,080,08
0,060,06
0,040,04
0,020,02IP P
rodu
ctio
nIP
Pro
duct
ion
-9-9 -8-8 -7-7 -6-6-5-5 -4-4 -3-3-10-10Log [GABA]Log [GABA]
The constitutive activity of The constitutive activity of the mGlu1a receptor allows the mGlu1a receptor allows
GABAGABABB-mediated IP -mediated IP productionproduction
Effector 1Effector 1
Effector 2Effector 2+/-+/-
Perspectives
+/-
+/-
PDZPDZ PDZPDZInteracting proteinInteracting protein
PDZPDZ
Interacting proteinInteracting protein
i/oi/o qq
New elements to study Signal Integration
Effector 1Effector 1
Effector 2Effector 2+/-+/-
PTX-AR
Inhibition of the -AR responsesdue to the constitutive activity of the GPR22 orphan receptor
cAM
P
Adams et al., 2008
Thanks
Cisbio - Bagnols / CézeCisbio - Bagnols / CézeNorbert TINEL Norbert TINEL
Eric TRINQUETEric TRINQUET
Department of Molecular PharmacologyDepartment of Molecular Pharmacology
Jean-Philippe PinJean-Philippe Pin’’s Teams TeamLaurent PREZEAULaurent PREZEAUPhilippe RONDARDPhilippe RONDARD
Cyril GOUDETCyril GOUDETJulie KNIAZEFFJulie KNIAZEFF
Claire VOLClaire VOLIsabelle BRABETIsabelle BRABET
Fanny MALHAIRE-FERREUXFanny MALHAIRE-FERREUX
Damien MAUREL Damien MAUREL (Kai Johnsson(Kai Johnsson’’s lab, Switzerland)s lab, Switzerland)Laetitia COMPS-AGRARLaetitia COMPS-AGRAR
Carine MONNIERCarine MONNIERNadia OUESLATI (Cisbio)Nadia OUESLATI (Cisbio)
Mohammed AYOUB Mohammed AYOUB Jean-Charles BOLOGNAJean-Charles BOLOGNA
Teresa DE VITATeresa DE VITAEtienne DOUMAZANEEtienne DOUMAZANE
Bruno VILARBruno VILAR
Thierry DURROUXThierry DURROUX
Department of NeurobiologyDepartment of Neurobiology
Philippe MARINPhilippe MARINAline DUMUISAline DUMUIS
Laurent FAGNILaurent FAGNI