The document discusses the functional interaction between mGlu1a and GABAB receptors. It first provides background on G protein-coupled receptors and their importance as drug targets. It then reviews evidence that mGlu1a and GABAB receptors physically interact in cortical neurons and Purkinje cells based on co-localization and co-immunoprecipitation studies. The study aims to further investigate the physical interaction between the receptors and the mechanism underlying their functional cross-talk using biophysical techniques like BRET, TR-FRET, and cell-surface co-immunoprecipitation. Preliminary data suggests mGlu1a and GABAB do not form complexes at the cell surface but may oligomerize intracellularly.
1. Functional interaction
between
mGlu1a and GABAB receptors
RIVES Marie-Laure
Institute of Functional GenomicsInstitute of Functional Genomics
Department of Molecular PharmacologyDepartment of Molecular Pharmacology
UNIVERSITE MONTPELLIER IIUNIVERSITE MONTPELLIER II
SCIENCES ET TECHNIQUES DU LANGUEDOCSCIENCES ET TECHNIQUES DU LANGUEDOC
T H E S E:T H E S E:
Discipline :Discipline : Pharmacologie Moléculaire – NeurosciencesPharmacologie Moléculaire – Neurosciences
Ecole Doctorale :Ecole Doctorale : Sciences Chimiques et Biologiques pour la SantéSciences Chimiques et Biologiques pour la Santé
February 12th, 2009February 12th, 2009
2. 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 genome
Around 800 human genesAround 800 human genes
ex: Isuprel® (Isoprenaline)
β-adrenergic agonist
Cardiac stimulant
Liorésal® (Baclofen)
GABAB agonist
Muscle relaxant and Antispastic
Alcool abstinence
3. 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 VFT
Venus FlytrapVenus Flytrap
AnxietyAnxiety
PainPain
SchizophreniaSchizophrenia
Epilepsy / Absence EpilepsyEpilepsy / Absence Epilepsy
SpasticitySpasticity
Neurodegenerative 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
cytosol
cytosol
extracellular
extracellular
Structure of 7-TM receptorsStructure of 7-TM receptors
4. Dimers …. And higher order oligomers
Rows of Dimers of rhodopsinRows of Dimers of rhodopsin
at the cell surfaceat the cell surface in vivoin vivo
(Fotiadis et al., 2003)(Fotiadis et al., 2003)
EXTRACELLULAR
INTRACELLULAR
CC CC
Romano et al., 1996Romano et al., 1996
mGluRsmGluRs
constitutive 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
7. Functional Interactions - Signal Integration
Signal IntegrationSignal Integration
Integration at the levelIntegration at the level
of the signalingof the signaling
pathwayspathways
Effector 1Effector 1
αα ββ
γγαα ββ
γγ
Effector 2Effector 2
+/-+/-
+/-+/-
a way to insurea way to insure specificityspecificity of the local integration?of the local integration?
Physical InteractionPhysical Interaction
+/-
+/-
γγ
αα ββ
8. Complex 5HT2a-mGlu2
HallucinogensHallucinogens
5HT2a5HT2a mGluR2mGluR2
Complex 5HT2a – mGluR2Complex 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 bothThe physical interaction between both
receptors seems to be responsible for thereceptors seems to be responsible for the
functional interaction observedfunctional interaction observed
ααqq ββ
γγ
Modulation of the Biological responses associated to theModulation of the Biological responses associated to the
hallucinogenshallucinogensααi/oi/o ββ
γγ
9. GABAGABA
(Hirono et al., 2001, Nature Neurosciences)(Hirono et al., 2001, Nature Neurosciences)
Cross-talk mGlu1a–GABACross-talk mGlu1a–GABABB in Purkinje cellsin Purkinje cells
Colocalization of mGlu1a andColocalization of mGlu1a and
GABAGABABB in Purkinje cellsin Purkinje cells
in dendritic spines:in dendritic spines:
(From Kamikubo et al., 2007)(From Kamikubo et al., 2007)
PKCPKC
AMPA NMDAAMPA NMDA
mGluR1amGluR1a
GABAGABABB
GluGlu
++ Mechanism ??Mechanism ??
FunctionalFunctional Cross talkCross talk::
Facilitation of the LTDFacilitation of the LTD
induction in Purkinje cellsinduction in Purkinje cells
LTDLTD
++
PLCPLCββ
++
??
Signal IntegrationSignal Integration
ααi/oi/o ββ
γγ
ααqq ββ
γγ
GqGq
Gi/oGi/o
10. 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 GABABB from cerebellarfrom cerebellar
membranesmembranes
From Kubo and Tateyama, 2006From Kubo and Tateyama, 2006
Some functional experimentsSome 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
11. 1- Is there a real1- Is there a real
physical interactionphysical interaction
between mGlu1abetween mGlu1a
and GABAand GABABB??
Aim of the study:
2- Mechanism of2- Mechanism of
the functionalthe functional
interactioninteractionGABAGABABBmGlu1amGlu1a
Functional cross talkFunctional cross talk
Physical interactionPhysical interaction
??
InIn Cortical NeuronsCortical Neurons::
Calciumrelease(a.u.)Calciumrelease(a.u.)
- 1 0 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
D R G lu .
D R G lu + 5 0 µ M G A B A .
D R G A B A .
lo g [ G lu t a m a te ] o r [G A B A ].
InIn HEK293 cellsHEK293 cells::
Calciumrelease(a.u.)Calciumrelease(a.u.)
Log [Glutamate] or [GABA]Log [Glutamate] or [GABA]
GluGlu
Glu + 50Glu + 50 µµM GABAM GABA
GABAGABA
0
2000
4000
6000
8000
D H P G
( 1 0 0 n M )
+
G A B A
(50 µM )
D H P G
( 1 0 0 n M )
G A B A
( 5 0 µM )
12. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ 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
??
13. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ 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
??
14. Technologies to study the physical interaction between receptors
1/ 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
Intensity
Wavelength (nm)
λ exc. λ em.
YFPRlucYFPCFP
15. Technologies to study the physical interaction between receptors
1/ 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 Measure
Time (µs)
Free
Donnor
RelativeIntensity
Wavelength nm
16. Technologies to study the physical interaction between receptors
1/ 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
18. BRET TechnologyBRET Technology
BRETratioBRETratio
YFP / RLucYFP / RLuc
YFP / RLucYFP / RLuc
BRETratioBRETratio
RLucRLuc RLucRLuc YFPYFP
RLucRLuc YFPYFP
VenusVenusRLucRLuc RLucRLuc
Homer3Homer3
0.000 0.025 0.050 0.075 0.100
0
10
20
30
40
50
60
70
80
m G lu 1 a - R L u c + V e n u s - H o m e r 3
m G lu 1 a - R L u c + G B 2 - YFP ( + G B 1 )
m G lu 1 a - R L u c + G B 1 - YFP ( + G B 2 )
m G lu 1 a - R L u c + m G lu 1 a - Y F P
Y F P / R L u c
0.0 0.1 0.2
0
100
200
300
400
500
G B 2 - R L u c + GB1-Y F P
G B 1 + G B 2 - R L u c + m G lu 1 a - Y F P
G B 1 + G B 2 - R L u c + PAR1-Y F P
Y F P / R L u c
YFPYFPRLucRLuc
YFPYFP RLucRLucYFPYFP
YFPYFP RLucRLuc
GABAGABABB
PAR1PAR1
mGlu1amGlu1a
20. TR-FRET technologiesdelta665delta665
normalizedonHAexpressionnormalizedonHAexpression
0
25
50
75
100
125
150
175
200
225
250
275
S T - G B 1 + Flag- G B 2
S T - G B 1 + Flag- G B 1 + G B 2
S T - 5 H T 2 a + Flag- m G lu 2
S T - G B 1 + G B 2 + Flag- m G lu 1 a
M o c k
FlagFlag
d2d2
STST
KK
FlagFlag
d2d2
STST
KK
d2d2d2d2
GABAGABABBmGlu1amGlu1a
FlagFlagFlagFlag
STST
KK
No significant FRET signal betweenNo 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
delta665delta665
ST-GB1 + GB2 + Flag-mGlu1aST-GB1 + GB2 + Flag-mGlu1a
Flag-GB2 or Flag-mGlu1a expression (MCps)Flag-GB2 or Flag-mGlu1a expression (MCps)
d2d2d2d2
GABAGABABBmGlu1amGlu1a
FlagFlagFlagFlag
STST
KK
21. TR-FRET technologies: Snap-Tags
22 11
STST
KK d2d2
STST
delta665delta665
0
2000
4000
6000
8000
S T - G B 1 + S T - G B 2
S T - G B 1 + G B 2
S T - m G lu 1 a C 1 + S T - G B 2
S T - m G lu 1 a C 2 + S T - G B 2
2211
d2d2
STST
22 11
STST
KK
2211
d2d2
STST STST
KK
No significant FRET signal between mGlu1aNo significant FRET signal between mGlu1a
and GB2and GB2
22. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ Do they oligomerize? Is the cross talk observed dependent of their potential
physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
Signal IntegrationSignal Integration
23. Results:
2/ Mechanism of the functional interaction: Involvement of the2/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ Do they oligomerize? Is the cross talk observed dependent of their potential
physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
Signal IntegrationSignal Integration
24. Mechanism of the functional interaction
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
C t l
+ G A B A
+ G A B A + P T X
lo g [ G lu t a m a te ]
PTXPTX
sensitivitysensitivity
Calciumrelease(a.u.)
-8 -7 -6 -5 -4 -3
0
2000
4000
600
0
800
0
-2
Log [Glutamate]
Cross Talk dependent on the Gi/o signalingCross Talk dependent on the Gi/o signaling
No potentiation with the GB2 mutant enableNo potentiation with the GB2 mutant enable
to couple Gi/oto couple Gi/o ((GB2-L686PGB2-L686P))
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
C t l.
+ G A B A .
w it h G B 2 - L 6 8 6 P ; C t l.
w it h G B 2 - L 6 8 6 P ; + G A B A .
lo g [ G lu ta m a t e ].
GABAGABAB1B1 GABAGABAB2B2
L686PL686P
Calciumrelease(a.u.)
-8 -7 -6 -5 -4 -3
0
1000
2000
300
0
400
0
-2
Log [Glutamate]
5000
ααi/oi/o ββγγ
ααqq ββ
γγ
25. ββARKARK
==
lessless
potentiationpotentiation
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2
Log [Glutamate]Log [Glutamate]
44
33
22
00
11
CaCa2+2+
release(AU*1000)release(AU*1000) Involvement of the βγ subunits of the Gαo protein
ΜοΜοrere βγβγ
==
betterbetter
potentiationpotentiation
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
1 0 0
2 0 0
3 0 0
C t l
+ G A B A
C t l
+ G A B A
m G lu 1 a + G A B A B + G o
+ 1 2
lo g [ G lu ta m a te ] .
NormalizedCaNormalizedCa2+2+
release(%)release(%)
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2
Log [Glutamate]Log [Glutamate]
300300
200200
00
100100
GABAGABABBmGlu1amGlu1a
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
26. Involvement of the βγ subunits of the Gαo proteinCaCa2+2+
release(AU*1000)release(AU*1000)
Log [Glutamate]Log [Glutamate]
FreeFree βγβγ
==
occlusionocclusion
GABAGABABBmGlu1amGlu1a
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
ββγγ
ββγγ
Basal [CaBasal [Ca2+2+
]i]i
27. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ Do they oligomerize? Is the cross talk observed dependent of their potential
physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
Only dependent on the Gi/o signalingOnly dependent on the Gi/o signaling
On theOn the βγβγ subunitssubunits
28. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ Do they oligomerize? Is the cross talk observed dependent of their potential
physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
Only dependent on the Gi/o signalingOnly dependent on the Gi/o signaling
On theOn the βγβγ subunitssubunits
29. Pre-stimulation of the GABAPre-stimulation of the GABABB
receptorreceptor
==
mGlu1a - more potentiated responsesmGlu1a - more potentiated responses
Kinetics of activation
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
2 5 0 0
5 0 0 0
7 5 0 0
1 0 0 0 0 C tl.
+ G A B A t 0 .
+ G A B A t - 1 0 0 .
lo g [G lu t a m a te ].
1010
7,57,5
55
00
2,52,5
CaCa2+2+
release(AU*1000)release(AU*1000)
Log [Glutamate]Log [Glutamate]
ACAC PLCPLC
Decrease in the BRET signalDecrease in the BRET signal
==
βγβγ available for potentiating theavailable for potentiating the
calcium signalingcalcium signaling
ααi/oi/o ββγγ
YFPYFP
RLucRLuc
ββγγ
YFPYFPααi/oi/o
RLucRLuc
0
10
20
30
40
50BRETratioBRETratio
Gales et al., 2006
30. Inhibition of the potentiation withInhibition of the potentiation with
the CGP54626the CGP54626
(GABA(GABABB Antagoniste)Antagoniste)
Cross Talk only dependent on the Gi/oCross Talk only dependent on the Gi/o
protein signaling and the kinetics ofprotein signaling and the kinetics of
activation of theactivation of the βγβγ subunitssubunits
Kinetics of activation
BRETratioBRETratio
TimeTime
CGP54626CGP54626GABAGABA
%of%of
mGlu1a–maximalmGlu1a–maximal
calciumresponsecalciumresponse
100
125
150
Re-association of theRe-association of the βγβγ
subunits after stimulation withsubunits after stimulation with
the CGP54626the CGP54626
ααi/oi/o ββγγ
YFPYFP
RLucRLuc
ββγγ
YFPYFP
ααi/oi/o
RLucRLuc
31. Results:
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
Only dependent on the kinetics ofOnly dependent on the kinetics of
activation of theactivation of the βγβγ subunitssubunits
Time integration of the signal at the level of the PLCTime integration of the signal at the level of the PLC
32. Priming effectPriming effect
BAY367620 = Inverse agonist ofBAY367620 = 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)
Fluorescence(RFU)Fluorescence(RFU)
TimeTime
GABAGABAGlutamateGlutamate
Time (s)Time (s)
Fluorescence(RFU)Fluorescence(RFU)
0 100 200
6000
7000
8000
9000
10000
11000
G lu 1 m M G A B A 1 0 µM
T im e ( s ) .
A ft e r P T X tr e a t m e n t
60006000
70007000
80008000
90009000
1000010000
1100011000
00 100100 200200
Time (s)Time (s)
Fluorescence(RFU)Fluorescence(RFU)
AfterAfter PTXPTX treatmenttreatment
33. TimeTime
mGlu1amGlu1a
activityactivity
Time IntegrationTime Integration
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
2 5 0 0
5 0 0 0
7 5 0 0
1 0 0 0 0 C tl.
+ G A B A t 0 .
+ G A B A t - 1 0 0 .
lo g [G lu ta m a te ].
1010
7,57,5
55
00
2,52,5
CaCa2+2+
release(AU*1000)release(AU*1000)
Log [Glutamate]Log [Glutamate]
GlutamateGlutamate
GABAGABAGABAGABA
No CalciumNo Calcium
responseresponse
0 2 5 5 0 7 5 1 0 0
1 3 0 0 0
1 5 5 0 0
1 8 0 0 0
T im e ( s ) .
G A B A 1 0 µ M .
Fluorescence(RFU)Fluorescence(RFU)
Time (s)Time (s)
34. TimeTime
mGlu1amGlu1a
activityactivity
Time IntegrationTime Integration
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
2 5 0 0
5 0 0 0
7 5 0 0
1 0 0 0 0 C tl.
+ G A B A t 0 .
+ G A B A t - 1 0 0 .
lo g [G lu ta m a te ].
1010
7,57,5
55
00
2,52,5
CaCa2+2+
release(AU*1000)release(AU*1000)
Log [Glutamate]Log [Glutamate]
GlutamateGlutamate
No CalciumNo Calcium
responseresponse
0 2 5 5 0 7 5 1 0 0
1 3 0 0 0
1 5 5 0 0
1 8 0 0 0
T im e ( s ) .
G A B A 1 0 µ M .
Fluorescence(RFU)Fluorescence(RFU)
Time (s)Time (s)
Fluorescence(RFU)Fluorescence(RFU)
No calcium responseNo calcium response
Calcium responseCalcium response
Time (s)Time (s)
Fluorescence(RFU)Fluorescence(RFU)
BAY367620BAY367620
GABAGABA
Time (s)Time (s)
36. 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 mGlu1a
IP productionIP production
-9 -8 -7 -6 -5 -4 -3 -2
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
C o n tr o l
+ 0 , 5 µM G A B A
+ 5 µM G A B A
lo g [G lu ta m a te ].
0,100,10
0,080,08
0,060,06
0,040,04
0,020,02
IPProductionIPProduction
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3-10-10
Log [Glutamate]Log [Glutamate]
0,120,12
00
mGlu1a +/- GABAmGlu1a +/- GABABB activationactivation
0,100,10
0,080,08
0,060,06
0,040,04
0,020,02
IPProductionIPProduction
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3-10-10
Log [GABA]Log [GABA]
-10 -9 -8 -7 -6 -5 -4 -3
0.00
0.02
0.04
0.06
0.08
0.10
0.12
C t l
+ m G lu 1 a
+ m G lu 1 a ; + B A Y 3 6 7 2 0
lo g [G A B A ]
0,120,12
00
GABAGABABB +/- mGlu1a+/- mGlu1a
+ mGlu1a, + BAY367620
37. Constitutive activity of mGlu1aConstitutive activity of mGlu1a
GABAGABABBmGlu1amGlu1a
ααi/oi/o ββγγ
CaCa2+2+
cytosol
cytosol
Endoplasmic reticulumEndoplasmic reticulum
Homer3 dimersHomer3 dimers
extracellular
extracellular
CaCa2+2+
GABAGABABBmGlu1amGlu1a
ααi/oi/o ββγγ
MonomericMonomeric
Homer1aHomer1a
IP3IP3
PLCPLC
ααqq ββ
γγ
ShankShank
Ango et al., 2001
38. Bettler et al., 2006Bettler et al., 2006
Time integration of the signalTime integration of the signal
Cross-talk mGlu1a–GABAB: Conclusion
PLCPLC
AMPA NMDAAMPA NMDA
mGluR1amGluR1a
GABAGABABB
GluGlu
FunctionalFunctional Cross talkCross talk::
Facilitation of the LTDFacilitation of the LTD
induction in Purkinje cellsinduction in Purkinje cells
LTDLTD
GqGq
Gi/oGi/o
ββ
γγ
GABAGABA
Gi/oGi/o
++
39. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ Do they oligomerize? Is the cross talk observed dependent of their potential
physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
Signal IntegrationSignal Integration
40. Results:
22/ Mechanism of the functional interaction: Involvement of the/ Mechanism of the functional interaction: Involvement of the βγβγ subunitssubunits
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 potential1/ Do they oligomerize? Is the cross talk observed dependent of their potential
physical interaction?physical interaction?
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
ααi/oi/o ββγγ
ααqq ββ
γγ
Signal IntegrationSignal Integration
41. Generalization to other pairs of GPCRs
mGlu1a +mGlu1a + mGlu2mGlu2 ++ GoGo
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2
0
5 0
1 0 0
1 5 0
2 0 0
C tl.
+ L Y 3 5 4 7 4 0 .
lo g [D H P G ].
ControlControl
+ LY354740+ LY354740
%Calciumincrease(a.u.)%Calciumincrease(a.u.)
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2
5HT2c5HT2c ++ GABAGABABB ++ GoGo
- 1 1 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4
0
1 0 0
2 0 0
C tl.
+ G A B A .
lo g [S e r o to n in e ].
ControlControl
+ GABA+ GABA
%Calciumincrease(a.u.)%Calciumincrease(a.u.)
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2
Log [5-HT]Log [5-HT]Log [DHPG]Log [DHPG]
Functional cross talkFunctional cross talk
exists between otherexists between other
Gq- and Gi/o-proteinGq- and Gi/o-protein
coupled receptors,coupled receptors,
even in native tissueeven in native tissue
mGlu3 and mGlu5mGlu3 and mGlu5
co-expressed inco-expressed in
cortical astrocytescortical astrocytes
Calciumrelease(a.u.*1000)Calciumrelease(a.u.*1000)
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
42. No physical interaction between these other pairs of GPCRsNo physical interaction between these other pairs of GPCRs
22 11
FlagFlag
STST
KK
d2d2 d2d2
STST
Flag-GB1 + GB2Flag-GB1 + GB2 ST-5-HT2cST-5-HT2c
delta665delta665
Expression of the Snap-taggedExpression of the Snap-tagged
subunit (MCps)subunit (MCps)
d2d2
STST FlagFlag FlagFlag
KK KK
Flag-mGlu2Flag-mGlu2ST-mGlu1aST-mGlu1a
(C1 ou C2KKXX)(C1 ou C2KKXX)
Expression of the Snap-taggedExpression of the Snap-tagged
subunit (MCps)subunit (MCps)
delta665delta665
No physicalNo physical
interactioninteraction
even foreven for
these pairs ofthese pairs of
receptorsreceptors
43. 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
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
L2L2
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
L3L3
ααi/oi/o ββγγ
ααqq ββ
γγ
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?
44. 5HT4 known to generate5HT4 known to generate
Ca responseCa response
Auto-potentiation when a receptor couples to both Go and Gq
C o n t r ô le N o n e 5 H T 4 5 H T 2 c A 1 G A B A B
0
5 0
1 0 0
1 5 0
B a s a l
S tim u la tio n
But 5HT4 also coupled to GoBut 5HT4 also coupled to Go
BRETratioBRETratio
150150
100100
00
5050
Auto-potentiationAuto-potentiation
==
can not be potentiated by the activation ofcan not be potentiated by the activation of
another Gi/oPCRanother Gi/oPCR
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
L4L4
Log [5-HT]Log [5-HT]
CaCa2+2+
releaserelease
(fluorescenceAU)(fluorescenceAU)
- 1 0 - 9 - 8 - 7 - 6 - 5 - 4 - 3
0
1 0 0 0
2 0 0 0
3 0 0 0
C t l
+ G A B A
+ G A B A ( + P T X )
lo g [S e r o to n in e ].
30003000
20002000
00
10001000
-9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3-10-10
PTXPTX
+ PTX
ααi/oi/o ββγγ
YFPYFP
RLucRLuc
45. 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 receptors
No 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
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
L2L2
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
L3L3
ααi/oi/o ββγγ
ααqq ββ
γγ
CaCa2+2+
L4L4
46. Functional Interactions - Signal Integration
Signal Integration atSignal Integration at
the membrane levelthe membrane level
+/-
+/-
αα
ββ
γγ
Signal IntegrationSignal Integration
Formation of protein complexes:Formation of protein complexes:
Scaffolding proteinsScaffolding proteins
PDZPDZ PDZPDZ
Interacting proteinInteracting protein
Signal IntegrationSignal IntegrationIntegration at theIntegration at the
level of the signalinglevel of the signaling
pathwayspathways
Effector 1Effector 1
αα ββ
γγ
αα ββ
γγ
Effector 2Effector 2
+/-+/-
47. Perspectives
Signal Integration atSignal Integration at
the membrane levelthe membrane level
+/-
+/-
αα
ββ
γγ
PDZPDZ PDZPDZ
Interacting proteinInteracting protein
Integration at theIntegration at the
level of the signalinglevel of the signaling
pathwayspathways
Effector 1Effector 1
αα ββ
γγ
αα ββ
γγ
Effector 2Effector 2
+/-+/-
1/1/
Are there scaffoldingAre there scaffolding
proteinsproteins in vivoin vivo to insureto insure
the specificity of the crossthe specificity of the cross
talk observedtalk observed betweenbetween
mGlu1a and GABAmGlu1a and GABABB??
48. Scaffolding proteins between mGlu1a and GABAB in vivo?
?
Ango et al., 2001
Kitano et al., 2002; Balasubramanian et al., 2007
GABAGABABBmGlu1amGlu1a
NoNo Physical interactionPhysical interaction
Specificity of the functional crosstalkSpecificity of the functional crosstalk
Mupp1
TamalineN P X X F
Homer3
49. Perspectives
+/-
+/-
αα
ββ
γγ
2/2/
Interacting proteins thatInteracting proteins that
regulate the Gi/o couplingregulate the Gi/o coupling
of the receptors and so,of the receptors and so,
calcium signaling?calcium signaling?
PDZPDZ PDZPDZ
Interacting protein
Interacting protein
PDZPDZ
Interacting protein
Interacting protein
ααi/oi/o ββγγ
ααqq ββγγ
Effector 1Effector 1
αα ββ
γγ
αα ββ
γγ
Effector 2Effector 2
+/-+/-
50. Regulation of the Gi/o coupling by Interacting proteins?
cytosol
cytosol
extracellular
extracellular
ααi/oi/o ββγγ
ααqq ββ
γγ
ααi/oi/o ββγγ
ααqq ββ
γγ
PDZPDZ
Interacting proteinInteracting protein
4.1G inhibits the Gs coupling of the
mGlu1a receptor
(Tateyama and Kubo, 2006)
CaCa2+2+
releaserelease
-10 -9 -8 -7 -6 -5 -4 -3
0
500
1000
1500
2000
2500
3000
3500
lo g [5 - H T ].
51. Perspectives
+/-
+/-
αα
ββ
γγ
3/3/
Orphan GPCRs:Orphan GPCRs:
potential rolespotential roles
PDZPDZ PDZPDZ
Interacting protein
Interacting protein
PDZPDZ
Interacting protein
Interacting protein
ααi/oi/o ββγγ
ααqq ββγγ
0,100,10
0,080,08
0,060,06
0,040,04
0,020,02
IPProductionIPProduction
-9-9 -8-8 -7-7 -6-6-5-5 -4-4 -3-3-10-10
Log [GABA]Log [GABA]
- 1 0 - 9 - 8 - 7 - 6 - 5 - 4 - 3
0 . 0 2
0 . 0 4
0 . 0 6
0 . 0 8
0 . 1 0
C t l
C t l
+ B A Y 3 6 7 2 0
G A B A B + m G lu 1 a
G A B A B
lo g [ G lu t a m a t e ] o r [ G A B A ] .
The constitutive activity ofThe constitutive activity of
the mGlu1a receptor allowsthe mGlu1a receptor allows
GABAGABABB-mediated IP-mediated IP
productionproduction
Effector 1Effector 1
αα ββ
γγ
αα ββ
γγ
Effector 2Effector 2
+/-+/-
52. Perspectives
+/-
+/-
αα
ββ
γγ
PDZPDZ PDZPDZ
Interacting protein
Interacting protein
PDZPDZ
Interacting protein
Interacting protein
ααi/oi/o ββγγ
ααqq ββγγ
New elements to study
Signal Integration
Effector 1Effector 1
αα ββ
γγ
αα ββ
γγ
Effector 2Effector 2
+/-+/-
PTX
β1-AR
Inhibition of the β-AR responses
due to the constitutive activity
of the GPR22 orphan receptor
cAMP
Adams et al., 2008
53. Thanks
Cisbio - Bagnols / CézeCisbio - Bagnols / Céze
Norbert TINELNorbert TINEL
Eric TRINQUETEric TRINQUET
Department of Molecular PharmacologyDepartment of Molecular Pharmacology
Jean-Philippe PinJean-Philippe Pin’s Team’s Team
Laurent PREZEAULaurent PREZEAU
Philippe RONDARDPhilippe RONDARD
Cyril GOUDETCyril GOUDET
Julie KNIAZEFFJulie KNIAZEFF
Claire VOLClaire VOL
Isabelle BRABETIsabelle BRABET
Fanny MALHAIRE-FERREUXFanny MALHAIRE-FERREUX
Damien MAURELDamien MAUREL (Kai Johnsson(Kai Johnsson’s lab, Switzerland)’s lab, Switzerland)
Laetitia COMPS-AGRARLaetitia COMPS-AGRAR
Carine MONNIERCarine MONNIER
Nadia OUESLATI (Cisbio)Nadia OUESLATI (Cisbio)
Mohammed AYOUBMohammed AYOUB
Jean-Charles BOLOGNAJean-Charles BOLOGNA
Teresa DE VITATeresa DE VITA
Etienne DOUMAZANEEtienne DOUMAZANE
Bruno VILARBruno VILAR
Thierry DURROUXThierry DURROUX
Department of NeurobiologyDepartment of Neurobiology
Philippe MARINPhilippe MARIN
Aline DUMUISAline DUMUIS
Laurent FAGNILaurent FAGNI
