PHd defense presentation Final RIVES

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

5. Diversity of the signaling pathways Diversity of the stimuli: Photons Ions Pheromones Amino acids Amines Peptides Proteins Nucleosides Lipids 1 2 3 4 5 6 7 Diversity of effectors: P P P GRK β-arrestin c-Src ERK p38 JNK RhoA PP2A Akt MEK1 RKIP Akt PI3K αα ββ γγ - GIRK - PLCβ - AC - Calcium channels N/P/Q - PI3K - ERK1/2, JNK… Effectors Effectors Effectors Effectors GGααq:q: - PLC; CaPLC; Ca2+2+ ; PKC; PKC -PI3KPI3K - ERK1/2ERK1/2 - JNK, p38MAPKJNK, p38MAPK Gαi/o: - AC (-) - PKA (-) Gαs: -AC (+) - PKA (+) Gα12/13: - Rho GTPases - ERK1/2, ERK5 - JNK - p38MAPK- ERK1/2, ERK5, p38MAPK Effector s

6. Diversity of the signaling pathways Diversity of the stimuli: Photons Ions Pheromones Amino acids Amines Peptides Proteins Nucleosides Lipids 1 2 3 4 5 6 7 Diversity of effectors: αα ββ γγ - GIRK - PLCβ - AC - Calcium channels N/P/Q - PI3K - ERK1/2, JNK… GGααq:q: - PLC; CaPLC; Ca2+2+ ; PKC; PKC -PI3KPI3K - ERK1/2ERK1/2 - JNK, p38MAPKJNK, p38MAPK Gαi/o: - AC (-) - PKA (-) Gαs: -AC (+) - PKA (+) Gα12/13: - Rho GTPases - ERK1/2, ERK5 - JNK - p38MAPK- ERK1/2, ERK5, p38MAPK

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

17. Cell-surface Co-immunoprecipitation ++ ++ ++ ++ ++ ++ ++ ++ HA-GB1HA-GB1 Flag-mGlu1aFlag-mGlu1a Flag-GB1Flag-GB1 Flag-GB2Flag-GB2 WB HAWB HA HA-GB1HA-GB1 Flag-GB1Flag-GB1 GB2GB2 Flag-GB2Flag-GB2 WB FlagWB Flag IP FlagIP Flag Flag-mGlu1aFlag-mGlu1a mGlu1a and GABAmGlu1a and GABABB seem not to be associated in complexes at theseem not to be associated in complexes at the cell surfacecell surface

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

19. TR-FRET technologies delta665delta665 KK HAHA FlagFlag d2d2 GABAGABABBmGlu1amGlu1a KK HAHA FlagFlag d2d2 KK HAHA KK HAHA FlagFlag d2d2 FlagFlag d2d2 mGlu2mGlu25-HT2a5-HT2a 0 500 1000 1500 2000 2500 3000 3500 F l a g - G B 1 + G B 2 G B 1 + F l a g - G B 2 H A - m G lu 1 a + Flag- m G lu 1 a H A - G B 1 + Flag- G B 2 H A -β 2 A R + Flag-β 2 A R H A -5 H T 2 a + Flag- m G lu 2 H A - m G lu 1 a H A - m G lu 3 H A - m G lu 4 H A - m G lu 1 a H A - m G lu 3 H A - m G lu 4 delta665delta665 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 mGlu1aNo significant FRET signal between mGlu1a and GABAand GABABB even for high expression leveleven for high expression level

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)

35. IP productionIP production GABAGABABBmGlu1amGlu1a NoNo Physical interactionPhysical interaction ααi/oi/o ββγγ ααqq ββ γγ IP3IP3PLCPLC CaCa2+2+ CaCa2+2+ CaCa2+2+ IP3IP3 cytosolcytosol EndoplasmicEndoplasmic reticulumreticulum extracellular extracellular

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 ++

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

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