phd defense presentation final rives

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

qq

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

??

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




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




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


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


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


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


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:






No No Physical interactionPhysical interaction

i/oi/o qq


Results:

2/ Mechanism of the functional interaction: Involvement of the 2/ Mechanism of the functional interaction: Involvement of the subunits subunits






i/oi/o qq


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


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


i/oi/o qq

CaCa2+2+

Basal [CaBasal [Ca2+2+]i]i

Results:







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)


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:



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)


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)


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



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


i/oi/o

CaCa2+2+

cytosolcytosol

Endoplasmic reticulumEndoplasmic reticulum

Homer3 dimersHomer3 dimers


CaCa2+2+


i/oi/o Monomeric Monomeric Homer1aHomer1a

IP3IP3

PLCPLC

qq

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:







i/oi/o qq


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



Perspectives

Signal Integration at Signal Integration at the membrane levelthe membrane level

+/-

+/-


Integration at the Integration at the level of the signaling level of the signaling

pathwayspathways



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



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

Interacting proteinInteracting protein

i/oi/o qq



Regulation of the Gi/o coupling by Interacting proteins?

cytosolcytosol


i/oi/o qq

i/oi/o qq

PDZPDZ


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


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



Perspectives

+/-

+/-


PDZPDZ


i/oi/o qq

New elements to study Signal Integration



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

phd defense presentation final rives

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