Basic concepts in G-protein-coupledreceptor homo- andheterodimerization

www.bq.ub.es/recep/franco.htmlwww.rafaelfranco.cat

RAFAEL FRANCOrfanco@ub.edu

NIDA minisymposium. SfNSan Diego. November 2007

STOCKHOLMK. FuxeC. IbáñezD. Marcellino

NIH/NIDA N. VolkowS. GoldbergS. FerréA. Woods

MODENAL. AgnatiS. TanganelliP. G. de BenedettiF. Fanelli

BERLINM. Bader

MONTREALM. Bouvier

WAKE-FOREST U.D. Roberts

COIMBRAR. Cunha

ALBACETER. Luján

GLASGOWG. MilliganM. Canals

Centro Investigación Médica Aplicada

Pamplona

Digna Biotech

GPCR classes

NOMENCLATURE

Receptor for Neurotransmitters (including neuromodulators and neuropeptides): - Ionotropic- Metabotropic (G-protein-coupled, GPCR)

Receptor homomerReceptor heteromer

Heteromeric receptor

Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

Heteromeric receptor: Ach receptor

NOMENCLATURE

Receptor for Neurotransmitters (including neuromodulators and neuropeptides): - Ionotropic- Metabotropic (G-protein-coupled, GPCR)

Receptor homomerReceptor heteromer

Heteromeric receptor

Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

Receptor homomers

Adenosine A2A

Dopamine D2Dopamine D2 Adenosine A2A Adenosine A2A

Receptor heteromers

Adenosine A2ADopamine D2 NMDADopamine D1

Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

HETEROMERS AS SENSORS

•1983: Evidence and formulation of the hypothesis: Agnati et al., Neurosc. Letters •1994: DR homomers: Ng et al., Eur. J. Pharmacol. (infected cells)•1995: A1R homomers: Ciruela et al., J. Neurosc. Res. (brain extracts)•Late nineties, early XXI Century: heteromers (opioid, GABA, Dopa/Ado)

Crude IP Avidina

0

20

40

60

80

100

RD

S (%

oft

otal

A2A

R)

Crudeextract

Cellsurface

**

*

**

*

100

50

MW(kDa)

150

100

50

MW(kDa)

150

The A2A

R dimer is the functional specie

Canals et al (2004) J Neurochem 88, 726-734

The C terminal tail of A2A

R is not required for dimerization

A2A R A2A R

Crude

Biotina

IP A2A R

HETERODIMERS: DR/AR

D2R D1R

D2

R-A2A

R receptor heteromers

D1

R-A1

R receptor heteromers

Hillion et al (2002) J Biol Chem

Gines et al (2000) PNAS

Rluc

A B

YFP

Coelenterazine H

470 nm

Rluc

AB

YFP

Coelenterazine H

530 nm

GFP2

A B

YFP

396 nm

510 nm

GFP2

AB

YFP 530 nm

396 nm

BRET FRET

The A2AR/D2 homodimers: BRET: Bioluminiscence Resonance Energy Transfer

0 10 20 30 400.0

0.05

0.1

YFP/Rluc

BR

ET in

crea

se A2A RRluc-D2 RYFP

A2A RRluc-GABABRYFP

BRET SATURATION CURVE

Canals et al (2003) J Biol Chem 278: 46741-49

LOOKING FOR THEBIOCHEMICAL/PHARMACOLOGICAL

“DIMER FINGERPRINT”

IUBCP: Recognition and nomenclature

Alternative nomenclature in:Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

1 A Coimmunolocalization1 B Coimmunoprecipitation/transgenics2 Specific functional property (Pharmacology/Signalling)3 Knockouts: Loss of function!!!??

IUBCP: Recognition 2 out of 3:

a b

Intramolecular cross-talk in the homomer:Cooperativity

2-R1-R 1-R

2-R1-R 3-R1-R

a b

dc

Intramolecular cross-talk in the heteromer:Cooperativity and/or ?

Looking for the “biochemical fingerprint”of a receptor heteromer

Fingerprint lost in knockout animals

Transfection Radioligand Displacer KD

A1R [3H]R-PIA

R-PIA

Caffeine

1.9±0.4 nM

90±20 M

A2AR [3H]CGS21680 CGS21680

Caffeine

110±30 nM

7±1 M

A1R+A2AR [3H]R-PIA

[3H]CGS21680

R-PIA

Caffeine

CGS21680

Caffeine

1.9±0.3 nM

90±20 M

120±40 nM

90±20 M*

A2AR+D2R [3H]CGS21680 CGS21680

Caffeine

110±40 nM

5±2 M

Different affinities for ligands: Caffeine as adenosine antagonist

A+A+(RR)

A+A(RR)

A(RR)A

A+A+(RR)*

A+A(RR)*

A(RR)*A

L

α L

θα L

α K

μθ K

K

μK

Franco et al (2005) Trends Biochem Sci 30: 360-66

RECEPTOR DIMER MODEL

A + R + G AR+G

A + RG ARG

TERNARY MODEL

A+A+(R–R) A+A(R–R) A(R–R)A

A+A+(R~R) A+A(R~R) A(R~R)A

L α L θα L

K μK

α K μθ K

[ ][ ]

[ ] [ ][ ] [ ]22

22

A)1(A)1(1A)1(2A)1(

RA

⋅+⋅+⋅+⋅++

⋅+⋅+⋅+⋅=

LKLKLLKLK

T

Bound

αθμααθμα

KD1 KD2A2A

RR

RR*

RR

RR*

RR

RR*

Abound = (KD2 A + 2 A2) RT /(KD1 KD2 + KD2 A + A2)

a

b

Dimer Cooperativity Index, Dc:It depends on KD1 and KD2

Casadó et al., Pharmacol Ther In the Press (Dec 2007)

D50 in Drug discovery:A more meaningful

parameter to measure binding potency

compared to IC50 .

Allosterism:Useful parameter to

“measure allosterism”

LOOKING FOR THEFUNCTIONAL

“DIMER FINGERPRINT”

Franco et al Trends Biochemical Sci 28 (2003) 238-243

Fig 4 Clustering adaptor/scaffolding proteins

Signalling A:

Signalling B:

Agonists for the two receptors

Blue agonist “sensitizes” yellow and pink receptors

Blue agonist “desensitizes” yellow and pink receptors

Blue agonist “sensitizes” pink and “desensitizes” yellow

SIGNALLING SIGNALLING

SIGNALLING

SIGNALLINGSIGNALLING

SIGNALLING

Signalling Signalling

Signalling

Fig 3

Franco et al Trends Biochemical Sci 28 (2003) 238-243

0

50

100

150

200

250

300

350

400

450

R-PIA SKF38393 R-PIA+SKF

Naive30 min60 min120 min

cAMP producion via D1R after pretreatment with agonists of A1R and/or D1R

(A1/D1 cotransfected mouse Ltk- fibroblasts)

The intracytoplasmic receptor-receptor cross-talk: ANTAGONISM (A1R/D1R)

Ginés et al (2000) PNAS 97, 8606-11

A Cyclase

GsGi

D2-R

A Cyclase

Gs

D1-R

Ca2+ IP3

D2-RD1-R

Gq

Ca2+ calmodulin kinase IIα(.: increase in accumbens)

Synaptic Plasticity Diacylglycerol

PKC

Rashid et al. (2007) Proc Natl Acad Sci 104, 654-9

Shift to Gq coupling in the D1-D2 receptor heteromer

1.-D1 receptor agonist affinity is enhanced by D3 agonists. This indicates the existence of a synergistic intramembrane receptor-receptor interaction.

2.-Experiments in reserpinized mice showed that D3 receptor stimulation potentiates D1receptor-mediated behavioral effects by a different mechanism than D2 receptor stimulation. Marcellino et al. (2007) Submitted

D3-RD1-R

D1-D3 Receptor heteromerization

-FRET-BRET-Fingerprint (Intramembrane cross-talk)

Dual regulatory role of Adenosine on Glu release via A1 R/A2A R

Ciruela et al (2006) J Neurosc 26:2080-87

CONCLUSIONS

1. Heteromers exist in natural tissues2. Heteromers are sensors for neurotransmitters 3. Heteromerization modifies:

- biochemical,- pharmacological,- and functional properties of receptors

FUNCTIONAL MODULES IN THE PLASMA MEMBRANE

ADA

mGluR1α

ADA

hsc73

A1

R D1

R

ßArrestincaveolin

A2A

R mGluR5D2

R

α-actinin

homerCa-calmodulin

filamin

ßArrestincaveolin

Class A Rhodopsin likeAminePeptideHormone protein(Rhodopsin

Rhodopsin VertebrateRhodopsin Vertebrate

type 1Rhodopsin Vertebrate

type 2Rhodopsin Vertebrate

type 3Rhodopsin Vertebrate

type 4Rhodopsin Vertebrate

type 5Rhodopsin ArthropodRhodopsin MolluscRhodopsin Other

OlfactoryProstanoidNucleotide-likeCannabisPlatelet activating factorGonadotropin-releasing hormoneThyrotropin-releasing hormone &

SecretagogueMelatoninViralLysosphingolipid & LPA (EDG)Leukotriene B4 receptorClass A Orphan/other

Class B Secretin likeClass C Metabotropic glutamate / pheromoneClass D Fungal pheromoneClass E cAMP receptors (Dictyostelium)Frizzled/Smoothened family