Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled....
Transcript of Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled....
![Page 1: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/1.jpg)
Basic concepts in G-protein-coupledreceptor homo- andheterodimerization
www.bq.ub.es/recep/franco.htmlwww.rafaelfranco.cat
RAFAEL [email protected]
NIDA minisymposium. SfNSan Diego. November 2007
![Page 2: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/2.jpg)
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
![Page 3: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/3.jpg)
Centro Investigación Médica Aplicada
Pamplona
Digna Biotech
![Page 4: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/4.jpg)
GPCR classes
![Page 5: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/5.jpg)
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
![Page 6: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/6.jpg)
Heteromeric receptor: Ach receptor
![Page 7: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/7.jpg)
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
![Page 8: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/8.jpg)
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
![Page 9: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/9.jpg)
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)
![Page 10: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/10.jpg)
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
![Page 11: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/11.jpg)
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
![Page 12: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/12.jpg)
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
![Page 13: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/13.jpg)
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
![Page 14: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/14.jpg)
LOOKING FOR THEBIOCHEMICAL/PHARMACOLOGICAL
“DIMER FINGERPRINT”
![Page 15: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/15.jpg)
IUBCP: Recognition and nomenclature
Alternative nomenclature in:Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446
![Page 16: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/16.jpg)
1 A Coimmunolocalization1 B Coimmunoprecipitation/transgenics2 Specific functional property (Pharmacology/Signalling)3 Knockouts: Loss of function!!!??
IUBCP: Recognition 2 out of 3:
![Page 17: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/17.jpg)
a b
Intramolecular cross-talk in the homomer:Cooperativity
![Page 18: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/18.jpg)
2-R1-R 1-R
2-R1-R 3-R1-R
a b
dc
Intramolecular cross-talk in the heteromer:Cooperativity and/or ?
![Page 19: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/19.jpg)
Looking for the “biochemical fingerprint”of a receptor heteromer
Fingerprint lost in knockout animals
![Page 20: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/20.jpg)
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
![Page 21: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/21.jpg)
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
![Page 22: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/22.jpg)
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
![Page 23: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/23.jpg)
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”
![Page 24: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/24.jpg)
LOOKING FOR THEFUNCTIONAL
“DIMER FINGERPRINT”
![Page 25: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/25.jpg)
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
![Page 26: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/26.jpg)
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
![Page 27: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/27.jpg)
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
![Page 28: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/28.jpg)
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
![Page 29: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/29.jpg)
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)
![Page 30: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/30.jpg)
Dual regulatory role of Adenosine on Glu release via A1 R/A2A R
Ciruela et al (2006) J Neurosc 26:2080-87
![Page 31: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/31.jpg)
CONCLUSIONS
1. Heteromers exist in natural tissues2. Heteromers are sensors for neurotransmitters 3. Heteromerization modifies:
- biochemical,- pharmacological,- and functional properties of receptors
![Page 32: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/32.jpg)
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
![Page 33: Basic concepts in G-protein-coupled receptor homo- and ...Basic concepts in G-protein-coupled. receptor homo- and. heterodimerization. ... Blue agonist “sensitizes” yellow and](https://reader034.fdocuments.in/reader034/viewer/2022051806/60027c0e74e190003624a315/html5/thumbnails/33.jpg)
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