1. Basic concepts in G-protein-coupled receptor homo- and heterodimerization www.bq.ub.es/recep/franco.html www.rafaelfranco.cat RAFAEL FRANCO rfanco@ub.edu NIDA minisymposium. SfN San Diego. November 2007

2. STOCKHOLM K. Fuxe C. Ibáñez D. Marcellino NIH/NIDA N. Volkow S. Goldberg S. Ferré A.Woods MODENA L. Agnati S. Tanganelli P. G. de Benedetti F. Fanelli BERLIN M. Bader MONTREAL M. Bouvier WAKE-FOREST U. D. Roberts COIMBRA R. Cunha ALBACETE R. Luján GLASGOW G. Milligan M. Canals

3. Centro Investigación Médica Aplicada Pamplona Digna Biotech

4. GPCR classes

5. NOMENCLATURE Receptor for Neurotransmitters (including neuromodulators and neuropeptides): - Ionotropic - Metabotropic (G-protein-coupled, GPCR) Receptor homomer Receptor heteromer Heteromeric receptor Ferr é, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

6. Heteromeric receptor: Ach receptor

7. NOMENCLATURE Receptor for Neurotransmitters (including neuromodulators and neuropeptides): - Ionotropic - Metabotropic (G-protein-coupled, GPCR) Receptor homomer Receptor heteromer Heteromeric receptor Ferr é, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

8. Receptor homomers Adenosine A 2A Dopamine D 2 Adenosine A 2A Receptor heteromers Adenosine A 2A Dopamine D 2 NMDADopamine D 1 Ferr é, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

9. 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)

10. Crude IP Avidina 0 20 40 60 80 100 RDS (% of total A 2A R) Crude extract Cell surface ** * * 100 50 MW (kDa) 150 100 50 MW (kDa) 150 The A 2A R dimer is the functional specie Canals et al (2004) J Neurochem 88, 726-734 The C terminal tail of A 2A R is not required for dimerization A 2A R Crude Biotina IP A 2A R

11. HETERODIMERS: DR/AR D 2 R-A 2A R receptor heteromers D 1 R-A 1 R receptor heteromers Hillion et al (2002) J Biol Chem Gines et al (2000) PNAS

12. Rluc A B YFP Coelenterazine H 470 nm Rluc A B YFP Coelenterazine H 530 nm GFP 2 A B YFP 396 nm 510 nm GFP 2 A B YFP 530 nm 396 nm BRETFRET

13. The A2AR/D2 homodimers: BRET: Bioluminiscence Resonance Energy Transfer 010203040 0.0 0.05 0.1 YFP/Rluc BRET increase A 2A RRluc-D 2 RYFP A 2A RRluc-GABABRYFP BRET SATURATION CURVE Canals et al (2003) J Biol Chem 278: 46741-49

14. LOOKING FOR THE BIOCHEMICAL/PHARMACOLOGICAL “DIMER FINGERPRINT”

15. IUBCP: Recognition and nomenclature Alternative nomenclature in: Ferr é, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

16. 1 A Coimmunolocalization 1 B Coimmunoprecipitation/transgenics 2 Specific functional property (Pharmacology/Signalling) 3 Knockouts: Loss of function!!!?? IUBCP: Recognition 2 out of 3:

17. a b Intramolecular cross-talk in the homomer: Cooperativity

18. 2-R1-R 2-R1-R3-R1-R a b dc Intramolecular cross-talk in the heteromer: Cooperativity and/or ?

19. Looking for the “biochemical fingerprint” of a receptor heteromer Fingerprint lost in knockout animals

20. Different affinities for ligands: Caffeine as adenosine antagonist

21. A+A+(RR) A+A(RR) A(RR)A A+A+(RR)* A+A(RR)* A(RR)*A L   L  L   K   K KK KK Franco et al (2005) Trends Biochem Sci 30: 360-66 RECEPTOR DIMER MODEL A + R + G AR+G A + RG ARG TERNARY MODEL

22. 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   L  L KK KK   K   K K D1 K D2 A2A2 A RR RR* RR RR* RR RR* A bound = (K D2 A + 2 A 2 ) R T /(K D1 K D2 + K D2 A + A 2 ) a b

23. Dimer Cooperativity Index, Dc: It depends on K D1 and K D2 Casadó et al., Pharmacol Ther In the Press (Dec 2007) D 50 in Drug discovery: A more meaningful parameter to measure binding potency compared to IC 50. Allosterism: Useful parameter to “measure allosterism”

24. LOOKING FOR THE FUNCTIONAL “DIMER FINGERPRINT”

25. 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

26. Blue agonist “sensitizes” yellow and pink receptors Blue agonist “desensitizes” yellow and pink receptors Blue agonist “sensitizes” pink and “desensitizes” yellow SIGNALLING Signalling Fig 3 Franco et al Trends Biochemical Sci 28 (2003) 238-243

27. 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

28. A Cyclase GsGi D2-R A Cyclase Gs D1-R Ca 2+ IP3 D2-R D1-R Gq Ca 2+ calmodulin kinase II  (.: 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

29. 1.-D 1 receptor agonist affinity is enhanced by D 3 agonists. This indicates the existence of a synergistic intramembrane receptor-receptor interaction. 2.-Experiments in reserpinized mice showed that D 3 receptor stimulation potentiates D 1 receptor-mediated behavioral effects by a different mechanism than D 2 receptor stimulation. Marcellino et al. (2007) Submitted D3-R D1-R D1-D3 Receptor heteromerization -FRET -BRET -Fingerprint (Intramembrane cross-talk)

30. Dual regulatory role of Adenosine on Glu release via A 1 R/A 2A R Ciruela et al (2006) J Neurosc 26:2080-87

31. CONCLUSIONS 1.Heteromers exist in natural tissues 2.Heteromers are sensors for neurotransmitters 3.Heteromerization modifies: -biochemical, -pharmacological, -and functional properties of receptors

32. FUNCTIONAL MODULES IN THE PLASMA MEMBRANE ADA mGluR1  ADA hsc73 A1RA1R D1RD1R ßArrestin caveolin A 2A R mGluR5D2RD2R  -actinin homer Ca-calmodulin filamin ßArrestin caveolin

33. Class A Rhodopsin like Amine Peptide Hormone protein (Rhodopsin Rhodopsin Vertebrate Rhodopsin Vertebrate type 1Rhodopsin Vertebrate type 1 Rhodopsin Vertebrate type 2Rhodopsin Vertebrate type 2 Rhodopsin Vertebrate type 3Rhodopsin Vertebrate type 3 Rhodopsin Vertebrate type 4Rhodopsin Vertebrate type 4 Rhodopsin Vertebrate type 5Rhodopsin Vertebrate type 5 Rhodopsin Arthropod Rhodopsin Mollusc Rhodopsin Other Olfactory Prostanoid Nucleotide-like Cannabis Platelet activating factor Gonadotropin-releasing hormone Thyrotropin-releasing hormone & SecretagogueThyrotropin-releasing hormone & Secretagogue Melatonin Viral Lysosphingolipid & LPA (EDG) Leukotriene B4 receptor Class A Orphan/other Class B Secretin like Class C Metabotropic glutamate / pheromone Class D Fungal pheromone Class E cAMP receptors (Dictyostelium) Frizzled/Smoothened family