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Basic concepts in G-protein-coupled receptor homo- and heterodimerization

Basic concepts in G-protein-coupled receptor homo- and heterodimerization. NIDA minisymposium. SfN San Diego. November 2007. www.bq.ub.es/recep/franco.html www.rafaelfranco.cat. RAFAEL FRANCO rfanco@ub.edu. STOCKHOLM K. Fuxe C. Ibáñez D. Marcellino. COIMBRA R. Cunha. WAKE-FOREST U.

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Basic concepts in G-protein-coupled receptor homo- and heterodimerization

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

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

  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) Heteromeric receptor Receptor homomer Receptor heteromer Ferré, 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) Heteromeric receptor Receptor homomer Receptor heteromer Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

  8. Receptor homomers Dopamine D2 Dopamine D2 Adenosine A2A Adenosine A2A Adenosine A2A Receptor heteromers Dopamine D2 Adenosine A2A Dopamine D1 NMDA Ferré, 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. 100 80 100 100 150 150 60 RDS (% of total A2AR) 40 50 50 20 0 Crude extract Cell surface The A2AR dimer is the functional specie IP Avidina IP A2AR MW (kDa) MW (kDa) Crude Crude ** ** * * A2AR A2AR Biotina The C terminal tail of A2AR is not required for dimerization Canals et al (2004) J Neurochem 88, 726-734

  11. HETERODIMERS: DR/AR D2R-A2AR receptor heteromers D1R-A1R receptor heteromers Hillion et al (2002) J Biol Chem Gines et al (2000) PNAS

  12. 396 nm Coelenterazine H 510 nm 470 nm Rluc GFP2 YFP YFP A A B B Rluc GFP2 Coelenterazine H 396 nm YFP YFP A A B B 530 nm 530 nm BRET FRET

  13. 0.1 0.05 0.0 0 10 20 30 40 The A2AR/D2 homodimers:BRET: Bioluminiscence Resonance Energy Transfer BRET SATURATION CURVE A2ARRluc-D2RYFP BRET increase A2ARRluc-GABABRYFP YFP/Rluc 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: Ferré, Ciruela, Woods, Lluis & Franco (2007) Trends Neurosci 30 (9) 440-446

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

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

  18. Intramolecular cross-talk in the heteromer: Cooperativity and/or ? 1-R 1-R 2-R a b c d 1-R 2-R 1-R 3-R

  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. L A+A+(RR) A+A+(RR)* a K K a L A+A(RR) A+A(RR)* mq K mK qa L A(RR)A A(RR)*A TERNARY MODEL RECEPTOR DIMER MODEL A + R + G AR+G A + RG ARG Franco et al (2005) Trends Biochem Sci 30: 360-66

  22. a K mK A+A+(R–R) A+A(R–R) A(R–R)A a L L qa L a K mq K A+A+(R~R) A+A(R~R) A(R~R)A b RR RR RR KD1 KD2 A2 A RR* RR* RR* Abound = (KD2 A + 2 A2) RT /(KD1 KD2 + KD2 A + A2)

  23. Dimer Cooperativity Index, Dc: It depends on KD1 and KD2 Allosterism: Useful parameter to “measure allosterism” D50 in Drug discovery: A more meaningful parameter to measure binding potency compared to IC50. Casadó et al., Pharmacol Ther In the Press (Dec 2007)

  24. LOOKING FOR THE FUNCTIONAL “DIMER FINGERPRINT”

  25. Clustering adaptor/scaffolding proteins Fig 4 Agonists for the two receptors Signalling A: Franco et al Trends Biochemical Sci 28 (2003) 238-243 Signalling B:

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

  27. The intracytoplasmic receptor-receptor cross-talk:ANTAGONISM (A1R/D1R) Ginés et al (2000) PNAS 97, 8606-11 cAMP producion via D1R after pretreatment with agonists of A1R and/or D1R (A1/D1 cotransfected mouse Ltk- fibroblasts)

  28. D1-R D2-R Gs Gi Gs A Cyclase A Cyclase Shift to Gq coupling in the D1-D2 receptor heteromer D2-R D1-R PKC Synaptic Plasticity Gq Diacylglycerol Ca2+ calmodulin kinase II (.: increase in accumbens) IP3 Ca2+ Rashid et al. (2007) Proc Natl Acad Sci 104, 654-9

  29. D1-D3 Receptor heteromerization D3-R D1-R • FRET • BRET • Fingerprint (Intramembrane cross-talk) 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 D1 receptor-mediated behavioral effects by a different mechanism than D2 receptor stimulation. Marcellino et al. (2007) Submitted

  30. Dual regulatory role of Adenosine on Glu release via A1R/A2AR Ciruela et al (2006) J Neurosc 26:2080-87

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

  32. FUNCTIONAL MODULES IN THE PLASMA MEMBRANE ADA ADA A2AR D2R mGluR5 A1R D1R mGluR1a ßArrestin caveolin ßArrestin caveolin homer Ca-calmodulin hsc73 filamin a-actinin

  33. Class A Rhodopsin like • Amine • Peptide • Hormone protein • (Rhodopsin • Rhodopsin Vertebrate • Rhodopsin Vertebrate type 1 • Rhodopsin Vertebrate type 2 • Rhodopsin Vertebrate type 3 • Rhodopsin Vertebrate type 4 • Rhodopsin Vertebrate type 5 • Rhodopsin Arthropod • Rhodopsin Mollusc • Rhodopsin Other • Olfactory • Prostanoid • Nucleotide-like • Cannabis • Platelet activating factor • Gonadotropin-releasing hormone • Thyrotropin-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

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