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CODA-RET reveals functional selectivity as a result of GPCR heteromerization

Nature Chemical Biology volume 7, pages 624–630 (2011)Cite this article

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Abstract

Here we present a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein–coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(−)-10,11-dihydroxy-N-n-propylnoraporphine (NPA) to change the Gαi conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a G protein–coupled receptor (GPCR) protomer depending on the identity of the second protomer participating in the formation of the signaling unit—opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling.

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Figure 1: D1R coexpression enhances NPA potency at D2R.
Figure 2: Characterization of homomeric receptors in G protein signaling complexes.
Figure 3: Characterization of heteromeric receptors in G protein signaling complexes.
Figure 4: CODA-RET reveals functional selectivity of NPA at the D2R-D1R heteromer.

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References

  1. Springael, J.Y., Urizar, E., Costagliola, S., Vassart, G. & Parmentier, M. Allosteric properties of G protein-coupled receptor oligomers. Pharmacol. Ther. 115, 410–418 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Han, Y., Moreira, I.S., Urizar, E., Weinstein, H. & Javitch, J.A. Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation. Nat. Chem. Biol. 5, 688–695 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ferré, S. et al. Building a new conceptual framework for receptor heteromers. Nat. Chem. Biol. 5, 131–134 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Albizu, L. et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol. 6, 587–594 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rivero-Müller, A. et al. Rescue of defective G protein-coupled receptor function in vivo by intermolecular cooperation. Proc. Natl. Acad. Sci. USA 107, 2319–2324 (2010).

    Article  PubMed  Google Scholar 

  6. Milligan, G. G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function. Br. J. Pharmacol. 158, 5–14 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Smith, N.J. & Milligan, G. Allostery at G protein-coupled receptor homo- and heteromers: uncharted pharmacological landscapes. Pharmacol. Rev. 62, 701–725 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Urban, J.D. et al. Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther. 320, 1–13 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Kilts, J.D. et al. Functional selectivity of dopamine receptor agonists. II. Actions of dihydrexidine in D2L receptor-transfected MN9D cells and pituitary lactotrophs. J. Pharmacol. Exp. Ther. 301, 1179–1189 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Masri, B. et al. Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction is a common property of clinically effective antipsychotics. Proc. Natl. Acad. Sci. USA 105, 13656–13661 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Mottola, D.M. et al. Functional selectivity of dopamine receptor agonists. I. Selective activation of postsynaptic dopamine D2 receptors linked to adenylate cyclase. J. Pharmacol. Exp. Ther. 301, 1166–1178 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Mailman, R.B. GPCR functional selectivity has therapeutic impact. Trends Pharmacol. Sci. 28, 390–396 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Urban, J.D., Vargas, G.A., von Zastrow, M. & Mailman, R.B. Aripiprazole has functionally selective actions at dopamine D2 receptor-mediated signaling pathways. Neuropsychopharmacology 32, 67–77 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Bosier, B. & Hermans, E. Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol. Sci. 28, 438–446 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Kenakin, T. Collateral efficacy in drug discovery: taking advantage of the good (allosteric) nature of 7TM receptors. Trends Pharmacol. Sci. 28, 407–415 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. George, S.R. et al. Oligomerization of mu- and delta-opioid receptors - Generation of novel functional properties. J. Biol. Chem. 275, 26128–26135 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Lee, S.P. et al. Dopamine D1 and D2 receptor co-activation generates a novel phospholipase C-mediated calcium signal. J. Biol. Chem. 279, 35671–35678 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Rives, M.L. et al. Crosstalk between GABA(B) and mGlu1a receptors reveals new insight into GPCR signal integration. EMBO J. 28, 2195–2208 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Guo, W. et al. Dopamine D2 receptors form higher order oligomers at physiological expression levels. EMBO J. 27, 2293–2304 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Galés, C. et al. Real time monitoring of receptor and G-protein interactions in living cells. Nat. Methods 2, 177–184 (2005).

    Article  PubMed  Google Scholar 

  21. Galés, C. et al. Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat. Struct. Mol. Biol. 13, 778–786 (2006).

    Article  PubMed  Google Scholar 

  22. O'Dowd, B.F. et al. Dopamine receptor oligomerization visualized in living cells. J. Biol. Chem. 280, 37225–37235 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Rashid, A.J. et al. D1–D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum. Proc. Natl. Acad. Sci. USA 104, 654–659 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. So, C.H. et al. D1 and D2 dopamine receptors form heterooligomers and cointernalize after selective activation of either receptor. Mol. Pharmacol. 68, 568–578 (2005).

    CAS  PubMed  Google Scholar 

  25. So, C.H., Verma, V., O'Dowd, B.F. & George, S.R. Desensitization of the dopamine D1 and D2 receptor hetero-oligomer mediated calcium signal by agonist occupancy of either receptor. Mol. Pharmacol. 72, 450–462 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Loening, A.M., Fenn, T.D., Wu, A.M. & Gambhir, S.S. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng. Des. Sel. 19, 391–400 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Hynes, T.R. et al. Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting. J. Biol. Chem. 279, 30279–30286 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Mervine, S.M., Yost, E.A., Sabo, J.L., Hynes, T.R. & Berlot, C.H. Analysis of G protein betagamma dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of beta1 for particular gamma subunits. Mol. Pharmacol. 70, 194–205 (2006).

    CAS  PubMed  Google Scholar 

  30. Paulmurugan, R., Umezawa, Y. & Gambhir, S.S. Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc. Natl. Acad. Sci. USA 99, 15608–15613 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, J., Xiong, B., Zhen, X. & Zhang, A. Dopamine D1 receptor ligands: where are we now and where are we going. Med. Res. Rev. 29, 272–294 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Guo, N. et al. Impact of D2 Receptor internalization on binding affinity of neuroimaging radiotracers. Neuropsychopharmacology 35, 806–817 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Mansour, A. et al. Site-directed mutagenesis of the human dopamine D2 receptor. Eur. J. Pharmacol. 227, 205–214 (1992).

    Article  CAS  PubMed  Google Scholar 

  34. Paulmurugan, R. & Gambhir, S.S. Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Anal. Chem. 75, 1584–1589 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Terrillon, S. & Bouvier, M. Roles of G-protein-coupled receptor dimerization—From ontogeny to signalling regulation. EMBO Rep. 5, 30–34 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dalrymple, M.B., Pfleger, K.D.G. & Eidne, K.A. G protein-coupled receptor dimers: functional consequences, disease states and drug targets. Pharmacol. Ther. 118, 359–371 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Selbie, L.A. & Hill, S.J. G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways. Trends Pharmacol. Sci. 19, 87–93 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Bygrave, F.L. & Roberts, H.R. Regulation of cellular calcium through signaling cross-talk involves an intricate interplay between the actions of receptors, G-proteins, and second messengers. FASEB J. 9, 1297–1303 (1995).

    Article  CAS  PubMed  Google Scholar 

  39. Kenakin, T. Agonist-receptor efficacy. 2. Agonist trafficking of receptor signals. Trends Pharmacol. Sci. 16, 232–238 (1995).

    Article  CAS  PubMed  Google Scholar 

  40. Meller, E., Puza, T., Diamond, J., Lieu, H.D. & Bohmaker, K. Comparative effects of receptor inactivation, 17 beta-estradiol and pertussis toxin on dopaminergic inhibition of prolactin secretion in vitro. J. Pharmacol. Exp. Ther. 263, 462–469 (1992).

    CAS  PubMed  Google Scholar 

  41. Spongier, D. et al. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365, 170–175 (1993).

    Article  Google Scholar 

  42. Wisler, J.W. et al. A unique mechanism of beta-blocker action: Carvedilol stimulates beta-arrestin signaling. Proc. Natl. Acad. Sci. USA 104, 16657–16662 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Hern, J.A. et al. Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc. Natl. Acad. Sci. USA 107, 2693–2698 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Fonseca, J.M. & Lambert, N.A. Instability of a class A G protein-coupled receptor oligomer interface. Mol. Pharmacol. 75, 1296–1299 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Dorsch, S., Klotz, K.N., Engelhardt, S., Lohse, M.J. & Bunemann, M. Analysis of receptor oligomerization by FRAP microscopy. Nat. Methods 6, 225–230 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Guo, W., Shi, L. & Javitch, J.A. The fourth transmembrane segment forms the interface of the dopamine D2 receptor homodimer. J. Biol. Chem. 278, 4385–4388 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Urizar, E. et al. Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity. EMBO J. 24, 1954–1964 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Costagliola, S., Khoo, D. & Vassart, G. Production of bioactive amino-terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor. FEBS Lett. 436, 427–433 (1998).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported in part by US National Institutes of Health (NIH) grants DA022413 and MH054137 (J.A.J.), GM078319 (N.L.) and NIH TL1 RR024158-04 (H.Y.) as well as by the Lieber Center for Schizophrenia Research and Treatment and EMBO Long-Term and Basque Country fellowships (E.U.).

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Author notes

  1. Eneko Urizar

    Present address: Present address: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Brussels, Belgium.,

  2. Eneko Urizar and Hideaki Yano: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York, USA

    Eneko Urizar, Hideaki Yano, Rachel Kolster & Jonathan A Javitch

  2. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA

    Eneko Urizar & Jonathan A Javitch

  3. Institut National de la Santé et de la Recherche Médicale, Toulouse, France

    Céline Galés

  4. Paul Sabatier University, Toulouse, France

    Céline Galés

  5. Department of Pharmacology and Toxicology, Georgia Health Sciences University, Augusta, Georgia, USA

    Nevin Lambert

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Contributions

E.U., H.Y. and R.K. performed experiments; E.U., H.Y. and J.A.J. planned experiments; J.A.J. supervised the project; C.G. and N.L. provided reagents and discussed the experimental findings and interpretation of results; E.U. and J.A.J. wrote the manuscript.

Corresponding author

Correspondence to Jonathan A Javitch.

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The authors declare no competing financial interests.

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Urizar, E., Yano, H., Kolster, R. et al. CODA-RET reveals functional selectivity as a result of GPCR heteromerization. Nat Chem Biol 7, 624–630 (2011). https://doi.org/10.1038/nchembio.623

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