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Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation

Nature Chemical Biology volume 5, pages 688–695 (2009)Cite this article

Abstract

A major obstacle to understanding the functional importance of dimerization between class A G protein–coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control, and we demonstrate it here for the human dopamine D2 receptor. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer. Inverse agonist binding to the second protomer enhances signaling, whereas agonist binding to the second protomer blunts signaling. Ligand-independent constitutive activation of the second protomer also inhibits signaling. Thus, GPCR dimer function can be modulated by the activity state of the second protomer, which for a heterodimer may be altered in pathological states. Our new methodology also makes possible the characterization of signaling from a defined heterodimer unit.

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Figure 1: Functional complementation of two 'nonfunctional receptors'.
Figure 2: Characterization of D2R mutants.
Figure 3: Asymmetric contributions of the protomers to signaling.
Figure 4: The second protomer allosterically modulates signaling.
Figure 5: Computational model of the complex between the rhodopsin dimer and heterotrimeric Gt.
Figure 6: Cartoon of different D2R dimer activation states, with activation data for these states, from the perspective of agonist-mediated activation of protomer A.

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References

  1. Pin, J.P. et al. International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers. Pharmacol. Rev. 59, 5–13 (2007).

    Article  CAS  Google Scholar 

  2. Sartania, N., Appelbe, S., Pediani, J.D. & Milligan, G. Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric beta2-adrenoceptor. Cell. Signal. 19, 1928–1938 (2007).

    Article  CAS  Google Scholar 

  3. Parenty, G., Appelbe, S. & Milligan, G. CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer. Biochem. J. 412, 245–256 (2008).

    Article  CAS  Google Scholar 

  4. Vilardaga, J.P. et al. Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling. Nat. Chem. Biol. 4, 126–131 (2008).

    Article  CAS  Google Scholar 

  5. Pin, J.P., Galvez, T. & Prezeau, L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacol. Ther. 98, 325–354 (2003).

    Article  CAS  Google Scholar 

  6. Brock, C. et al. Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement. J. Biol. Chem. 282, 33000–33008 (2007).

    Article  CAS  Google Scholar 

  7. Ji, I.H., Lee, C., Song, Y.S., Conn, P.M. & Ji, T.H. Cis- and trans-activation of hormone receptors: the LH receptor. Mol. Endocrinol. 16, 1299–1308 (2002).

    Article  CAS  Google Scholar 

  8. Bayburt, T.H., Leitz, A.J., Xie, G., Oprian, D.D. & Sligar, S.G. Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J. Biol. Chem. 282, 14875–14881 (2007).

    Article  CAS  Google Scholar 

  9. Whorton, M.R. et al. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl. Acad. Sci. USA 104, 7682–7687 (2007).

    Article  CAS  Google Scholar 

  10. 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  Google Scholar 

  11. 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  Google Scholar 

  12. 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  Google Scholar 

  13. Mesnier, D. & Baneres, J.L. Cooperative conformational changes in a G-protein-coupled receptor dimer, the leukotriene B4 receptor BLT1. J. Biol. Chem. 279, 49664–49670 (2004).

    Article  CAS  Google Scholar 

  14. Damian, M., Mary, S., Martin, A., Pin, J.P. & Baneres, J.L. G protein activation by the leukotriene B4 receptor dimer: evidence for an absence of trans-activation. J. Biol. Chem. 283, 21084–21092 (2008).

    Article  CAS  Google Scholar 

  15. Carrillo, J.J., Pediani, J. & Milligan, G. Dimers of class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins. J. Biol. Chem. 278, 42578–42587 (2003).

    Article  CAS  Google Scholar 

  16. Molinari, P. et al. Promiscuous coupling at receptor-Galpha fusion proteins. The receptor of one covalent complex interacts with the alpha -subunit of another. J. Biol. Chem. 278, 15778–15788 (2003).

    Article  CAS  Google Scholar 

  17. Pascal, G. & Milligan, G. Functional complementation and the analysis of opioid receptor homodimerization. Mol. Pharmacol. 68, 905–915 (2005).

    CAS  PubMed  Google Scholar 

  18. Seifert, R., Wenzel-Seifert, K. & Kobilka, B.K. GPCR-G[alpha] fusion proteins: molecular analysis of receptor-G-protein coupling. Trends Pharmacol. Sci. 20, 383–389 (1999).

    Article  CAS  Google Scholar 

  19. Lee, T.W., Seifert, R., Guan, X. & Kobilka, B.K. Restricting the mobility of Gs alpha: impact on receptor and effector coupling. Biochemistry 38, 13801–13809 (1999).

    Article  CAS  Google Scholar 

  20. Wenzel-Seifert, K., Lee, T.W., Seifert, R. & Kobilka, B.K. Restricting mobility of Gsalpha relative to the beta2-adrenoceptor enhances adenylate cyclase activity by reducing Gsalpha GTPase activity. Biochem. J. 334, 519–524 (1998).

    Article  CAS  Google Scholar 

  21. Rizzuto, R., Simpson, A.W.M., Brini, M. & Pozzan, T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358, 325–327 (1992).

    Article  CAS  Google Scholar 

  22. Blanpain, C. et al. Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity. J. Biol. Chem. 274, 18902–18908 (1999).

    Article  CAS  Google Scholar 

  23. Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D. & Bourne, H.R. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363, 274–276 (1993).

    Article  CAS  Google Scholar 

  24. Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B. & Bourne, H.R. A mutation that prevents GTP-dependent activation of the alpha chain of Gs. Nature 334, 712–715 (1988).

    Article  CAS  Google Scholar 

  25. Xu, W. et al. Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor. FEBS Lett. 447, 318–324 (1999).

    Article  CAS  Google Scholar 

  26. Ballesteros, J.A. et al. Activation of the beta(2)-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J. Biol. Chem. 276, 29171–29177 (2001).

    Article  CAS  Google Scholar 

  27. Moro, O., Lameh, J., Hogger, P. & Sadee, W. Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling. J. Biol. Chem. 268, 22273–22276 (1993).

    CAS  PubMed  Google Scholar 

  28. Strader, C.D. et al. Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity. J. Biol. Chem. 262, 16439–16443 (1987); erratum 263, 3050 (1988).

    CAS  PubMed  Google Scholar 

  29. Neve, K.A. et al. Modeling and mutational analysis of a putative sodium-binding pocket on the dopamine D-2 receptor. Mol. Pharmacol. 60, 373–381 (2001).

    Article  CAS  Google Scholar 

  30. Urizar, E. et al. An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor. J. Biol. Chem. 280, 17135–17141 (2005).

    Article  CAS  Google Scholar 

  31. Simpson, M.M. et al. Dopamine D4/D2 receptor selectivity is determined by a divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments. Mol. Pharmacol. 56, 1116–1126 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Wilson, J., Lin, H., Fu, D., Javitch, J.A. & Strange, P.G. Mechanisms of inverse agonism of antipsychotic drugs at the D2 dopamine receptor: use of a mutant D2 dopamine receptor that adopts the activated conformation. J. Neurochem. 77, 493–504 (2001).

    Article  CAS  Google Scholar 

  34. Guo, W., Shi, L., Filizola, M., Weinstein, H. & Javitch, J.A. From the cover: crosstalk in G protein-coupled receptors: changes at the transmembrane homodimer interface determine activation. Proc. Natl. Acad. Sci. USA 102, 17495–17500 (2005).

    Article  CAS  Google Scholar 

  35. Kota, P., Reeves, P.J., RajBhandary, U.L. & Khorana, H.G. Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface. Proc. Natl. Acad. Sci. USA 103, 3054–3059 (2006).

    Article  CAS  Google Scholar 

  36. Goudet, C. et al. Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators. J. Biol. Chem. 280, 24380–24385 (2005).

    Article  CAS  Google Scholar 

  37. Hlavackova, V. et al. Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR. EMBO J. 24, 499–509 (2005).

    Article  CAS  Google Scholar 

  38. Kniazeff, J. et al. Closed state of both binding domains of homodimeric mGlu receptors is required for full activity. Nat. Struct. Mol. Biol. 11, 706–713 (2004).

    Article  CAS  Google Scholar 

  39. Kaupmann, K. et al. GABAB-receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683–687 (1998).

    Article  CAS  Google Scholar 

  40. Damian, M., Martin, A., Mesnier, D., Pin, J.P. & Baneres, J.L. Asymmetric conformational changes in a GPCR dimer controlled by G-proteins. EMBO J. 25, 5693–5702 (2006).

    Article  CAS  Google Scholar 

  41. 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  Google Scholar 

  42. Springael, J.Y. et al. Allosteric modulation of binding properties between units of chemokine receptor homo- and hetero-oligomers. Mol. Pharmacol. 69, 1652–1661 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Javitch, J.A. et al. The fourth transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle. Biochemistry 39, 12190–12199 (2000).

    Article  CAS  Google Scholar 

  45. Costagliola, S. et al. Structure-function relationships of two loss-of-function mutations of the thyrotropin receptor gene. Thyroid 9, 995–1000 (1999).

    Article  CAS  Google Scholar 

  46. Brini, M. et al. Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). J. Biol. Chem. 270, 9896–9903 (1995).

    Article  CAS  Google Scholar 

  47. Niv, M.Y., Skrabanek, L., Filizola, M. & Weinstein, H. Modeling activated states of GPCRs: the rhodopsin template. J. Comput. Aided Mol. Des. 20, 437–448 (2006).

    Article  CAS  Google Scholar 

  48. van Dijk, A.D. et al. Data-driven: HADDOCK's adventures in CAPRI. Proteins 60, 232–238 (2005).

    Article  CAS  Google Scholar 

  49. van Dijk, M., van Dijk, A.D., Hsu, V., Boelens, R. & Bonvin, A.M. Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility. Nucleic Acids Res. 34, 3317–3325 (2006).

    Article  CAS  Google Scholar 

  50. Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C. Galás for discussion and comments on the manuscript. Plasmids encoding apo-aequorin were a gift from V. Dupriez (Euroscreen). This work was supported in part by US National Institutes of Health grants DA022413 and MH054137 (to J.A.J.) and DA012923 (to H.W.), by the Lieber Center for Schizophrenia Research and Treatment, and by a European Molecular Biology Organization long-term fellowship (to E.U.). Computational resources of the David A. Cofrin Center for Biomedical Information (Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University) are gratefully acknowledged.

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Authors and Affiliations

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

    Yang Han, Eneko Urizar & Jonathan A Javitch

  2. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Yang Han & Eneko Urizar

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

    Yang Han, Eneko Urizar & Jonathan A Javitch

  4. Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, New York, USA

    Irina S Moreira & Harel Weinstein

  5. The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, New York, New York, USA

    Harel Weinstein

  6. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Jonathan A Javitch

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Contributions

Y.H. created all the mutant constructs and cell lines, helped to design the experiments, carried out the experimental assays and analyzed the results. I.S.M. performed the computational analysis. E.U., H.W. and J.A.J. helped to design experiments and interpret results. All the authors participated in the writing and editing of the manuscript.

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Correspondence to Jonathan A Javitch.

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Han, Y., Moreira, I., Urizar, E. et al. Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation. Nat Chem Biol 5, 688–695 (2009). https://doi.org/10.1038/nchembio.199

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