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In vivo effects of a GPR30 antagonist

Nature Chemical Biology volume 5, pages 421–427 (2009)Cite this article

Abstract

Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein–coupled receptor GPR30 (also known as GPER), in addition to classical nuclear estrogen receptors (ERα and ERβ), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized G-1 (1), a selective agonist of GPR30. To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

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Figure 1: Structures and synthesis of G-1 and G15.
Figure 2: Ligand binding properties of G15.
Figure 3: G15 antagonism of intracellular calcium mobilization by GPR30.
Figure 4: G15 antagonism of PI(3)K activation by GPR30.
Figure 5: Effects of G15 on physiological responses mediated by GPR30.

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References

  1. Edwards, D.P. Regulation of signal transduction pathways by estrogen and progesterone. Annu. Rev. Physiol. 67, 335–376 (2005).

    Article  CAS  Google Scholar 

  2. Lange, C.A., Gioeli, D., Hammes, S.R. & Marker, P.C. Integration of rapid signaling events with steroid hormone receptor action in breast and prostate cancer. Annu. Rev. Physiol. 69, 171–199 (2007).

    Article  CAS  Google Scholar 

  3. Fu, X.D. & Simoncini, T. Extra-nuclear signaling of estrogen receptors. IUBMB Life 60, 502–510 (2008).

    Article  CAS  Google Scholar 

  4. Prossnitz, E.R. et al. Estrogen signaling through the transmembrane G protein-coupled receptor GPR30. Annu. Rev. Physiol. 70, 165–190 (2008).

    Article  CAS  Google Scholar 

  5. Prossnitz, E.R., Sklar, L.A., Oprea, T.I. & Arterburn, J.B. GPR30: a novel therapeutic target in estrogen-related disease. Trends Pharmacol. Sci. 29, 116–123 (2008).

    Article  CAS  Google Scholar 

  6. Revankar, C.M., Cimino, D.F., Sklar, L.A., Arterburn, J.B. & Prossnitz, E.R. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307, 1625–1630 (2005).

    Article  CAS  Google Scholar 

  7. Thomas, P., Pang, Y., Filardo, E.J. & Dong, J. Identity of an estrogen membrane receptor coupled to a G-protein in human breast cancer cells. Endocrinology 146, 624–632 (2005).

    Article  CAS  Google Scholar 

  8. Filardo, E.J., Quinn, J.A., Bland, K.I. & Frackelton, A.R. Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol. Endocrinol. 14, 1649–1660 (2000).

    Article  CAS  Google Scholar 

  9. Ariazi, E.A., Ariazi, J.L., Cordera, F. & Jordan, V.C. Estrogen receptors as therapeutic targets in breast cancer. Curr. Top. Med. Chem. 6, 181–202 (2006).

    Article  CAS  Google Scholar 

  10. Bologa, C.G. et al. Virtual and biomolecular screening converge on a selective agonist for GPR30. Nat. Chem. Biol. 2, 207–212 (2006).

    Article  CAS  Google Scholar 

  11. Noel, S.D., Keen, K.L., Baumann, D.I., Filardo, E.J. & Terasawa, E. Involvement of G-protein coupled receptor 30 (GPR30) in rapid action of estrogen in primate LHRH neurons. Mol. Endocrinol. 23, 349–359 (2009).

    Article  CAS  Google Scholar 

  12. Brailoiu, E. et al. Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system. J. Endocrinol. 193, 311–321 (2007).

    Article  CAS  Google Scholar 

  13. Dun, S.L. et al. Expression of estrogen receptor GPR30 in the rat spinal cord and in autonomic and sensory ganglia. J. Neurosci. Res. 87, 1610–1619 (2009).

    Article  CAS  Google Scholar 

  14. Kuhn, J. et al. GPR30 estrogen receptor agonists induce mechanical hyperalgesia in the rat. Eur. J. Neurosci. 27, 1700–1709 (2008).

    Article  Google Scholar 

  15. Albanito, L. et al. G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells. Cancer Res. 67, 1859–1866 (2007).

    Article  CAS  Google Scholar 

  16. Pandey, D.P. et al. Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF. EMBO J. 28, 523–532 (2009).

    Article  Google Scholar 

  17. Teng, J., Wang, Z.Y., Prossnitz, E.R. & Bjorling, D.E. The G protein-coupled receptor GPR30 inhibits human urothelial cell proliferation. Endocrinology 149, 4024–4034 (2008).

    Article  CAS  Google Scholar 

  18. Pang, Y., Dong, J. & Thomas, P. Estrogen signaling characteristics of Atlantic croaker G protein-coupled receptor 30 (GPR30) and evidence it is involved in maintenance of oocyte meiotic arrest. Endocrinology 149, 3410–3426 (2008).

    Article  CAS  Google Scholar 

  19. Wang, C., Prossnitz, E.R. & Roy, S.K. G protein-coupled receptor 30 expression is required for estrogen stimulation of primordial follicle formation in the hamster ovary. Endocrinology 149, 4452–4461 (2008).

    Article  CAS  Google Scholar 

  20. Wang, C. et al. GPR30 contributes to estrogen-induced thymic atrophy. Mol. Endocrinol. 22, 636–648 (2008).

    Article  CAS  Google Scholar 

  21. Wang, C. et al. Membrane estrogen receptor regulates experimental autoimmune encephalomyelitis through up-regulation of programmed death 1. J. Immunol. 182, 3294–3303 (2009).

    Article  CAS  Google Scholar 

  22. Haas, E. et al. Regulatory role of G protein-coupled estrogen receptor for vascular function and obesity. Circ. Res. 104, 288–291 (2009).

    Article  CAS  Google Scholar 

  23. Kobayashi, S., Ishitani, H. & Nagayama, S. Lanthanide triflate catalyzed imino Diels-Alder reactions; convenient synthesis of pyridine and quinoline derivatives. Synthesis 1195–1202 (1995).

  24. Balla, T. & Varnai, P. Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci. STKE 2002, PL3 (2002).

    PubMed  Google Scholar 

  25. Owens, J.W. & Ashby, J. Critical review and evaluation of the uterotrophic bioassay for the identification of possible estrogen agonists and antagonists: in support of the validation of the OECD uterotrophic protocols for the laboratory rodent. Organisation for Economic Co-operation and Development. Crit. Rev. Toxicol. 32, 445–520 (2002).

    Article  CAS  Google Scholar 

  26. Hewitt, S.C., Harrell, J.C. & Korach, K.S. Lessons in estrogen biology from knockout and transgenic animals. Annu. Rev. Physiol. 67, 285–308 (2005).

    Article  CAS  Google Scholar 

  27. Epperson, C.N., Wisner, K.L. & Yamamoto, B. Gonadal steroids in the treatment of mood disorders. Psychosom. Med. 61, 676–697 (1999).

    Article  CAS  Google Scholar 

  28. Genazzani, A.R., Spinetti, A., Gallo, R. & Bernardi, F. Menopause and the central nervous system: intervention options. Maturitas 31, 103–110 (1999).

    Article  CAS  Google Scholar 

  29. Porsolt, R.D., Anton, G., Blavet, N. & Jalfre, M. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47, 379–391 (1978).

    Article  CAS  Google Scholar 

  30. Willner, P. Animal models of depression: an overview. Pharmacol. Ther. 45, 425–455 (1990).

    Article  CAS  Google Scholar 

  31. Estrada-Camarena, E., Fernandez-Guasti, A. & Lopez-Rubalcava, C. Antidepressant-like effect of different estrogenic compounds in the forced swimming test. Neuropsychopharmacology 28, 830–838 (2003).

    Article  CAS  Google Scholar 

  32. Steru, L. et al. The automated Tail Suspension Test: a computerized device which differentiates psychotropic drugs. Prog. Neuropsychopharmacol. Biol. Psychiatry 11, 659–671 (1987).

    Article  CAS  Google Scholar 

  33. Steru, L., Chermat, R., Thierry, B. & Simon, P. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology (Berl.) 85, 367–370 (1985).

    Article  CAS  Google Scholar 

  34. Cryan, J.F., Mombereau, C. & Vassout, A. The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice. Neurosci. Biobehav. Rev. 29, 571–625 (2005).

    Article  CAS  Google Scholar 

  35. Tanenbaum, D.M., Wang, Y., Williams, S.P. & Sigler, P.B. Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains. Proc. Natl. Acad. Sci. USA 95, 5998–6003 (1998).

    Article  CAS  Google Scholar 

  36. Wada-Hiraike, O. et al. Role of estrogen receptor beta in uterine stroma and epithelium: insights from estrogen receptor beta−/− mice. Proc. Natl. Acad. Sci. USA 103, 18350–18355 (2006).

    Article  CAS  Google Scholar 

  37. Otto, C. et al. G protein-could receptor 30 localizes to the endoplasmic reticulum and is not activated by estradiol. Endocrinology 149, 4846–4856 (2008).

    Article  CAS  Google Scholar 

  38. Grunert, G., Porcia, M. & Tchernitchin, A.N. Differential potency of oestradiol-17 beta and diethylstilboestrol on separate groups of responses in the rat uterus. J. Endocrinol. 110, 103–114 (1986).

    Article  CAS  Google Scholar 

  39. Diel, P. et al. The differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and proliferation in the rat is associated with a substance specific modulation of uterine gene expression. Mol. Cell. Endocrinol. 221, 21–32 (2004).

    Article  CAS  Google Scholar 

  40. Frasor, J. et al. Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus. Endocrinology 144, 3159–3166 (2003).

    Article  CAS  Google Scholar 

  41. Thomas, P. & Dong, J. Binding and activation of the seven-transmembrane estrogen receptor GPR30 by environmental estrogens: a potential novel mechanism of endocrine disruption. J. Steroid Biochem. Mol. Biol. 102, 175–179 (2006).

    Article  CAS  Google Scholar 

  42. Ramirez, S., Aiken, C.T., Andrzejewski, B., Sklar, L.A. & Edwards, B.S. High-throughput flow cytometry: validation in microvolume bioassays. Cytometry A 53, 55–65 (2003).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grants CA118743 and CA127731 (to E.R.P.), by grants from the Oxnard and Stranahan Foundations (to E.R.P.), by the University of New Mexico Center for Molecular Discovery (NIH U54 MH084690; to L.A.S.) by the New Mexico Tobacco Settlement fund (to T.I.O.), by the New Mexico Cowboys for Cancer Research (to J.B.A.) and by NIH grants R37 NS18710 (to N.J.D.) and HL90804 (to E.B.). Flow cytometry data and confocal images in this study were generated in the Flow Cytometry and Fluorescence Microscopy Facilities, which received support from the University of New Mexico Health Sciences Center and the University of New Mexico Cancer Center (as detailed at http://hsc.unm.edu/crtc/microscopy/facility.html). In vivo data were generated with the support of the University of New Mexico Cancer Center Animal Models & Imaging Core.

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Authors and Affiliations

  1. Department of Cell Biology & Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA

    Megan K Dennis, Whitney K Petrie, Sara N Alcon, Tapan K Nayak, Helen J Hathaway & Eric R Prossnitz

  2. Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico, USA

    Ritwik Burai, Chinnasamy Ramesh & Jeffrey B Arterburn

  3. Division of Biocomputing, Department of Biochemistry & Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA

    Cristian G Bologa, Andrei Leitao & Tudor I Oprea

  4. Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

    Eugen Brailoiu, Elena Deliu & Nae J Dun

  5. University of New Mexico Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA

    Larry A Sklar, Helen J Hathaway, Jeffrey B Arterburn, Tudor I Oprea & Eric R Prossnitz

  6. Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA

    Larry A Sklar

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Contributions

M.K.D. designed, performed and analyzed biomolecular screening, ligand binding and cell biology experiments. W.K.P., S.N.A. and H.J.H. designed, performed and analyzed animal uterine experiments. T.K.N. designed, performed and analyzed ligand binding experiments. E.B., E.D. and N.J.D. designed, performed and analyzed animal depression experiments. C.G.B., A.L. and T.I.O. designed, performed and analyzed virtual screening experiments. R.B., C.R. and J.B.A. synthesized G-1 and G15. L.A.S. provided integration with the University of New Mexico MLSC. E.R.P. designed and analyzed experiments and, with contributions from all authors, wrote the paper.

Corresponding authors

Correspondence to Jeffrey B Arterburn, Tudor I Oprea or Eric R Prossnitz.

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Dennis, M., Burai, R., Ramesh, C. et al. In vivo effects of a GPR30 antagonist. Nat Chem Biol 5, 421–427 (2009). https://doi.org/10.1038/nchembio.168

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