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A Simple Method for Visual Assessment and Quantification of Altered Subcellular Localization of Nuclear Receptors

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Nuclear Receptors

Abstract

All nuclear receptors reside in the nuclear compartment when transcriptionally active. However, their subcellular localization can vary when unliganded, functionally inactive, or dysregulated in diseased conditions. This property of differential localization and inducible nuclear translocation of nuclear receptors has proven to be useful tool in characterizing their transcription functions, discovery of novels drugs, ligands (agonists, antagonists, selective modulators of receptor action, endocrine disruptors, endobiotics, xenobiotics, etc.), partner-mediated interactions and coregulators. To efficiently study these ligand-modulated transcription factors, we describe a simple method for visual assessment and quantification of subcellular localization achieved by these receptors under varying physiological conditions. Contrary to some of the emerging high-end instrument-based assessments, the current method is simple, economical and highly reproducible, giving options to conduct similar studies in less sophisticated settings. It is expected that the current assessment approach will help investigators in their discovery of mechanisms of actions of these receptors in health and disease, and also in defining novel small molecule modulators to overcome physiological perturbations.

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References

  1. Dash AK, Tyagi RK. A compendium of nuclear receptors: the superfamily of ligand-modulated transcription factors. J Endocrinol Reprod. 2016;20(1):1–27.

    Google Scholar 

  2. Escriva H, Bertrand S, Laudet V. The evolution of the nuclear receptor superfamily. Essays Biochem. 2015;40(3):11–26.

    Google Scholar 

  3. Mazaira GI, Zgajnar NR, Lotufo CM, Daneri-Becerra C, Sivils JC, Soto OB, et al. The nuclear receptor field: a historical overview and future challenges. Nucl Recept Res. 2018;5:101320.

    Article  CAS  Google Scholar 

  4. Mullican SE, DiSpirito JR, Lazar MA. The orphan nuclear receptors at their 25-year Reunion. J Mol Endocrinol. 2013;51(3):T115–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ning L, Lou X, Zhang F, Xu G. Nuclear receptors in the pathogenesis and management of inflammatory bowel disease. Hindawi Mediat Inflammatio. 2019;2019:2624941.

    Google Scholar 

  6. Dhiman VK, Bolt MJ, White KP. Nuclear receptors in cancer — uncovering new and evolving roles through genomic analysis. Nat Rev Genet. 2017;19:160.

    Article  PubMed  CAS  Google Scholar 

  7. Kumar S, Saradhi M, Chaturvedi NK, Tyagi RK. Intracellular localization and nucleocytoplasmic trafficking of steroid receptors: an overview. Mol Cell Endocrinol. 2006;246(1–2):147–56.

    Article  CAS  PubMed  Google Scholar 

  8. Fadel L, Rehó B, Volkó J, Bojcsuk D, Kolostyák Z, Nagy G, et al. Agonist binding directs dynamic competition among nuclear receptors for heterodimerization with retinoid X receptor. J Biol Chem. 2020;295(29):10045–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Griekspoor A, Zwart W, Neefjes J, Michalides R. Visualizing the action of steroid hormone receptors in living cells. Nucl Recept Signal. 2007;5:1–9.

    Article  CAS  Google Scholar 

  10. Guiochon-Mantel A, Lescop P, Christin-Maitre S, Loosfelt H, Perrot-Applanat M, Milgrom E. Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J. 1991;10(12):3851–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Guiochon-Mantel A, Delabre K, Lescop P, Milgrom E. Intracellular traffic of steroid hormone receptors. J Steroid Biochem Mol Biol. 1996;56(1–6):3–9.

    Article  CAS  PubMed  Google Scholar 

  12. Tyagi RK, Y L, Ahn S, Song C, Chatterjee B, Roy A. Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol Endocrinol. 2000;14(8):1162–74.

    Article  CAS  PubMed  Google Scholar 

  13. Roy AK, Tyagi RK, Song CS, Lavrovsky Y, Ahn SC, Oh TS, et al. Androgen receptor: structural domains and functional dynamics after ligand-receptor interaction. Ann N Y Acad Sci. 2001;949:44–57.

    Article  CAS  PubMed  Google Scholar 

  14. Kumar S, Chaturvedi NK, Kumar S, Tyagi RK. Agonist-mediated docking of androgen receptor onto the mitotic chromatin platform discriminates intrinsic mode of action of prostate cancer drugs. Biochim Biophys Acta, Mol Cell Res. 2008;1783(1):59–73.

    Article  CAS  PubMed  Google Scholar 

  15. Savory JGA, Préfontaine GG, Lamprecht C, Liao M, Walther RF, Lefebvre YA, et al. Glucocorticoid receptor homodimers and glucocorticoid-mineralocorticoid receptor heterodimers form in the cytoplasm through alternative dimerization interfaces. Mol Cell Biol. 2001;21(3):781–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Poukka H, Karvonen U, Yoshikawa N, Tanaka H, Palvimo JJ, Janne OA. The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor. J Cell Sci. 2000;113(17):2991–3001.

    Article  CAS  PubMed  Google Scholar 

  17. Schnell U, Dijk F, Sjollema KA, Giepmans BNG. Immunolabeling artifacts and the need for live-cell imaging. Nat Methods. 2012;9(2):152–8.

    Article  CAS  PubMed  Google Scholar 

  18. Cole R. Live-cell imaging: the cell’s perspective. Cell Adhes Migr. 2014;8(5):452–9.

    Article  Google Scholar 

  19. Hung MC, Link W. Protein localization in disease and therapy. J Cell Sci. 2011;124(20):3381–92.

    Article  CAS  PubMed  Google Scholar 

  20. Mathew SP, Thakur K, Kumar S, Yende AS, Singh SK, Dash AK, et al. A comprehensive analysis and prediction of sub-cellular localization of human nuclear receptors. Nucl Recept Res. 2018;5:1–20.

    Article  CAS  Google Scholar 

  21. Davis JR, Kakar M, Lim CS. Controlling protein compartmentalization to overcome disease. Pharm Res. 2007;24(1):17–27.

    Article  CAS  PubMed  Google Scholar 

  22. Walther RF, Atlas E, Carrigan A, Rouleau Y, Edgecombe A, Visentin L, et al. A serine/threonine-rich motif is one of three nuclear localization signals that determine unidirectional transport of the mineralocorticoid receptor to the nucleus. J Biol Chem. 2005;280(17):17549–61.

    Article  CAS  PubMed  Google Scholar 

  23. Guiochon-Mantel A, Loosfelt H, Lescop P, Sar S, Atger M, Perrot-Applanat M, et al. Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers. Cell. 1989;57(7):1147–54.

    Article  CAS  Google Scholar 

  24. Chaturvedi NK, Kumar S, Negi S, Tyagi RK. Endocrine disruptors provoke differential modulatory responses on androgen receptor and pregnane and xenobiotic receptor: potential implications in metabolic disorders. Mol Cell Biochem. 2010;345(1–2):291–308.

    Article  CAS  PubMed  Google Scholar 

  25. Kumar S, Jaiswal B, Kumar S, Negi S, Tyagi RK. Cross-talk between androgen receptor and pregnane and xenobiotic receptor reveals existence of a novel modulatory action of anti-androgenic drugs. Biochem Pharmacol. 2010;80(7):964–76.

    Article  CAS  PubMed  Google Scholar 

  26. Yende AS, Tyagi RK. Influence of signaling kinases on functional dynamics of nuclear receptor CAR. Mol Cell Biochem. 2019;461(1–2):127–39.

    Article  CAS  PubMed  Google Scholar 

  27. Amazit L, Alj Y, Tyagi RK, Chauchereau A, Loosfelt H, Pichon C, et al. Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1. J Biol Chem. 2003;278(34):32195–203.

    Article  CAS  PubMed  Google Scholar 

  28. Elbi C, Walker DA, Romero G, Sullivan WP, Toft DO, Hager GL, et al. Molecular chaperones function as steroid receptor nuclear mobility factors. Proc Natl Acad Sci U S A. 2004;101(9):2876–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kumar S, Vijayan R, Dash AK, Gourinath S, Tyagi RK. Nuclear receptor SHP dampens transcription function and abrogates mitotic chromatin association of PXR and ERα via intermolecular interactions. Biochim Biophys Acta - Gene Regul Mech. 2021; 1864(3):194683. 

    Article  CAS  Google Scholar 

  30. Singh SK, Yende AS, Ponnusamy K, Tyagi RK. A comprehensive evaluation of anti-diabetic drugs on nuclear receptor PXR platform. Toxicol Vitr. 2019;60:347–58.

    Article  CAS  Google Scholar 

  31. Feige JN, Gelman L, Tudor C, Engelborghs Y, Wahli W, Desvergne B. Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand. J Biol Chem. 2005;280(18):17880–90.

    Article  CAS  PubMed  Google Scholar 

  32. Dash AK, Yende AS, Jaiswal B, Tyagi RK. Heterodimerization of retinoid X receptor with xenobiotic receptor partners occurs in the cytoplasmic compartment: mechanistic insights of events in living cells. Exp Cell Res. 2017;360(2):337–46.

    Google Scholar 

  33. Li H, Fidler ML, Lim CS. Effect of initial subcellular localization of progesterone receptor on import kinetics and transcriptional activity. Mol Pharm. 2005;2(6):509–18.

    Article  CAS  Google Scholar 

  34. Kumar S, Tyagi RK. Androgen receptor association with mitotic chromatin - analysis with introduced deletions and disease-inflicting mutations. FEBS J. 2012;279(24):4598–614.

    Article  CAS  PubMed  Google Scholar 

  35. Rana M, Devi S, Gourinath S, Goswami R, Tyagi RK. A comprehensive analysis and functional characterization of naturally occurring non-synonymous variants of nuclear receptor PXR. Biochim Biophys Acta - Gene Regul Mech. 2016;1859(9):1183–97.

    Article  CAS  Google Scholar 

  36. Deeble PD, Murphy DJ, Parsons SJ, Cox ME. Interleukin-6- and cyclic AMP-mediated signaling potentiates neuroendocrine differentiation of LNCaP prostate tumor cells. Mol Cell Biol. 2001;21(24):8471–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Stossi F, Mistry RM, Singh PK, Johnson HL, Mancini MG, Szafran AT, et al. Single-cell distribution analysis of AR levels by high-throughput microscopy in cell models: application for testing endocrine-disrupting chemicals. SLAS Discov. 2020;25(7):684–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Xiao X, Wang P, Chou KC. iNR-physchem: a sequence-based predictor for identifying nuclear receptors and their subfamilies via physical-chemical property matrix. PLoS One. 2012;7(2):e30869.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen X, Velliste M, Murphy RF. Automated interpretation of subcellular patterns in fluorescence microscope images for location proteomics. Cytom Part A. 2006;69(7):631–40.

    Article  CAS  Google Scholar 

  40. Krylova I, Kumar RR, Kofoed EM, Schaufele F. A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging. PLoS One. 2013;8(5):e63286.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Dimauro I, Pearson T, Caporossi D, Jackson MJ. A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue. BMC Res Notes. 2012;5:513.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Carrigan A, Walther RF, Salem HA, Wu D, Atlas E, Lefebvre YA, et al. An active nuclear retention signal in the glucocorticoid receptor functions as a strong inducer of transcriptional activation. J Biol Chem. 2007;282(15):10963–71.

    Article  CAS  PubMed  Google Scholar 

  43. Madan AP, Defranco DB. Bidirectional transport of glucocorticoid receptors across the nuclear envelope. Proc Natl Acad Sci U S A. 1993;90(8):3588–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tyagi RK, Amazit L, Lescop P, Milgrom E, Guiochon-Mantel A. Mechanisms of progesterone receptor export from nuclei: role of nuclear localization signal, nuclear export signal, and ran guanosine triphosphate. Mol Endocrinol. 1998;12(11):1684–95.

    Article  CAS  PubMed  Google Scholar 

  45. Liu J, DeFranco DB. Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway. Mol Endocrinol. 2000;14(1):40–51.

    Article  PubMed  Google Scholar 

  46. Sackey FNA, Haché RJG, Reich T, Kwast-Welfeld J, Lefebvre YA. Determinants of subcellular distribution of the glucocorticoid receptor. Mol Endocrinol. 1996;10(10):1191–205.

    CAS  PubMed  Google Scholar 

  47. Hua Y, Shun TY, Strock CJ, Johnston PA. High-content positional biosensor screening assay for compounds to prevent or disrupt androgen receptor and transcriptional intermediary factor 2 protein-protein interactions. Assay Drug Dev Technol. 2014;12(7):395–418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hartig SM, Newberg JY, Bolt MJ, Szafran AT, Marcelli M, Mancini MA. Automated microscopy and image analysis for androgen receptor function. Methods Mol Biol. 2011;776(1):313–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Jones JO, An WF, Diamond MI. AR inhibitors identified by high-throughput microscopy detection of conformational change and subcellular localization. ACS Chem Biol. 2009;4(3):199–208.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Dash AK, Yende AS, Tyagi RK. Novel application of red fluorescent protein (DsRed-express) for the study of functional dynamics of nuclear receptors. J Fluoresc. 2017;27(4):1225–31.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The method presented herein was financially supported by a current research grant to RKT by National Agricultural Science Fund (NASF) - Indian Council of Agricultural Research (F.No. NASF/ ABA-7006/2018–2019). Financial assistance to our Centre by the Department of Science and Technology – Promotion of University Research and Scientific Excellence (DST-PURSE) and University Grants Commission – Special Assistance Programme (UGC-SAP) is duly acknowledged.

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The cell images shown are prepared exclusively for representation purposes.

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Authors and Affiliations

  1. Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India

    Sudhir Kumar, Jyoti Kashyap, Keshav Thakur & Rakesh K. Tyagi

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  1. Sudhir Kumar
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  2. Jyoti Kashyap
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  3. Keshav Thakur
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  4. Rakesh K. Tyagi
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Correspondence to Rakesh K. Tyagi .

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  1. School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA

    Mostafa Z. Badr

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Kumar, S., Kashyap, J., Thakur, K., Tyagi, R.K. (2021). A Simple Method for Visual Assessment and Quantification of Altered Subcellular Localization of Nuclear Receptors. In: Badr, M.Z. (eds) Nuclear Receptors. Springer, Cham. https://doi.org/10.1007/978-3-030-78315-0_2

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