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. 1992 Jul 1;89(13):5946–5950. doi: 10.1073/pnas.89.13.5946

Characterization of human androgen receptor overexpressed in the baculovirus system.

C Chang 1, C Wang 1, H F DeLuca 1, T K Ross 1, C C Shih 1
PMCID: PMC402115  PMID: 1631078

Abstract

An essential step in the process of understanding the structure and function of the human androgen receptor (hAR) involves the production of large quantities of the hAR. For this purpose, the full-length hAR has been overproduced in insect cells by using a baculovirus genetic expression system. The recombinant hAR is produced in Sf21 insect cells at approximately 7 pmol/mg of protein (1 x 10(6) AR molecules per cell), which is 70-150 times greater than levels detected in androgen target tissues. Androgen can bind to the baculovirus-expressed hAR with high affinity (Kd = 0.46 nM), and the specificity of hormone binding in baculovirus-expressed hAR is essentially identical to that of bona fide hAR. An anti-AR monoclonal antibody can recognize the baculovirus-expressed hAR at approximately 100 kDa upon Western blot analysis. Sucrose gradient analysis shows that baculovirus-expressed hAR complexes sediment at 4 S in a high salt medium and these complexes can interact with anti-AR monoclonal antibody to form complexes that sediment at 8-10 S. Therefore, production of recombinant hAR from the baculovirus expression system will provide an alternative source of biologically active hAR for studies on the molecular mechanisms of androgen action.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alnemri E. S., Maksymowych A. B., Robertson N. M., Litwack G. Characterization and purification of a functional rat glucocorticoid receptor overexpressed in a baculovirus system. J Biol Chem. 1991 Feb 25;266(6):3925–3936. [PubMed] [Google Scholar]
  2. Binart N., Lombes M., Rafestin-Oblin M. E., Baulieu E. E. Characterization of human mineralocorticosteroid receptor expressed in the baculovirus system. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10681–10685. doi: 10.1073/pnas.88.23.10681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brown M., Sharp P. A. Human estrogen receptor forms multiple protein-DNA complexes. J Biol Chem. 1990 Jul 5;265(19):11238–11243. [PubMed] [Google Scholar]
  5. Chang C. S., Kokontis J., Liao S. T. Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science. 1988 Apr 15;240(4850):324–326. doi: 10.1126/science.3353726. [DOI] [PubMed] [Google Scholar]
  6. Chang C. S., Kokontis J., Liao S. T. Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7211–7215. doi: 10.1073/pnas.85.19.7211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang C. S., Liao S. S. Topographic recognition of cyclic hydrocarbons and related compounds by receptors for androgens, estrogens, and glucocorticoids. J Steroid Biochem. 1987;27(1-3):123–131. doi: 10.1016/0022-4731(87)90303-7. [DOI] [PubMed] [Google Scholar]
  8. Chang C. S., Whelan C. T., Popovich T. C., Kokontis J., Liao S. T. Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies. Endocrinology. 1989 Aug;125(2):1097–1099. doi: 10.1210/endo-125-2-1097. [DOI] [PubMed] [Google Scholar]
  9. De Vos P., Claessens F., Winderickx J., Van Dijck P., Celis L., Peeters B., Rombauts W., Heyns W., Verhoeven G. Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli. J Biol Chem. 1991 Feb 25;266(6):3439–3443. [PubMed] [Google Scholar]
  10. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaillard-Moguilewsky M. Pharmacology of antiandrogens and value of combining androgen suppression with antiandrogen therapy. Urology. 1991;37(2 Suppl):5–12. doi: 10.1016/0090-4295(91)80095-o. [DOI] [PubMed] [Google Scholar]
  12. Geller J., Albert J., Yen S. S., Geller S., Loza D. Medical castration of males with megestrol acetate and small doses of diethylstilbestrol. J Clin Endocrinol Metab. 1981 Mar;52(3):576–580. doi: 10.1210/jcem-52-3-576. [DOI] [PubMed] [Google Scholar]
  13. Gustafasson J., Pousette K. Demonstration and partial characterization of cytosol receptors for testosterone. Biochemistry. 1975 Jul 15;14(14):3094–3101. doi: 10.1021/bi00685a009. [DOI] [PubMed] [Google Scholar]
  14. Imperato-McGinley J., Ip N. Y., Gautier T., Neuweiler J., Gruenspan H., Liao S., Chang C., Balazs I. DNA linkage analysis and studies of the androgen receptor gene in a large kindred with complete androgen insensitivity. Am J Med Genet. 1990 May;36(1):104–108. doi: 10.1002/ajmg.1320360121. [DOI] [PubMed] [Google Scholar]
  15. Kemppainen J. A., Lane M. V., Sar M., Wilson E. M. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem. 1992 Jan 15;267(2):968–974. [PubMed] [Google Scholar]
  16. Kitts P. A., Ayres M. D., Possee R. D. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Res. 1990 Oct 11;18(19):5667–5672. doi: 10.1093/nar/18.19.5667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lubahn D. B., Joseph D. R., Sullivan P. M., Willard H. F., French F. S., Wilson E. M. Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science. 1988 Apr 15;240(4850):327–330. doi: 10.1126/science.3353727. [DOI] [PubMed] [Google Scholar]
  19. Luckow V. A., Summers M. D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989 May;170(1):31–39. doi: 10.1016/0042-6822(89)90348-6. [DOI] [PubMed] [Google Scholar]
  20. Mooradian A. D., Morley J. E., Korenman S. G. Biological actions of androgens. Endocr Rev. 1987 Feb;8(1):1–28. doi: 10.1210/edrv-8-1-1. [DOI] [PubMed] [Google Scholar]
  21. Neumann F. Pharmacology and potential use of cyproterone acetate. Horm Metab Res. 1977 Jan;9(1):1–13. doi: 10.1055/s-0028-1093574. [DOI] [PubMed] [Google Scholar]
  22. Nishimura R., Shida K. Antiandrogenic therapy for the treatment of early stage prostatic cancer. Prostate Suppl. 1981;1:27–34. doi: 10.1002/pros.2990020506. [DOI] [PubMed] [Google Scholar]
  23. Ojasoo T., Raynaud J. P. Unique steroid congeners for receptor studies. Cancer Res. 1978 Nov;38(11 Pt 2):4186–4198. [PubMed] [Google Scholar]
  24. Riegman P. H., Vlietstra R. J., van der Korput J. A., Brinkmann A. O., Trapman J. The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. Mol Endocrinol. 1991 Dec;5(12):1921–1930. doi: 10.1210/mend-5-12-1921. [DOI] [PubMed] [Google Scholar]
  25. Ross T. K., Moss V. E., Prahl J. M., DeLuca H. F. A nuclear protein essential for binding of rat 1,25-dihydroxyvitamin D3 receptor to its response elements. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):256–260. doi: 10.1073/pnas.89.1.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ross T. K., Prahl J. M., DeLuca H. F. Overproduction of rat 1,25-dihydroxyvitamin D3 receptor in insect cells using the baculovirus expression system. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6555–6559. doi: 10.1073/pnas.88.15.6555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rushmere N. K., Parker M. G., Davies P. Androgen receptor-binding regions of an androgen-responsive gene. Mol Cell Endocrinol. 1987 Jun;51(3):259–265. doi: 10.1016/0303-7207(87)90036-0. [DOI] [PubMed] [Google Scholar]
  28. Sai T. J., Seino S., Chang C. S., Trifiro M., Pinsky L., Mhatre A., Kaufman M., Lambert B., Trapman J., Brinkmann A. O. An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity. Am J Hum Genet. 1990 Jun;46(6):1095–1100. [PMC free article] [PubMed] [Google Scholar]
  29. Sompayrac L., Danna K. J. Method to identify genomic targets of DNA binding proteins. Proc Natl Acad Sci U S A. 1990 May;87(9):3274–3278. doi: 10.1073/pnas.87.9.3274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Takeda H., Chang C. Immunohistochemical and in-situ hybridization analysis of androgen receptor expression during the development of the mouse prostate gland. J Endocrinol. 1991 Apr;129(1):83–89. doi: 10.1677/joe.0.1290083. [DOI] [PubMed] [Google Scholar]
  31. Takeda H., Chodak G., Mutchnik S., Nakamoto T., Chang C. Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J Endocrinol. 1990 Jul;126(1):17–25. doi: 10.1677/joe.0.1260017. [DOI] [PubMed] [Google Scholar]
  32. Takeda H., Nakamoto T., Kokontis J., Chodak G. W., Chang C. Autoregulation of androgen receptor expression in rodent prostate: immunohistochemical and in situ hybridization analysis. Biochem Biophys Res Commun. 1991 May 31;177(1):488–496. doi: 10.1016/0006-291x(91)92010-h. [DOI] [PubMed] [Google Scholar]
  33. Vialard J., Lalumière M., Vernet T., Briedis D., Alkhatib G., Henning D., Levin D., Richardson C. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene. J Virol. 1990 Jan;64(1):37–50. doi: 10.1128/jvi.64.1.37-50.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wilson E. M., French F. S. Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate. J Biol Chem. 1976 Sep 25;251(18):5620–5629. [PubMed] [Google Scholar]
  35. Yamamoto K. R. Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet. 1985;19:209–252. doi: 10.1146/annurev.ge.19.120185.001233. [DOI] [PubMed] [Google Scholar]

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