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. 1992 Nov 1;89(21):10282–10286. doi: 10.1073/pnas.89.21.10282

Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor.

T K Ross 1, J M Prahl 1, I M Herzberg 1, H F DeLuca 1
PMCID: PMC50322  PMID: 1332041

Abstract

The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.

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Selected References

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

  1. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  2. Benbrook D., Lernhardt E., Pfahl M. A new retinoic acid receptor identified from a hepatocellular carcinoma. Nature. 1988 Jun 16;333(6174):669–672. doi: 10.1038/333669a0. [DOI] [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. Brand N., Petkovich M., Krust A., Chambon P., de Thé H., Marchio A., Tiollais P., Dejean A. Identification of a second human retinoic acid receptor. Nature. 1988 Apr 28;332(6167):850–853. doi: 10.1038/332850a0. [DOI] [PubMed] [Google Scholar]
  5. Brockes J. Developmental biology. Reading the retinoid signals. Nature. 1990 Jun 28;345(6278):766–768. doi: 10.1038/345766a0. [DOI] [PubMed] [Google Scholar]
  6. Dame M. C., Pierce E. A., DeLuca H. F. Identification of the porcine intestinal 1,25-dihydroxyvitamin D3 receptor on sodium dodecyl sulfate/polyacrylamide gels by renaturation and immunoblotting. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7825–7829. doi: 10.1073/pnas.82.23.7825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Delescluse C., Cavey M. T., Martin B., Bernard B. A., Reichert U., Maignan J., Darmon M., Shroot B. Selective high affinity retinoic acid receptor alpha or beta-gamma ligands. Mol Pharmacol. 1991 Oct;40(4):556–562. [PubMed] [Google Scholar]
  8. Demay M. B., Gerardi J. M., DeLuca H. F., Kronenberg H. M. DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1990 Jan;87(1):369–373. doi: 10.1073/pnas.87.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eichele G., Tickle C., Alberts B. M. Studies on the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects. J Cell Biol. 1985 Nov;101(5 Pt 1):1913–1920. doi: 10.1083/jcb.101.5.1913. [DOI] [PMC free article] [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. Giguere V., Ong E. S., Segui P., Evans R. M. Identification of a receptor for the morphogen retinoic acid. Nature. 1987 Dec 17;330(6149):624–629. doi: 10.1038/330624a0. [DOI] [PubMed] [Google Scholar]
  12. Green S., Chambon P. Nuclear receptors enhance our understanding of transcription regulation. Trends Genet. 1988 Nov;4(11):309–314. doi: 10.1016/0168-9525(88)90108-4. [DOI] [PubMed] [Google Scholar]
  13. Hamada K., Gleason S. L., Levi B. Z., Hirschfeld S., Appella E., Ozato K. H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8289–8293. doi: 10.1073/pnas.86.21.8289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hashida S., Imagawa M., Inoue S., Ruan K. H., Ishikawa E. More useful maleimide compounds for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. J Appl Biochem. 1984 Feb-Apr;6(1-2):56–63. [PubMed] [Google Scholar]
  15. Hoffmann B., Lehmann J. M., Zhang X. K., Hermann T., Husmann M., Graupner G., Pfahl M. A retinoic acid receptor-specific element controls the retinoic acid receptor-beta promoter. Mol Endocrinol. 1990 Nov;4(11):1727–1736. doi: 10.1210/mend-4-11-1727. [DOI] [PubMed] [Google Scholar]
  16. Kessel M., Gruss P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell. 1991 Oct 4;67(1):89–104. doi: 10.1016/0092-8674(91)90574-i. [DOI] [PubMed] [Google Scholar]
  17. Kliewer S. A., Umesono K., Mangelsdorf D. J., Evans R. M. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature. 1992 Jan 30;355(6359):446–449. doi: 10.1038/355446a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krust A., Kastner P., Petkovich M., Zelent A., Chambon P. A third human retinoic acid receptor, hRAR-gamma. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5310–5314. doi: 10.1073/pnas.86.14.5310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leid M., Kastner P., Lyons R., Nakshatri H., Saunders M., Zacharewski T., Chen J. Y., Staub A., Garnier J. M., Mader S. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 1992 Jan 24;68(2):377–395. doi: 10.1016/0092-8674(92)90478-u. [DOI] [PubMed] [Google Scholar]
  20. Liao J., Ozono K., Sone T., McDonnell D. P., Pike J. W. Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9751–9755. doi: 10.1073/pnas.87.24.9751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liotta L. A., Rao C. N., Barsky S. H. Tumor invasion and the extracellular matrix. Lab Invest. 1983 Dec;49(6):636–649. [PubMed] [Google Scholar]
  22. Litman R. M. A deoxyribonucleic acid polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acid-cellulose. J Biol Chem. 1968 Dec 10;243(23):6222–6233. [PubMed] [Google Scholar]
  23. 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]
  24. Maden M. Vitamin A and pattern formation in the regenerating limb. Nature. 1982 Feb 25;295(5851):672–675. doi: 10.1038/295672a0. [DOI] [PubMed] [Google Scholar]
  25. Mangelsdorf D. J., Ong E. S., Dyck J. A., Evans R. M. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990 May 17;345(6272):224–229. doi: 10.1038/345224a0. [DOI] [PubMed] [Google Scholar]
  26. O'Malley B. The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol. 1990 Mar;4(3):363–369. doi: 10.1210/mend-4-3-363. [DOI] [PubMed] [Google Scholar]
  27. Oro A. E., McKeown M., Evans R. M. Relationship between the product of the Drosophila ultraspiracle locus and the vertebrate retinoid X receptor. Nature. 1990 Sep 20;347(6290):298–301. doi: 10.1038/347298a0. [DOI] [PubMed] [Google Scholar]
  28. Petkovich M., Brand N. J., Krust A., Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 1987 Dec 3;330(6147):444–450. doi: 10.1038/330444a0. [DOI] [PubMed] [Google Scholar]
  29. Rosa F. W., Wilk A. L., Kelsey F. O. Teratogen update: vitamin A congeners. Teratology. 1986 Jun;33(3):355–364. doi: 10.1002/tera.1420330315. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Sone T., McDonnell D. P., O'Malley B. W., Pike J. W. Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies. J Biol Chem. 1990 Dec 15;265(35):21997–22003. [PubMed] [Google Scholar]
  33. Strickland S., Smith K. K., Marotti K. R. Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell. 1980 Sep;21(2):347–355. doi: 10.1016/0092-8674(80)90471-7. [DOI] [PubMed] [Google Scholar]
  34. Sucov H. M., Murakami K. K., Evans R. M. Characterization of an autoregulated response element in the mouse retinoic acid receptor type beta gene. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5392–5396. doi: 10.1073/pnas.87.14.5392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Summerbell D. The effect of local application of retinoic acid to the anterior margin of the developing chick limb. J Embryol Exp Morphol. 1983 Dec;78:269–289. [PubMed] [Google Scholar]
  36. Timpl R. Structure and biological activity of basement membrane proteins. Eur J Biochem. 1989 Apr 1;180(3):487–502. doi: 10.1111/j.1432-1033.1989.tb14673.x. [DOI] [PubMed] [Google Scholar]
  37. Umesono K., Evans R. M. Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell. 1989 Jun 30;57(7):1139–1146. doi: 10.1016/0092-8674(89)90051-2. [DOI] [PubMed] [Google Scholar]
  38. Vasios G. W., Gold J. D., Petkovich M., Chambon P., Gudas L. J. A retinoic acid-responsive element is present in the 5' flanking region of the laminin B1 gene. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9099–9103. doi: 10.1073/pnas.86.23.9099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vaughn J. L., Goodwin R. H., Tompkins G. J., McCawley P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro. 1977 Apr;13(4):213–217. doi: 10.1007/BF02615077. [DOI] [PubMed] [Google Scholar]
  40. Yu V. C., Delsert C., Andersen B., Holloway J. M., Devary O. V., När A. M., Kim S. Y., Boutin J. M., Glass C. K., Rosenfeld M. G. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell. 1991 Dec 20;67(6):1251–1266. doi: 10.1016/0092-8674(91)90301-e. [DOI] [PubMed] [Google Scholar]
  41. Zelent A., Krust A., Petkovich M., Kastner P., Chambon P. Cloning of murine alpha and beta retinoic acid receptors and a novel receptor gamma predominantly expressed in skin. Nature. 1989 Jun 29;339(6227):714–717. doi: 10.1038/339714a0. [DOI] [PubMed] [Google Scholar]
  42. Zhang X. K., Hoffmann B., Tran P. B., Graupner G., Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature. 1992 Jan 30;355(6359):441–446. doi: 10.1038/355441a0. [DOI] [PubMed] [Google Scholar]
  43. de Thé H., Vivanco-Ruiz M. M., Tiollais P., Stunnenberg H., Dejean A. Identification of a retinoic acid responsive element in the retinoic acid receptor beta gene. Nature. 1990 Jan 11;343(6254):177–180. doi: 10.1038/343177a0. [DOI] [PubMed] [Google Scholar]

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