The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.

Annals of the New York Academy of SciencesVolume 585, Issue 1 p. 438-451 Pyridoxal Phosphate as a Regulator of the Glucocorticoid Receptora ANDREW B. MAKSYMOWYCH, ANDREW B. MAKSYMOWYCH The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 Trainee, Training Grant T32 DK07162 from the National Institutes of Health to Department of Biochemistry.Search for more papers by this authorVIOLET DANIEL, VIOLET DANIEL The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 Permanent address: Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.Search for more papers by this authorGERALD LITWACK, Corresponding Author GERALD LITWACK The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140dAuthor to whom correspondence should be addressed.Search for more papers by this author ANDREW B. MAKSYMOWYCH, ANDREW B. MAKSYMOWYCH The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 Trainee, Training Grant T32 DK07162 from the National Institutes of Health to Department of Biochemistry.Search for more papers by this authorVIOLET DANIEL, VIOLET DANIEL The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 Permanent address: Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.Search for more papers by this authorGERALD LITWACK, Corresponding Author GERALD LITWACK The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140dAuthor to whom correspondence should be addressed.Search for more papers by this author First published: May 1990 https://doi.org/10.1111/j.1749-6632.1990.tb28075.xCitations: 4 a Work in this laboratory related to vitamin B6 is supported by research grants: 87A19 from American Institute for Cancer Research, DK 13531 from the National Institutes of Health, and Core Grant CA 12227 to the Fels Institute from the National Institutes of Health. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Cake, M. H. & G. Litwack 1977. Interaction of pyridoxal-P with the DNA binding site of activated glucocorticoid receptor from rat liver. Proc. Austral. Biochem. Soc. 10: 51. 2 Litwack, G. & M. H. Cake 1977. DNA binding site of activated glucocorticoid receptor. Interaction with pyridoxal-P. Fed. Proc. 36: 911. 3 Cake, M. H. & G. Litwack 1978. The effect of methylxanthines on binding of the glucocorticoid receptor to DNA-cellulose and nuclei. Eur. J. 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Recombinant plasmids containing the double-stranded cDNA sequences of mRNA for the Mr 22,000 ligandin (glutathione S-transferase B) subunit (Ya) have been constructed. The DNA sequence of an insert corresponding to the middle and 3' regions of the mRNA was determined and an amino acid sequence was proposed for the ligandin Ya subunit. The proposed sequence reveals a high content of basic amino acids (Arg and Lys) and Leu, is consistent with the amino acid composition, and predicts the correct number of peptides derived from tryptic digests reported for ligandin.

Poly(A)-containing rat liver mRNA isolated from animals injected with phenobarbital and uninjected controls was translated efficiently in a wheat-germ system. The synthesis of ligandin (glutathione S-transferase B; glutathione transferase; RX-gluathione R-transferase, EC 2.5.1.18) was detected by immunoprecipitation with a highly purified monospecific ligandin antibody and analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The extent of incorporation of [35S]methionine into ligandin in the translation system was similar for poly(A)-containing messages from un-infected animals and those treated with phenobarbital.

Cultured mouse lymphosarcoma cells are killed on exposure to 0.1 mM N(6),O(2')-dibutyryl-adenosine 3':5'-cyclic monophosphate. A population of cells resistant to the killing effect of dibutyryl cyclic AMP at concentrations as high as 1 mM was selected. The growth characteristics of the resistant cells were similar to those of the sensitive parental line. However, the resistant cells contain less cytoplasmic cyclic AMP-binding proteins and decreased cyclic AMP-stimulated protein kinase activity. It is proposed that transition from sensitivity to resistance to dibutyryl cyclic AMP in lymphoma cells is connected with a modification of the cyclic AMP-binding protein, which appears to be the regulatory subunit of the cyclic AMP-activated protein kinase.