Glucocorticoid receptor from developing rat brain

-261 GLUCOCORTICOID RECEPTOR FROM DEVELOPING RAT BRAIN. Alexis M.N., Kitraki, E., Stylianopoulou F. and Sekeris C.E. Inst. of Biol. Res. The National 1Hellenic Research Foundation , 40 Vas. Con/nou Ave., Athens - GREECE. Glucocorticoid binding to the receptor could be detected in developing rat brain since the 17th day of gestation. A 94KDa specific receptor was detected throughout development by covalent dexamethasone mesylate receptor labelling in the cy-tosolicfraction, followed by SDS PAGE and gel fluorography. However , binding to the receptor was characterized by an equilibrium dissociation contact (Kd) of about 20 nM, up to the end of the first week of life whereas the adult values never exceeded 5 nM. Analysis of receptor heat stability demonstrated an increased receptor lability in the cytosol from early neonatal animals. The Arrhenius plot of heat inactivation gave a &i value of 24,4 KJ/mole for the neonatal receptor, whereas the re- , spective value for the adult was 31 KJ/mole. Evidence, of increased receptor proteolysis in the embryonic and neonatal cytosol was clearly present in the corresponding fluorographs of dexamethasone mesylate receptor complex. Receptor lability and high Kd values in the cytosol. could thus be due to enchanced proteolysis. Receptor translocation and binding to the nucleus, measured in intact or in adrenalect mized animals after injection of saturating amounts of dexamethasone, was significantly re- -i duced during embryonic and early neonatal life. However, purification of dexamethasone mesylate receptor complex by DNA-cellulose binding showed that the 90 KDa receptor protein is capable of binding to DNA at all stages of development. These results suggest that the impaired translocation during early stages of brain development may reflect the action of traslocation inhibitors. 262 i-MOLECULAR DETAILS OF GLUCOCORTICOID MBCBANISM OF ACTION MAY BE REVEALED BY CONSIDERATION OF DNA STEREOCHEMISTRY: Lehner, Departments of Endocrinology* and Department of PhysiologyO,

A. F.*t; Muldoon, T. G.*; Cidlowski, J. A.$; Nahesh, V. B.* and Medicinet, Medical College of Georgia, Augusta, Georgia 3091Zi University of North Carolina, Chapel Hill, N.C. 27514; USA I

Two observations may be useful in elucidating molecular details of steroid hormone mechFirst, a number of groups have observed the occurrence of the consensus sequ-’ anism of action. ence 5’-d(TGTTCT)-3’ in published DNA binding sites for mammalian glucocorticoid receptors. Additional features of these sites may have relevance, e.g. partial symmetry of the overall binding region inclusive of the 5’-d(TGTTCT)-3’ element, as well as the capacity for the d(TG) portion of the element to base pair with an upstream d(CA) base sequence. This combination of base pairing could contribute to the formation of hairpins in vlvo. ) symmetry and “same strand” -Second, mammalian steroid hormones including glucocorticoids display precise topographical complementarity to the space between base pairs in the partially unwound DNA sequence 5’-d(TG)-3’. 5’-d(CA)-3’; e.g., cortisol can undergo five stereospecific hydrogen bonds to donor/acceptor groups in this DNA cavity (Hendry et al., 18th Miami Winter Sump. [1986], p.292). Recep- i -Proc. --tor-DNA binding could be envisioned to occur with simultaneous insertion of the ligand between this base sequence and the related the T-A and G-C base pairs of the consensus d(TG) sequence; upstream d(CA) might be of particular importance for responsiveness to glucocorticoids, perhaps, to enable functional hairpin formation. A ternary receptor-ligand-DNA complex should occur unwinding may influence gene transcription via with partial unwinding of the DNA. Such local transmission of conformational changes along the DNA, consistent with recent findings by M. of glucocorticoid receptors to affect DNA supercoiling. Beato (Ibid. . p.250) on the ability

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263 THE STRUCTURES OF ANTAGONISTS TO CORTICOSTEROIDS CAN BE PREDICTED BY THEIR STEREOCHEMICAL COMFLEMENTARITYTO THE STRUCTURE OF DNA; Bransome, Departments of Medicine and Endocrinology, Medical Genetics, Inc.; Augusta, Georgia 30912; USA

E. D., College

Jr. of

and Hendry, L. B. Georgia and Stereochemical

We have already indicated that the hormonal activity of steroids can be predicted with molecular models by their stereochemical complementarlty to-the DNA double helix when inserted between base pairs at 5’-dTdG-3’*5’-dCdA-3’. Antagonists must fit between the same base pairs as agonists but differ either in hydrogen bonding pattern- and/or possess structural components capable of extending .beyond base pairs and hydrogen bonding with the outside surface of DNA (Hendry et al., Steroid Biochem., in press). These relatlonshlps provide a way of identifying novel structures of potential antagonists (US Patent 4,461,619). Change in H-bonds Extend out H-Bond to Relative Antagonist Steroid/Analog 3 118 170 20 21 of cavity helix surface Activity CORTISOL 00 0 00 0 0 0 CORTEXOLONE o+ 0 00 0 0 1 PROGESTERONE o+ + o+ 0 0 1 CORTISOL-1700 + ++ + 0 2 OXETANONE A-l-l l-OXAo+ 0 00 0 0 2 II-DESOXYCORTISOL o+ + ++ RU-38486 + + 4

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