PHOTOAFFINITY-LABELING OF THE HUMAN MINERALOCORTICOID RECEPTOR WITH STEROIDS HAVING A REACTIVE GROUP AT POSITION-3, POSITION-18 OR POSITION-21

The ability of a glucocorticoid (triamcinolone acetonide: TA) and thre e progesterone derivatives with photoreactive groups at different posi tions (promegestone: R5020; 18-oxo-18-vinylprogesterone : 18OVP; 21-di azoprogesterone : 21DP) to bind covalently to the human mineralocortic oid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimen tation gradient analysis and exchange assays with aldosterone showed t hat [H-3]TA, a partial mineralocorticoid agonist, and [H-3]R5020, a pu rl antimineralocorticoid, were covalently bound to hMR after UV irradi ation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradi ated [H-3]TA- and [H-3]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyac rylamide gel electrophoresis revealed a band labelled by [H-3]TA or [H -3]R5020, having a molecular mass of 120 kDa. This band was not detect ed in the presence of an excess of the corresponding unlabelled steroi d or when the cytosol was recovered from non-infected Sf9 cells. Elect rophoresis of a truncated hMR (hMR Delta(1-351)) photolabelled with [H -3]TA revealed a 80 kDa band, compatible with the molecular mass of th e truncated hMR. Limited chymotrypsin proteolysis of the [H-3]TA photo labelled hMR generated a 30 kDa fragment covalently associated with [H -3]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S, Jalaguier, M. Lombes, A. Souque, M.E. Rafestin-Oblin, B iochem. J. 315 (1996) 421-427), the present experiments provide eviden ce that [H-3]TA is covalently bound to the ligand binding domain of th e hMR. Exchange assays with [H-3]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are cova lently bound to hMR with an approx. 30-35% labelling efficiency. (C) 1 998 Elsevier Science B.V. All rights reserved.