GLUCOCORTICOID-RECOGNIZING AND GLUCOCORTICOID-EFFECTOR SITES IN RAT-LIVER PLASMA-MEMBRANE, KINETICS OF CORTICOSTERONE UPTAKE BY ISOLATED MEMBRANE-VESICLES - III - SPECIFICITY AND STEREOSPECIFICITY

In previous papers we provided evidence for a glucocorticoid (GC) resp onsive site in a highly purified rat liver plasma membrane (PM) fracti on, which has proved to be osmotically active, 'right side-out' vesicl es, free of CBG, glucocorticoid receptors (GR) and ATP CI. Steroid Bio chem. Molec. Biol. 42 (1992) 737-756 and 757-771). This site, now call ed 'GC importer', mediates active transmembrane transport of corticost erone (B). Pronounced specificity, including stereo-and enantiomeric s pecificity, of ligand-GC importer interaction was demonstrated by comp etition assays using 54 different steroidal hormones and molecules. Im portant structural prerequisites for ligands with high specificity for the GC importer are plane C-21-steroid hormones with 1-ene and/or 4-e ne or 5 alpha-reduced configuration, and/or OH-group(s) at C11 beta>C1 7 alpha>C21. Unexpectedly, other preferred ligands are C17 alpha-ethyn yl steroids like estrogens with an OH- or OCH3-group at C3 (EE2, mestr anol) as well as progestins with C3-OH and 4-ene configuration (ethyno diol). C-21-steroids with 11 alpha-OH, 11-keto, 16 alpha-CH3, 16 beta- CH3, 16 alpha-OH or 5 beta-reduced configuration are low specificity l igands. The importer even displays different specificity for enantiome rs (levonorgestrel>L-norgestrel). Altogether, the GC importer preferen tially recognizes active GC and natural progestins which act as GC-ant agonist (e.g. prednisolone>11 beta-cortisol=B greater than or equal to progestins). Synthetic GC-agonists (e.g. dexamethasone, betamethasone , triamcinolone), most synthetic progestins, biologically inactive GC (e.g. 11 alpha-cortisol, prednisone, cortisone, 11-dehydro-B), mineral ocorticoids (aldosterone), natural estrogens (e.g. E-1, E-2, E-3), DES and vitamin D-3 derivatives do not interact with the GC importer. Osm otic shrinkage experiments revealed that interaction of high as well a s low specificity ligands with the GC importer comprises reversible bi nding and transport through the PM. The Ligand specificity profile of the GC importer and the GR exhibit pronounced differences, suggesting that both GC recognizing sites are different proteins. Performing immu noblotting, using specific mono-and polyclonal antibodies directed aga inst the intracellular rat GR, of the PM pretreated with the membrane protein solubilizing detergent CHAPSO, we found that specific steroid binding to the PM site is not due to contamination with GR. Colchicine , daunorubicine, quinine, reserpine, verapamil and vinblastine, repres entatives of Lipophilic xenobiotics which are known to be transported out of cells by the glycoprotein P170, did not compete with B for upta ke into PM-vesicles, indicating that the GC importer is not a member o f the ABC/mdr superfamily. The GC importer seems to be an additional L ink in the chain of steroid signal transduction and may be functionall y involved in the action of natural GC-agonists and GC-antagonists. (C ) 1998 Elsevier Science Ltd. All rights reserved.