SUBCELLULAR-LOCALIZATION OF THE TYPE-2 11-BETA-HYDROXYSTEROID DEHYDROGENASE - A GREEN FLUORESCENT PROTEIN STUDY

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is thought to confe r aldosterone specificity to mineralocorticoid target cells by protect ing the inherently nonselective mineralocorticoid receptor (MR) from o ccupancy by endogenous glucocorticoids. Recently, we characterized a n ovel isoform of 11 beta-HSD in aldosterone target cells, which has hig h affinity for its substrate, is unidirectional, and prefers NAD as co factor, In this study we utilized a green fluorescent protein (GFP) te chnique to determine the subcellular localization of this isoform, 11 beta-HSD2, We generated a chimeric gene encoding the full-length rabbi t 11 beta-HSD2 and, fused to its C terminus, the coding sequence of GF P. This construct was stably transfected into CHO cells, The enzymatic characteristics of the expressed 11 beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (K-M 8-10 nar), NAD dependence, and lack of reductase a ctivity, The intracellular location of the recombinant protein was det ermined by fluorescence microscopy. 11 beta-HSD2-associated fluorescen ce was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, s uggesting endoplasmic reticulum (ER) localization, Staining of CHO cel ls expressing 11 beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11 beta-HSD2/GFP only with ER mar kers and tubulin, To examine the orientation of 11 beta-HSD2 within th e ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody, Fluorescence microscopy indicated that the C-termin al region of 11 beta-HSD2 is on the cytoplasmic surface of the ER memb rane, since it was accessible to the GFP antibody, This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Wes tern blotting, The C-terminal region of 11 beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER memb rane, These results indicate that 11 beta-HSD2 is localized exclusivel y to the ER, Since 11 beta-HSD2 does not contain any known ER retrieva l signal, experiments are currently under way to determine what struct ural motifs are responsible for its FR localization.