Subcellular localization of the type 2 11beta-hydroxysteroid dehydrogenase. A green fluorescent protein study.
11beta-Hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11beta-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11beta-HSD2. We generated a chimeric gene encoding the full-length rabbit 11beta-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8-10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11beta-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11beta-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11beta-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11beta-HSD2 is localized exclusively to the ER. Since 11beta-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.
Náray-Fejes-Tóth
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Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
Fejes-Tóth
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D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002451:Cell Compartmentation; D003345:Corticosterone / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D018871:Endoplasmic Reticulum, Smooth / Q000201:enzymology*; D049452:Green Fluorescent Proteins; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D003345:Corticosterone