Direct protein-protein interaction of 11 beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11 beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17 beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11 beta-HSD1 but not with the related enzymes 11 beta-HSD2 and 17 beta-HSD2, suggesting a specific interaction between H6PDH and 11 beta-HSD1. The use of the 11 beta-HSD1/11 beta-HSD2 chimera indicates that the N-terminal 39 residues of 11 beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11 beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11 beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11 beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11 beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11 beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins. (C) 2008 Elsevier B.V. All rights reserved.