Figure 6.

(A) MLS and NLS are located within different domains of GR. GR-negative 293 epithelial cells were transfected with plasmids encoding the indicated human GR variants. After 20 h, the cells were subjected to subcellular fractionation and GR was detected on Western blot using the PA1-511 antibody to GR. (B) α-Helix wheel model of the putative MLS located within aa 558–580 of human GR. The positive-charged arginine and lysine (red) and the hydrophilic threonine (orange) are located on the one side of the α-helix, whereas the hydrophobic aa leucine, isoleucine, valine, and tryptophane (blue) are located on the opposite side of the α-helix. Amino acids interacting with GC are labeled with gray numbers. This putative MLS is the α-Helix 3 of LBD. (C) The α-Helix 3 (aa 558–580) as it appears in the 1M2Z crystal structure. The same color labeling of aa is used as in B. (D) The MLS of GR resembles that of cytochrome C oxidase (COX). Amino acid sequence alignment between MLS of GR and MLS of COX. (E) The R564G and R575G mutants show reduced ability to enter the mitochondria. Mouse GFP-GR was point-mutated at aa 564 or 575 corresponding to the human R558 and R569, and the ability of these mutants to enter the mitochondria was determined as described in A. (F) The MLS (H3) of GR in the LBD crystal structure. The putative MLS is labeled in red in the 1M2Z crystal structure of a dimer complex of the human GR LBD (aa 521–777) bound to Dex and a Tif2 coactivator motif (reference 6).