Here, we also show that native GR⅐hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR⅐hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52.Steroid receptors move continuously into and out of the nucleus (Refs. 1-4; for review, see Ref. 5), and, depending upon the receptor, the hormone-free, untransformed receptor may have a predominantly nuclear or cytoplasmic localization. The hormone-free glucocorticoid receptor (GR) 1 is localized to the cytoplasm of most cells, and after steroid binding and transformation, it translocates to the nucleus (6 -8). Several studies with inhibitors suggest that the multiprotein hsp90-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in movement of the GR along microtubular tracks to the nucleus (for review, see Ref. 9). Assembly of receptors into heterocomplexes with hsp90 is a dynamic process (10), and it has been shown that the GR and hsp90 can move together from the cytoplasm to the nucleus (11). A couple of observations suggest that the role of hsp90 in receptor movement is likely to involve dynamic assembly and disassembly of GR⅐hsp90 heterocomplexes. For example, Yang and DeFranco (12) showed that molybdate, which binds to hsp90 and stabilizes GR⅐hsp90 heterocomplexes in vivo (13), traps the GR in the cytoplasm of cells continuously exposed to hormone. Molybdate in this case was thought to inhibit reimport of the GR into the nucleus by inhibiting the dynamic cycling of receptors into and out of their complexes with the hsp90 chaperone. Also, geldanamycin, an antibiotic that binds to the nucleotide binding site on hsp90 (14) and prevents formation of normal receptor⅐hsp90 heterocomplexes (15), impedes steroid-induced movement of the GR from the cytoplasm to the nucleus (16, 17).Some localization studies have shown the untransformed GR to colocalize with microtubules (for review, see Ref. 18), but the evidence supporting movement along microtubular tracks is indirect. Although microtubule disrupting agents, such as colcemid, do not affect the overall rate of steroid-dependent receptor translocation to the nucleus (8, 19), they eliminate the hsp90-dependent mode of receptor movement (17). Using a fusion protein of murine GR with Aequorea green fluorescent protein (GFP), we found that steroid-dependent GFP-GR translocation to the nucleus is rapid (t1 ⁄2 ϭ ϳ5 min) both in cells with intact cytoskeleton and in cells with disrupted cytoskeletal networks (17). However, in cells with normal cytoskeleton, the hsp90 inhibitor geldanamycin slowed translocation of the GFP-GR by an order of magnitude (t1 ⁄2 ϭ ϳ45 min), whereas in cells with colcemid-disrupted microtubules, geldanamycin had no effect on the translocation rate (t1 ⁄2 ϭ ϳ5 min). This suggests two mechanisms of GR movement. Under physiological conditions where the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery in which the hsp90 heter...

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor⅐hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.