Evidence That the Peptidylprolyl Isomerase Domain of the hsp90-binding Immunophilin FKBP52 Is Involved in Both Dynein Interaction and Glucocorticoid Receptor Movement to the Nucleus
We have previously shown that immunoadsorption of the FKBP52 immunophilin component of steroid receptor·hsp90 heterocomplexes is accompanied by coadsorption of cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. Coimmunoadsorption of dynein is competed by an expressed fragment of FKBP52 comprising its peptidylprolyl isomerase (PPIase) domain (Silverstein, A. M., Galigniana, M. D., Kanelakis, K. C., Radanyi, C., Renoir, J.-M., and Pratt, W. B. (1999)J. Biol. Chem. 52, 36980–36986). Here we show that cotransfection of 3T3 cells with the FKBP52 PPIase domain and a green fluorescent protein (GFP) glucocorticoid receptor (GR) chimera inhibits dexamethasone-dependent movement of the GFP-GR from the cytoplasm to the nucleus. Cotransfection with FKBP12 does not affect GFP-GR movement. Inhibition of movement by the FKBP52 PPIase domain is abrogated in cells treated with colcemid to eliminate microtubules prior to steroid addition. After withdrawal of colcemid, microtubules reform, and PPIase inhibition of GFP-GR movement is restored. These observations are consistent with the notion that FKBP52 targets retrograde movement of the GFP-GR along microtubules by linking the receptor to the dynein motor. Here, we also show that native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR·hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52. We have previously shown that immunoadsorption of the FKBP52 immunophilin component of steroid receptor·hsp90 heterocomplexes is accompanied by coadsorption of cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. Coimmunoadsorption of dynein is competed by an expressed fragment of FKBP52 comprising its peptidylprolyl isomerase (PPIase) domain (Silverstein, A. M., Galigniana, M. D., Kanelakis, K. C., Radanyi, C., Renoir, J.-M., and Pratt, W. B. (1999)J. Biol. Chem. 52, 36980–36986). Here we show that cotransfection of 3T3 cells with the FKBP52 PPIase domain and a green fluorescent protein (GFP) glucocorticoid receptor (GR) chimera inhibits dexamethasone-dependent movement of the GFP-GR from the cytoplasm to the nucleus. Cotransfection with FKBP12 does not affect GFP-GR movement. Inhibition of movement by the FKBP52 PPIase domain is abrogated in cells treated with colcemid to eliminate microtubules prior to steroid addition. After withdrawal of colcemid, microtubules reform, and PPIase inhibition of GFP-GR movement is restored. These observations are consistent with the notion that FKBP52 targets retrograde movement of the GFP-GR along microtubules by linking the receptor to the dynein motor. Here, we also show that native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR·hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52. glucocorticoid receptor heat shock protein green fluorescent protein FK506 binding protein peptidylprolyl isomerase tetratricopeptide repeat Dulbecco's modified Eagle's medium dexamethasone protein phosphatase 5 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid Steroid receptors move continuously into and out of the nucleus (Refs. 1Guichon-Mantel A. Lescop P. Christin-Maitre S. Loosfelt H. Perrot-Applanat M. Milgrom E. EMBO J. 1991; 10: 3851-3859Crossref PubMed Scopus (246) Google Scholar, 2Chandran U.R. DeFranco D.B. Mol. Endocrinol. 1992; 6: 837-844PubMed Google Scholar, 3Madan A.P. DeFranco D.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3588-3592Crossref PubMed Scopus (144) Google Scholar, 4Dauvois S. White R. Parker M.G. J. Cell Sci. 1993; 106: 1377-1388Crossref PubMed Google Scholar; for review, see Ref. 5DeFranco D.B. Madan A.P. Tang Y. Chandran U.R. Xiao N. Yang J. Vitam. Horm. 1995; 51: 315-338Crossref PubMed Scopus (81) Google Scholar), 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 (6Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (722) Google Scholar, 7Qi M. Hamilton B.J. DeFranco D.B. Mol. Endocrinol. 1989; 3: 1279-1288Crossref PubMed Scopus (72) Google Scholar, 8Czar M.J. Lyons R.H. Welsh M.J. Renoir J.M. Pratt W.B. Mol. Endocrinol. 1995; 9: 1549-1560PubMed Google Scholar). 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. 9Pratt W.B. Silverstein A.M. Galigniana M.D. Cell. Signal. 1999; 11: 839-851Crossref PubMed Scopus (149) Google Scholar). Assembly of receptors into heterocomplexes with hsp90 is a dynamic process (10Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar), and it has been shown that the GR and hsp90 can move together from the cytoplasm to the nucleus (11Kang K.I. Devin J. Cadepond F. Jibard N. Guichon-Mantel A. Baulieu E.E. Catelli M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 340-344Crossref PubMed Scopus (121) Google Scholar). A couple of observations suggest that the role of hsp90 in receptor movement is likely to involve dynamic assembly and disassembly of GR·hsp90 heterocomplexes. For example, Yang and DeFranco (12Yang J. DeFranco D.B. Mol. Endocrinol. 1996; 10: 3-13Crossref PubMed Scopus (87) Google Scholar) showed that molybdate, which binds to hsp90 and stabilizes GR·hsp90 heterocomplexes in vivo (13Raaka B.M. Finnerty M. Sun E. Samuels H.H. J. Biol. Chem. 1985; 260: 14009-14015Abstract Full Text PDF PubMed Google Scholar), 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 (14Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar) and prevents formation of normal receptor·hsp90 heterocomplexes (15Smith D.F. Whitesell L. Nair S.C. Chen S. Prapapanich V. Rimerman R.A. Mol. Cell. Biol. 1995; 15: 6804-6812Crossref PubMed Scopus (271) Google Scholar), impedes steroid-induced movement of the GR from the cytoplasm to the nucleus (16Czar M.J. Galigniana M.D. Silverstein A.M. Pratt W.B. Biochemistry. 1997; 36: 7776-7785Crossref PubMed Scopus (134) Google Scholar, 17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar). Some localization studies have shown the untransformed GR to colocalize with microtubules (for review, see Ref. 18Akner G. Wikstrom A.C. Gustafsson J.A. J. Steroid Biochem. Mol. Biol. 1995; 52: 1-16Crossref PubMed Scopus (57) Google Scholar), 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 (8Czar M.J. Lyons R.H. Welsh M.J. Renoir J.M. Pratt W.B. Mol. Endocrinol. 1995; 9: 1549-1560PubMed Google Scholar, 19Perrot-Applanat M. Lescop P. Milgrom E. J. Cell Biol. 1992; 119: 337-348Crossref PubMed Scopus (51) Google Scholar), they eliminate the hsp90-dependent mode of receptor movement (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar). 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 (t12 = ∼5 min) both in cells with intact cytoskeleton and in cells with disrupted cytoskeletal networks (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar). However, in cells with normal cytoskeleton, the hsp90 inhibitor geldanamycin slowed translocation of the GFP-GR by an order of magnitude (t12 = ∼45 min), whereas in cells with colcemid-disrupted microtubules, geldanamycin had no effect on the translocation rate (t12 = ∼5 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 heterocomplex assembly machinery plays a role. In cells where the microtubules are disrupted with colcemid, movement is still steroid-dependent, but the transformed GR moves through the cytoplasm by diffusion (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar, 20Galigniana M.D. Housley P.R. DeFranco D.B. Pratt W.B. J. Biol. Chem. 1999; 274: 16222-16227Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In normal cells, the retrograde movement of vesicles toward the nucleus is known to occur on cytoskeletal tracks in a process requiring the molecular motor protein cytoplasmic dynein (for review, see Refs. 21Vallee R.B. Bloom G.S. Annu. Rev. Neurosci. 1991; 14: 59-92Crossref PubMed Scopus (229) Google Scholarand 22Langford G.M. Curr. Opin. Cell Biol. 1995; 7: 82-88Crossref PubMed Scopus (209) Google Scholar). However, like GFP-GR translocation to the nucleus, short range vesicle movement still occurs by diffusion when microtubules are disrupted (23Bloom G.S. Goldstein L.S.B. J. Cell Biol. 1998; 140: 1277-1280Crossref PubMed Scopus (97) Google Scholar). Using retrograde vesicular movement as a model, we have looked for potential links between the GR and cytoplasmic dynein. In addition to hsp90, steroid receptor heterocomplexes contain hsp90-binding immunophilins (for review, see Ref. 24Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1529) Google Scholar), and we have shown that cytoplasmic dynein is coimmunoadsorbed from cytosols with FKBP52, one of the major immunophilins in GR·hsp90 heterocomplexes. The immunophilins are proteins possessing peptidylprolyl isomerase (PPIase) domains that bind immunosuppressant drugs of the FK506 group (the FKBPs) or of the cyclosporin A group (the cyclosporin A-binding proteins). Three high molecular weight immunophilins, FKBP52, FKBP51, and cyclosporin A-binding protein-40, exist in receptor·hsp90 heterocomplexes (24Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1529) Google Scholar). In addition to their PPIase domains, these high molecular weight immunophilins contain three tetratricopeptide repeats (TPRs), which are degenerative sequences of 34 amino acids that determine their binding to a common TPR acceptor site on hsp90 (25Radanyi C. Chambraud B. Baulieu E.-E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11197-11201Crossref PubMed Scopus (155) Google Scholar, 26Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffman K. Pratt W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. N. M. Pratt W.B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We have shown that FKBP52 also binds to the GR and that a acid the localization that the nuclear localization of the GR Ref. 5DeFranco D.B. Madan A.P. Tang Y. Chandran U.R. Xiao N. Yang J. Vitam. Horm. 1995; 51: 315-338Crossref PubMed Scopus (81) Google for is for FKBP52 binding A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar). FKBP52 binds or a protein with cytoplasmic dynein M.J. J.K. Welsh M.J. Pratt W.B. Mol. Endocrinol. 1994; Google Scholar), and of dynein is competed by a fragment of FKBP52 comprising its PPIase domain A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar). These binding studies the notion W.B. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, W.B. Czar M.J. J.K. J. Steroid Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar) that FKBP52, which with microtubules M.J. J.K. Welsh M.J. Pratt W.B. Mol. Endocrinol. 1994; Google Scholar, M. C. G. Renoir Baulieu E.-E. J. Cell Sci. 1995; Google Scholar), may receptor movement toward the nucleus by its to the retrograde dynein motor the in linking FKBP52 to GR movement is the that of an a of amino between domains (PPIase) and of FKBP52 Renoir N. A. Baulieu E.-E. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar) impedes steroid-dependent translocation of the GR in L cells (8Czar M.J. Lyons R.H. Welsh M.J. Renoir J.M. Pratt W.B. Mol. Endocrinol. 1995; 9: 1549-1560PubMed Google Scholar). In this we show that cotransfection of 3T3 with GFP-GR and the PPIase domain of FKBP52 steroid-dependent translocation of the GFP-GR to the nucleus (t12 = ∼45 min) to the as with the hsp90 The effect is as cotransfection with FKBP12 does not affect GFP-GR movement. cells are treated with colcemid to microtubules prior to steroid the of PPIase domain does not inhibit movement. when colcemid is and microtubules reform, GFP-GR translocation is slowed by the of the PPIase The observations are consistent with an of the receptor from movement along microtubules by for the binding of FKBP52 to the dynein motor. 3T3 from the Dulbecco's modified Eagle's medium was from and colcemid from The for cell was from The the of cytoplasmic dynein was from reticulocyte lysate was from and geldanamycin was from the and of the inhibitor from The was by of and the FKBP52 J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar) was a from and The for the GR was from and the for GR immunoadsorption was by The hsp90 was from and was from of the GFP-GR was previously (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar). The for FKBP52, FKBP52 FKBP52 and FKBP12 previously B. N. C. K. Baulieu E.-E. Biochem. 1993; PubMed Scopus Google Scholar, B. C. K. K. Baulieu E.-E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and into the from The for GR was from of and was previously Y. Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The for the TPR domain of Silverstein A.M. Pratt W.B. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) was by of 3T3 on in in of a the cells the medium was by of with and the was for with of GFP-GR and of domain or domain or or The cotransfection was by with of of and of for The was to the cell and for prior to of with out the for After the medium was cells of with and cells for an as with and and cells with and by in for with and the a with 5 of in with a for GFP-GR translocation as we have previously (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar), a of for nuclear cytoplasmic for nuclear cytoplasmic for nuclear to cytoplasmic for nuclear cytoplasmic and for nuclear cytoplasmic The translocation the from three in which cells of was by of by For immunoadsorption of FKBP52, of reticulocyte lysate immunoadsorbed for to of protein with of FKBP52. three by in of with prior to and to for FKBP52. GR was in cells as Y. Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and cytosol was by in of and of inhibitor of The lysate was for and the was immunoadsorbed from of cytosol by for with of protein and of and the with of to with reticulocyte immunoadsorbed receptors of hsp90 by for with of and The three with of and with GR·hsp90 heterocomplexes assembled by these GR with of reticulocyte lysate and 5 of an system and of The for with of the by the the of the the three with of with and in GR and on and proteins to The with for for hsp90, for FKBP52, and of for dynein. The a with the to was from by in of molybdate, and of inhibitor of and for immunoadsorbed from of cytosol by for with of protein and of and the three with of with was to The fusion protein was by binding to and the PPIase domain of FKBP52 was by with the FKBP52 domain fragment for FKBP52 with cytoplasmic dynein in A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar), we of domain the rate of GFP-GR movement from the cytoplasm to the nucleus in In the of the GFP-GR expressed in 3T3 cells is predominantly localized to the cytoplasm and and upon to it moves to the nucleus we previously (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar), nuclear translocation of the GFP-GR is when hsp90 is by geldanamycin shown in cotransfection with FKBP52 domain also inhibits GFP-GR The on the in the translocation from cells where a of a cytoplasmic localization and a of a nuclear a system we have previously with the GFP-GR (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar, 20Galigniana M.D. Housley P.R. DeFranco D.B. Pratt W.B. J. Biol. Chem. 1999; 274: 16222-16227Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). After the of steroid nuclear translocation of the GFP-GR is to the by of FKBP52 domain as it is by with the rate of GFP-GR but by of steroid of the receptors have to the nucleus 17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google and shown in cotransfection with FKBP52 domain GFP-GR translocation to the as geldanamycin, and geldanamycin of cells FKBP52 domain does not a rate of translocation that with shown in of FKBP52 domain with geldanamycin GFP-GR translocation in cells that have been treated with colcemid to We have shown that with colcemid these conditions of microtubules in 3T3 cells (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar). cells are in medium colcemid for the microtubular is and geldanamycin inhibits GFP-GR movement (17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar). In the of cells that treated with colcemid, such that steroid-dependent GFP-GR movement was not by FKBP52 domain or geldanamycin from colcemid for prior to steroid addition. In the cells, GFP-GR translocation was by FKBP52 domain and by These observations suggest normal physiological conditions where the microtubular is intact, the GFP-GR is an such as FKBP52, to a system for movement along microtubular The FKBP52 domain expressed in the of and of domain and a that domains and that the PPIase domain was for inhibition of receptor we the GFP-GR with FKBP52 domain which does not contain the shown in GFP-GR translocation is to the in cells FKBP52 domain as in cells FKBP52 domain We by that the of both FKBP52 domain and domain the FKBP52 in cells not Although domain has the PPIase as the FKBP52 B. N. C. K. Baulieu E.-E. Biochem. 1993; PubMed Scopus Google Scholar), that is not to inhibition of GFP-GR movement. This is from the that the immunosuppressant which binds in the site and PPIase does not affect the rate of steroid-dependent GFP-GR translocation or inhibition of the rate of translocation by FKBP52 domain not We have also shown previously that FK506 does not affect FKBP52 binding to cytoplasmic dynein A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar). Inhibition of GFP-GR movement by FKBP52 domain does not an inhibition by PPIase domains in We have shown previously that FKBP12 does not affect FKBP52 binding to cytoplasmic dynein A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar). In it is shown that cotransfection of 3T3 cells with FKBP12 does not affect steroid-dependent GFP-GR The of FKBP12 in cells was by binding to binding in The of three that affect the binding of FKBP52 to hsp90 or dynein. In this of reticulocyte lysate with and FKBP52 was of FKBP52 is accompanied by coadsorption of both hsp90 and cytoplasmic dynein The TPR domain fragment of FKBP52 binding to hsp90 but not to dynein whereas FKBP52 domain FKBP52 binding to dynein but does not affect its binding to hsp90 FKBP52 binding to dynein is not by FK506 which suggests that the binding is of PPIase A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar). of reticulocyte lysate with geldanamycin FKBP52 binding to hsp90 lysate the chaperone machinery that GR·hsp90 heterocomplexes in Ref. 24Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1529) Google Scholar). In the of we the GR·hsp90 heterocomplex in reticulocyte dynein. GR that was of its hsp90 was with reticulocyte and after the the GR and proteins by and shown the GR is assembled into heterocomplexes hsp90, FKBP52, and cytoplasmic dynein. the lysate is with geldanamycin it is with the is a in hsp90, and is no FKBP52 or dynein FKBP52 is to hsp90 its TPR domain (25Radanyi C. Chambraud B. Baulieu E.-E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11197-11201Crossref PubMed Scopus (155) Google Scholar), and with a TPR domain fragment of a GR·hsp90 that does not contain FKBP52 or dynein shown in 5 with FKBP52 domain dynein the of hsp90 or FKBP52 in the GR These observations are consistent with a in which the GR is to the motor protein an such as FKBP52. In 5 the GR was immunoadsorbed from cytosol from L dynein was that it is a component of native GR·hsp90 heterocomplexes in the Inhibition of GFP-GR translocation by of the PPIase domain of FKBP52 is consistent with a in which rapid receptor movement in cells with intact cytoskeleton of one or of the high molecular hsp90-binding immunophilins (8Czar M.J. Lyons R.H. Welsh M.J. Renoir J.M. Pratt W.B. Mol. Endocrinol. 1995; 9: 1549-1560PubMed Google Scholar). We have previously that the rate of GR translocation is by geldanamycin (16Czar M.J. Galigniana M.D. Silverstein A.M. Pratt W.B. Biochemistry. 1997; 36: 7776-7785Crossref PubMed Scopus (134) Google Scholar, 17Galigniana M.D. Scruggs J.L. Herrington J. Welsh M.J. Carter-Su C. Housley P.R. Pratt W.B. Mol. Endocrinol. 1998; 12: 1903-1913Crossref PubMed Scopus (156) Google Scholar), which GR·hsp90 heterocomplex but the in which dynamic GR·hsp90 heterocomplex assembly GR movement along cytoskeletal was FKBP52 a between the receptor heterocomplex and cytoplasmic dynein, a motor protein for retrograde movement along of reticulocyte lysate with geldanamycin inhibits FKBP52 binding to hsp90 and inhibits GR·hsp90 receptors that are not with FKBP52 and dynein 5 A effect in vivo rapid GR movement in normal cells with intact microtubules is by The of both geldanamycin and FKBP52 domain to GR to dynein the two the rate of GFP-GR movement to the and is no with the two together In this we show that dynein is in native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol and that GR·hsp90 heterocomplexes are in reticulocyte These complexes contain FKBP52 complexes immunophilins, such as FKBP51, are also Nair S.C. Y. Rimerman R.A. J. Y. D.F. Mol. Endocrinol. 1998; 12: PubMed Google Scholar). Assembly of GR·hsp90 heterocomplexes in the of the TPR domain fragment the binding of of the immunophilins J.K. Czar M.J. Hutchison K.A. Hoffman K. Pratt W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). this we can that the of dynein in the GR·hsp90 heterocomplex a TPR domain with FKBP52 and of that The common of immunophilins is the of their PPIase domains, and the PPIase domain of FKBP52 PPIase when it is expressed as a domain fragment B. N. C. K. Baulieu E.-E. Biochem. 1993; PubMed Scopus Google Scholar). However, the PPIase inhibitor FK506 does not affect FKBP52 binding to cytoplasmic dynein and Ref. A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar), or the rate of GFP-GR or inhibition of translocation by the FKBP52 PPIase the PPIase of FKBP52 does not to involved in the with cytoplasmic dynein or retrograde movement of The PPIase domain of FKBP52 as a dynein domain of binding by In this it from the of FKBP12 with where binding the immunophilin J. J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). is that the PPIase domains of immunophilins are involved in and For example, the molecular weight immunophilin FKBP12 does not affect GFP-GR whereas the PPIase domain of FKBP52 does FKBP12 also does not for FKBP52 binding to cytoplasmic dynein, whereas the PPIase domain of FKBP52 does A.M. Galigniana M.D. C. Renoir Pratt W.B. J. Biol. Chem. 1999; 52: Full Text Full Text PDF Scopus Google Scholar). Although FKBP12 binds to and inhibits the phosphatase J. J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar), FKBP52 its expressed PPIase domain fragment inhibits in N. Baulieu E.-E. Biochem. 1994; PubMed Scopus Google Scholar). is that the PPIase domains may a of domains, with no of binding targets between FKBP12 and the high molecular hsp90-binding we see the rate of GFP-GR nuclear translocation in the and of microtubules it may that nuclear and not receptor movement through the cytoplasm is for nuclear both the microtubules are intact, it that receptor movement occurs predominantly on a movement system that dynamic of the receptor with hsp90 and the cytoskeleton is the GR may move through the cytoplasm by Although movement of the receptor through the cytoplasm is not the in translocation physiological conditions in cells with intact cytoskeleton, it when geldanamycin or the PPIase domain of FKBP52 are to the receptor from the movement in the normal rapid movement of protein by diffusion may by the cytoskeleton, and to a movement system rapid to their of a movement system may not for protein movement in in movement by diffusion is not and a movement such as that In this we have the in vivo evidence that the immunophilin PPIase domain links the glucocorticoid receptor to a retrograde movement We also show for the that heterocomplexes are to the retrograde motor protein cytoplasmic dynein the PPIase domain of the immunophilin We and for and and for