THE CYCLOSPORINE A-BINDING IMMUNOPHILIN CYP-40 AND THE FK506-BINDING IMMUNOPHILIN HSP56 BIND TO A COMMON SITE ON HSP90 AND EXIST IN INDEPENDENT CYTOSOLIC HETEROCOMPLEXES WITH THE UNTRANSFORMED GLUCOCORTICOID RECEPTOR

We have recently shown that hsp56, the FKBOG-binding immunophilin comp onent of both the heat shock protein (hsp90 . hsp70 . hsp56) heterocom plex and the untransformed glucocorticoid receptor heterocomplex, is b ound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., an d Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 hete rocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A -binding class. CyP-40 is present in both native glucocorticoid recept or heterocomplexes and receptor heterocomplexes reconstituted with rab bit reticulocyte lysate, and the presence of CyP-40 in the receptor he terocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showi ng that both immunophilins are in native heterocomplex with hsp90. How ever, immunoadsorption of hsp56 does not yield coimmunoadsorption of C yP-40; thus, the two immunophilins do not exist in the same heterocomp lex with hsp90. Both purified CyP-40 and hsp56 bind directly to purifi ed hsp90, and excess CyP-40 blocks the binding of hsp56, consistent wi th the presence of a common immunophilin binding site on hsp90. Our da ta also suggest that there are at least two types of untransformed glu cocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 an d another that contains CyP-40. The role played by the immunophilins i n steroid receptor action is unknown, but it is clear that the peptidy lprolyl isomerase activity of immunophilins is not required for glucoc orticoid receptor-hsp90 heterocomplex assembly and proper folding of t he hormone binding domain by the hsp90-associated protein folding syst em of reticulocyte lysate.