IMMUNOLOGICAL IDENTIFICATION OF A 50 KDA MR FK506-BINDING IMMUNOPHILIN AS A COMPONENT OF THE NON-DNA BINDING, HSP90 AND HSP70 CONTAINING, HETEROOLIGOMERIC FORM OF THE CHICK OVIDUCT PROGESTERONE-RECEPTOR

p53/HBI, a Heat shock protein (hsp90)-Binding-Immunophilin of similar to 59 kDa, was originally detected in the heterooligomeric, non-DNA bi nding form of numerous mammalian steroid hormone receptors. P59/HBI be longs to the FKBP family since it binds the immunosuppressants FK506 a nd Rapamycin. Using immobilized FK506, we purified [H-3]Org 2058-proge sterone receptor (PR)-hsp90-complexes from chick oviduct cytosol in a species migrating at 9S in density gradient. This led to suppose that a protein present in these complexes is a FK506 binding protein. Follo wing incubation of this FK506-affinity purified 9S-PR with BF4, a spec ific anti-chick hsp90 monoclonal antibody, a shift of the [H-3]Org 205 8-PR complexes from 9S to 11S has been observed, indicating the presen ce of hsp90. hsp70 also is included in the 9S-PR complexes as demonstr ated by Western blotting and density gradient experiments. Two lines o f evidence support the notion that an immunophilin of 50 kDa also asso ciates to heterooligomeric 9S-PR complexes. Firstly, a shift at 11S of the FK506 eluted 9S-PR occurs in sucrose gradient after incubation wi th 419, a new polyclonal antibody raised against a peptide correspondi ng to the Hinge I region [Callebaut I., Renoir J. M., Lebeau M. C., Ma ssol N., Burny A., Baulieu E. E., Mornon J. P. (1992) Proc. Natl. Acad . Sci. USA 89, 6270-6274] of the rabbit p59/HBI (amino acids Phe(135) to Gly(149)). Secondly, in Western blotting experiments, both 419 and another anti-FKBP antibody, raised against purified Streptomyces Cryso mallus FKBP12 [Pahl A. and Keller U. (1992) J. Bacteriol, 3, 325-333], allow detection of the same 50 kDa protein in the affinity purified P R This 50 kDa protein (as well as PR and hsps) is absent in the eluate s obtained after addition of 5 mu M FK506 in the cytosol prior purific ation by FK506-affinity column, but it is still present if a washing s tep with a 0.3 M NaCl containing buffer is included in the classical p urification. This suggests that the 50 kDa protein binds the immunosup presant FK506 and is responsible of the binding of [H-3]Org2058-PR com plexes to the affinity resin. Altogether, these results demonstrate th e presence of a 50 kDa immunophilin of the FKBP family in the heterool igomeric chicken 9S-PR, which represent likely the chicken p53/HBI hom ologue.