EFFECTS OF IMMUNOSUPPRESSANTS FK506 AND RAPAMYCIN ON THE HETEROOLIGOMERIC FORM OF THE PROGESTERONE-RECEPTOR

The non-DNA binding form of the rabbit uterus cytosol progesterone rec eptor (PR) contains, in addition to the hormone binding unit and heat shock protein M(r) 90kDa (hsp90), a Heat shock protein Binding Immunop hilin (p59/HBI) which interacts with hsp90. P59/HBI binds the immunosu ppressants FK506 and Rapamycin (RAP) and belongs to the FK506 binding protein family. A recombinant p59/HBI-glutathione-S-transferase fusion protein, purified by Sephadex LH-20 filtration of tritiated drug-p59/ HBI complexes, binds FK506 and RAP with apparent K-d values of 75+/-40 and 40+/-15 nM, respectively. Immunopurification from cytosol of [H-3 ]steroid-labeled tungstate-stabilized PR with anti-PR immunoadsorbent yielded ''9S''-PR species in which hsp90, hsp70 and p59/HBI were prese nt. In the absence of tungstate ions, only the 4-6S PR was eluted, and Western blot analysis demonstrated the absence of hsps and p59/HBI. I n contrast 30 to 50% of the original 9S-PR species containing hsps and p59/HBI, was eluted in the absence of tungstate ions but after exposu re of cytosol to 5 mu M FK506 or RAP. Other experiments showed that cy tosol fractions incubated for 2 h at 25 degrees C with 0.05 to 10 mu M FK506 or RAP, then with [H-3]steroids (the agonist [H-3]Org 2058 or t he anti-progestin [H-3]RU486), contains greater amounts of 9S-PR speci es than that detected in non-immunosuppressant exposed control cytosol . Scatchard analysis showed an up to 2-fold decrease of the K-d value for both hormones following exposure to drugs, without modification of the number of steroid binding sites. Purification of cytosol PR on im mobilized FK506 yields a 9S form still containing hsp90, hsp70 and p59 /HBI associated to PR units. Altogether, these results suggest that bi nding of immunosuppressants to p59/HBI does not promote hsps dissociat ion from the receptor and, as a consequence, that inhibition of peptid yl-prolyl isomerase activity of p59/HBI by immunosuppressants binding does not transform (activate) PR in vitro. However, given the assumpti on that hsp90 binds to receptor and that p59/HBI binds hsp90 but not d irectly to receptor, immunosuppressants affect hormone binding by an u nknown mechanism involving receptor associated proteins. In addition, we show that the chick oviduct cytosol 9S-PR, not displaced with the E C1 antibody specific for several mammalian p59/HBI, also binds to FK50 6 columns and can be eluted by exchange with either FK506 or RAP, sugg esting that there is an avian HBI homolog. These findings extend the n otion of immunophilin-steroid receptor association and suggest that, b esides the already described molecular chaperone hsp90, other associat ed proteins might be involved in steroid receptor function.