Effects of immunosuppressants FK506 and rapamycin on the heterooligomeric form of the progesterone receptor

Abstract

The non-DNA binding form of the rabbit uterus cytosol progesterone receptor (PR) contains, in addition to the hormone binding unit and heat shock protein M r 90kDa (hsp90), a H eat shock protein B inding I mmunophilin (p59/HBI) which interacts with hsp90. P59/HBI binds the immunosuppressants 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 [ 3H]steroid-labeled tungstate-stabilized PR with anti-PR immunoadsorbent yielded “9S”-PR species in which hsp90, hsp70 and p59/HBI were present. 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. In 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 exposure of cytosol to 5 μM FK506 or RAP. Other experiments showed that cytosol fractions incubated for 2 h at 25°C with 0.05 to 10 μM FK506 or RAP, then with [ 3H]steroids (the agonist [ 3H]Org 2058 or the anti-progestin [ 3H]RU486), contains greater amounts of 9S-PR species than that detected in non-immunosuppressant exposed control cytosol. Scatchard analysis showed an up to 2-fold decrease of the K d values for both hormones following exposure to drugs, without modification of the number of steroid binding sites. Purification of cytosol PR on immobilized FK506 yields a 9S form still containing hsp90, hsp70 and p59/HBI associated to PR units. Altogether, these results suggest that binding of immunosuppressants to p59/HBI does not promote hsps dissociation from the receptor and, as a consequence, that inhibition of peptidyl-prolyl isomerase activity of p59/HBI by immunosuppressants binding does not transform (activate) PR in vitro. However, given the assumption that hsp90 binds to receptor and that p59/HBI binds hsp90 but not directly to receptor, immunosuppressants affect hormone binding by an unknown mechanism involving receptor associated proteins. In addition, we show that the chick oviduct cytosol 9S-PR, not displaced with the EC1 antibody specific for several mammalian p59/HBI, also binds to FK506 columns and can be eluted by exchange with either FK506 or RAP, suggesting that there is an avian HBI homolog. These findings extend the notion of immunophilin-steroid receptor association and suggest that, besides the already described molecular chaperone hsp90, other associated proteins might be involved in steroid receptor function.