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 receptor (PR) contains, in addition to the hormone binding unit and heat shock protein M(r) 90kDa (hsp90), a Heat shock protein Binding Immunophilin (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 Kd 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 microM FK506 or RAP. Other experiments showed that cytosol fractions incubated for 2 h at 25 degrees C with 0.05 to 10 microM 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 Kd value 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Renoir
J M
JM
Lab. Hormones, INSERM U33, Le Kremlin-Bicêtre, France.
Le Bihan
S
S
Mercier-Bodard
C
C
Gold
A
A
Arjomandi
M
M
Radanyi
C
C
Baulieu
E E
EE
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006360:Heat-Shock Proteins / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D011090:Polyenes / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D011817:Rabbits; D011980:Receptors, Progesterone / Q000737:chemistry / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020123:Sirolimus; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins; D014599:Uterus / Q000378:metabolism
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D011090:Polyenes; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus; D016559:Tacrolimus