Abstract
In the absence of hormone, the avian progesterone receptor exists in a large multiprotein complex that is inactive but able to bind and respond to progestins. This inactive complex can be reconstituted in vitro by incubation of receptor monomer in rabbit reticulocyte lysate in the presence of ATP and magnesium. This results in receptor binding to the two heat shock proteins, hsp90 and hsp70, the FK506 binding-proteins, FKBP54 and FKBP52, the cyclosporin A-binding protein, cyclophilin-40, and the recently characterized protein p23. Immune isolation of p23 from rabbit reticulocyte lysate in the absence of receptor reveals an ATP-dependent complex containing the major proteins associated with steroid receptors. Depletion of p23 from lysate prevents the assembly of progesterone receptor complexes, and the addition of purified p23 restores this activity, indicating that the p23 protein complex is an essential precursor to the formation of progesterone receptor complexes.
Highlights
P-40; and p23 [2, 14, 15]. p23 is a recently described, novel receptor exists in a large multiprotein complex thatis protein that shows no sequencehomology to either heat shock inactive but able to bind and respond to progestinsp.roTtehiniss or immunophilins [16]
Thisresults in plexes in rabbit reticulocyte lysateand their rolein the assemreceptor binding to the two heat shock proteihnssp,9O bly of steroid receptor complexes. andhsp70,the FKS06 binding-proteins, FKBP54 and
ATPyS is as effective as ATP, the ticulocyte lysate on protein A-Sepharose yields p23 in complex analog AMP-PCP does not support the formation of the p23 with hsp90, and a 40-kDa protein (Fig.1, lane2).The identity complex.The addition of 10 mM EDTA (Fig. 2, of hsp90 was verified by Westernblotting, and the 40-kDa lane 8 )sharply reduces threcovery of both hsp90 andCyP-40, protein comigrates with a protein seen in chick progesterone indicating a requirement for magnesium in p23 complex asreceptor complexes reconstituted in reticulocyte lysate
Summary
P23 Complex Reconstitution-Untreated rabbit reticulocyte lysate (Green Hectares, Oregon, WI) wasadjusted to 0.5 M KC1, incubated on ice for 30 min, and dialyzedin 10m~ ”is-HC1 (pH7.5),50mM KC1, In the absence of hormone, the avian progesterone receptor (PR)’ exists as a large multiprotein complexthat is inactive but able t o bind and respond to progestins. The inactive PR complex can be reconstituted in vitro by incubation of receptor monomer in rabbit reticulocyte lysiante the presence of magnesium, ATP, and elevated temperature [14, 15] Under these conditions, isolated chickPR will reassociate with hsp, hsp, FKBP52, FKBP54, Cy-.
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