title: Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.
abstract: To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905.
authors.forename: D F
authors.initials: DF
authors.lastname: Smith
authors.forename: B A
authors.initials: BA
authors.lastname: Stensgard
authors.forename: W J
authors.initials: WJ
authors.lastname: Welch
authors.forename: D O
authors.initials: DO
authors.lastname: Toft
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism*; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D006360:Heat-Shock Proteins; D011980:Receptors, Progesterone; D000255:Adenosine Triphosphate; D008274:Magnesium