Composition, assembly and activation of the avian progesterone receptor.
When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, 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 ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system.
Smith
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Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, MN 55905.
Toft
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DO
D000818:Animals; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010057:Oviducts / Q000187:drug effects / Q000378:metabolism*; D014176:Protein Biosynthesis; D011817:Rabbits; D011980:Receptors, Progesterone / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012156:Reticulocytes / Q000378:metabolism
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011980:Receptors, Progesterone; D004958:Estradiol