Mutational analysis of hsp90 binding to the progesterone receptor.
The 90-kDa heat shock protein, hsp90, is known to associate with steroid receptors that are in the inactive state. While the biochemical function of hsp90 is unclear, this association is believed to be significant because dissociation of hsp90 occurs when receptors are activated by hormone. Complexes between hsp90 and the progesterone receptor can be formed in vitro in rabbit reticulocyte lysate. This has been shown to be an ATP-dependent process, and dissociation of the complex occurs when progesterone is added to the system. We now show that hsp90 synthesized by in vitro translation in reticulocyte lysate can form complexes with progesterone receptor that are sensitive to hormone. This system was used to analyze several mutant forms of hsp90. A series of NH2-terminal deletions showed that amino acids 1-380 can be removed from hsp90 without substantial loss of receptor binding activity. However, several deletions in the COOH-terminal half of hsp90 resulted in a partial or complete loss of this activity. Two regions, amino acids 381-441 and 601-677, appear to be particularly important for receptor binding. These studies describe a convenient and reliable method for the initial screening of hsp90 mutants, and they provide important clues to the identification of domains on hsp90 that interact with other proteins.
Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010057:Oviducts / Q000187:drug effects / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011374:Progesterone / Q000494:pharmacology; D011485:Protein Binding; D014176:Protein Biosynthesis; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects
D006360:Heat-Shock Proteins; D009838:Oligodeoxyribonucleotides; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D011374:Progesterone; D004958:Estradiol