title: Structural characterization of the trypsinized estrogen receptor.
abstract: Structural differences between the unoccupied and ligand-occupied rat uterine estrogen receptors (ERs) were investigated using partial proteolysis followed by immunoblotting, affinity labeling, and gel filtration chromatography. Trypsin digestion of the unoccupied ER at 4 degrees C resulted in retention of 70-80% of high-affinity [3H]estradiol binding. Only two fragments of the rat ER were detected after prolonged trypsin treatment of the unoccupied ER followed by affinity labeling with [3H]tamoxifen aziridine. One fragment represents the intact steroid binding domain (28 kDa), and the other fragment is about 10 kDa. The small 10-kDa fragment of the ER detected by denaturing gel electrophoresis is shown to be held in a large oligomeric complex in solution using gel filtration chromatography. This oligomeric complex probably represents the steroid binding domain, which has its tertiary structure maintained predominantly by noncovalent interactions between the trypsin-generated fragments. The estrogen, anti-estrogen, and unoccupied trypsinized ERs all result in similar patterns of fragments after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection by immunoblotting. Although no new trypsin cleavage sites were exposed, the sensitivity of the available trypsin sites was altered by heating the ER and, to a lesser extent, by hormone treatment. Gel filtration chromatography of the trypsinized estradiol- and 4-hydroxytamoxifen-occupied ERs demonstrates similar, diffuse peaks centered at about the correct size for the intact steroid binding domain (28 kDa), whereas the trypsinized unoccupied ER results in a sharp, discrete peak centered at about 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison 53706-1569.
authors.forename: M
authors.initials: M
authors.lastname: Fritsch
authors.forename: I
authors.initials: I
authors.lastname: Anderson
authors.forename: J
authors.initials: J
authors.lastname: Gorski
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D011960:Receptors, Estrogen; D004958:Estradiol; D014357:Trypsin