STRUCTURAL CHARACTERIZATION OF THE TRYPSINIZED ESTROGEN-RECEPTOR

Structural differences between the unoccupied and ligand-occupied rat uterine estrogen receptors (ERs) were investigated using partial prote olysis followed by immunoblotting, affinity labeling, and gel filtrati on chromatography. Trypsin digestion of the unoccupied ER at 4-degrees -C resulted in retention of 70-80% of high-affinity [H-3]estradiol bin ding. Only two fragments of the rat ER were detected after prolonged t rypsin treatment of the unoccupied ER followed by affinity labeling wi th [H-3]tamoxifen aziridine. One fragment represents the intact steroi d binding domain (28 kDa), and the other fragment is about 10 kDa. The small 10-kDa fragment of the ER detected by denaturing gel electropho resis is shown to be held in a large oligomeric complex in solution us ing gel filtration chromatography. This oligomeric complex probably re presents the steroid binding domain, which has its tertiary structure maintained predominantly by noncovalent interactions between the tryps in-generated fragments. The estrogen, anti-estrogen, and unoccupied tr ypsinized ERs all result in similar patterns of fragments after separa tion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection by immunoblotting. Although no new trypsin cleavage sites we re 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-hydroxyt amoxifen-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 pe ak centered at about 80 kDa. We conclude that the unoccupied steroid b inding domain has a different conformation than either the estradiol- or the 4-hydroxytamoxifen-occupied steroid binding domains.