ESTROGEN RECEPTORS AS REAGENTS FOR ASSESSING THERAPEUTIC ESTROGEN ACTIVITY. E. Jisa1 , A. Thalhammer1, T. T. Nguyen1 , M. D. Grant1 , W. Raffelsberger2 and J. L. Wittliff1,

1 Brown Cancer Center, University of Louisville, Louisville, Ky. 40202 &

2 IGBMC, Cedex - Strasbourg, France.

Estrogen action in target cells is communicated via intracellular receptors which are transcriptional regulatory proteins. Our approach utilizes cell-free preparations of recombinant human (hER) and calf uterine wild-type (wtER) estrogen receptors to rapidly assess estrogen mimicry of a candidate therapeutic compound. hER, expressed via a ubiquitin-fusion vector in Saccheromyces cerevisiae (YEpE12) under the control of a copper-methionine promoter, and wtER were extracted with either 10 mM or 50 mM K2HPO4 buffer (pH 6.5), respectively, containing 10 mM sodium molybdate, 1.5mM EDTA and 10% glycerol. Specific binding capacities were 6-9 pmol/mg protein (Kd = 1-6 x 10-10M) and 230-262 fmol/mg protein (Kd = 1-7 x 10-10M) for hER and wtER, respectively. Either hER or wtER was titrated in the presence of [3H-estradiol-17b] with increasing concentrations of a model therapeutic estrogen mimic and binding affinity (Kd) & capacity were calculated using One-SiteÒ and CompeteÒ Software (Lundon Software, Inc.). Neither tamoxifen nor 4-hydroxytamoxifen (4OH-TAM) altered the affinity of [3H]estradiol-17b binding to either hER or wtER. Tamoxifen exhibited a Kd = 3-4 x 10-6 M from titration data and 0.7-8 x 10-6 M from competition data with wtER while 4OH-TAM gave a Kd = 4-5 x 10-8 M and Kd = 0.3-4 x 10-8 M. Using hER, tamoxifen exhibited a Kd = 4-5 x 10-7 M from titration data and 2-5 x 10-7 M from competition data while 4OH-TAM gave a Kd = 0.5-8 x 10-9 M and Kd = 5-9 x 10-9 M. There was good agreement between the two methods of calculation using a single receptor type. However hER recognized both therapeutic anitestrogens with higher affinity compared to wtER. These data suggest that at least some of the ER species in calf uterus are different from hER. It is unclear if the difference in binding properties is related to the expression of ER-b in calf uterus since the recombinant hER is known to be the expression product of the ER-a gene. Supported in part by a grant from Department of Veterans Affairs (#Hendler 0006). EJ & AT are Research Fellows in the International Scholars Program.