Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors a and b

The rat estrogen receptor (ER) exists as two subtypes, ERa and ERb, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ERa and rat ERb protein revealed a single binding component for 16a-iodo-17b-estradiol with high affinity [dissociation constant (Kd) 5 0.1 nM for ERa protein and 0.4 nM for ERb protein]. Most estrogenic substances or estrogenic antagonists compete with 16a-[I]iodo17b-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol . hexestrol . dienestrol . 4-OH-tamoxifen . 17b-estradiol . coumestrol, ICI-164384 . estrone, 17a-estradiol . nafoxidine, moxestrol . clomifene . estriol, 4-OH-estradiol . tamoxifen, 2-OH-estradiol, 5-androstene-3b,17bdiol, genistein for the ERa protein and dienestrol . 4-OH-tamoxifen . diethylstilbestrol . hexestrol . coumestrol, ICI-164384 . 17b-estradiol . estrone, genistein . estriol . nafoxidine, 5-androstene-3b,17b-diol. 17a-estradiol, clomifene, 2-OH-estradiol. 4-OHestradiol, tamoxifen, moxestrol for the ERb protein. The rat tissue distribution and/or the relative level of ERa and ERb expression seems to be quite different, i.e.moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ERa and prostate, ovary, lung, bladder, brain, uterus, and testis for ERb. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues. (Endocrinology 138: 863–870, 1997) E INFLUENCE the growth, differentiation and functioning of many target tissues. These include tissues of the male and female reproductive systems such as mammary gland, uterus, ovary, testis, and prostate. Estrogens also play an important role in bone maintenance and in the cardiovascular system, where estrogens have certain cardioprotective effects (1). Estrogens are mainly produced in the ovaries and testis. They diffuse in and out of all cells, but are retained with high affinity and specificity in target cells by an intranuclear binding protein, termed the estrogen receptor (ER). Once bound by estrogens, the ER undergoes a conformational change, allowing the receptor to bind with high affinity to chromatin and to modulate transcription of target genes (2). Steroid hormone receptors consist of a hypervariable N-terminal domain that contributes to the transactivation function; a highly conserved central domain responsible for specific DNA binding, dimerization, and nuclear localization, and a C-terminal domain involved in ligand binding and ligand-dependent transactivation function (1). The rat ER cDNAwas cloned from uterus and found to be highly homologous to the ER complementary DNAs (cDNAs) cloned from mouse, human, and chicken (3). We recently cloned a novel rat ER cDNA fromprostate (4), which we suggested be named rat ERb subtype to distinguish it from the previously cloned ER cDNA (consequently ERa subtype). The rat ERb cDNA encodes a protein of 485 amino acid residues with a calculated mol wt of 54200. Rat ERb protein is highly homologous to rat ERa protein, particularly in theDNAbinding domain (. 90% amino acid identity) and in the C-terminal ligand binding domain (LBD) (55%). Saturation ligand binding experiments with in vitro synthesized ERb protein revealed a single binding component for 17bestradiol (E2) with high affinity [dissociation constant (Kd)5 0.6 nm]. Expression of ERb was investigated by in situ hybridization, and prominent expressionwas found in rat prostate (secretory epithelial cells) and ovary (granulosa cells). In cotransfection experiments of Chinese hamster ovary (CHO) cells with an ERb expression vector and an estrogen-regulated reporter gene, maximal stimulation of reporter gene activity was found during incubation with 1 nm E2 (4). The biological significance of the existence of two ER subtypes is at this moment unclear. Perhaps the existence of two ER subtypes provides, at least in part, an explanation for the selective actions of estrogens in different target tissues (5). In fact, the high degree of interspecies conservation of the individual ER subtypes throughout vertebrate evolution (Ref. 6 and our unpublished observations) could suggest that the basis for the selective effects of estrogens resides in the control of different subsets of estrogen-responsive promoters by Received September 27, 1996. Address all correspondence and requests for reprints to: George Kuiper, Center for Biotechnology, Karolinska Institute, NOVUM, S-14186 Huddinge, Sweden. E-mail: [email protected]. * Supported in part by grants from The Netherlands Organization for Scientific Research (NWO) and from the Karolinska Institute. † Supported by grants from the Swedish Cancer Society and from the European Union (EU-PL95-1223) Climate and Environment Program. 0013-7227/97/$03.00/0 Vol. 138, No. 3 Endocrinology Printed in U.S.A. Copyright © 1997 by The Endocrine Society