The transcriptional activity of the estrogen receptor (ER) is known to be highly modulated by the character and amount of coregulator proteins present in the cells. TR2 orphan receptor (TR2), a member of the nuclear receptor superfamily without identified ligands, is found to be expressed in the breast cancer cell lines and to function as a repressor to suppress ER-mediated transcriptional activity. Utilizing an interaction blocker, ER-6 (amino acids 312-340), responsible for TR2 interaction, the suppression of ER by TR2 could be reversed, suggesting that this suppression is conferred by the direct protein-protein interaction. Administration of antisense TR2, resulting in an enhancement of ER transcriptional activity in MCF7 cells, indicates that endogenous TR2 normally suppresses ER-mediated signaling. To gain insights into the molecular mechanism by which TR2 suppresses ER, we found that TR2 could interrupt ER DNA binding via formation of an ER-TR2 heterodimer that disrupted the ER homodimerization. The suppression of ER transcription by TR2 consequently caused the inhibition of estrogen-induced cell growth and G 1 /S transition in estrogen-dependent breast cancer cells. Taken together in addition to the potential roles in spermatogenesis and neurogenesis, our data provide a novel biological function of TR2 that may exert an important repressor in regulating ER activity in mammary glands.The human TR2 orphan receptor (TR2), 1 a member of the nuclear hormone receptor superfamily, was cloned from human testis and prostate cDNA libraries and has no identified ligand(s) (1, 2). TR2 is mapped to locate on chromosome 12q22 (3), which is known to be frequently deleted in various tumors, including testicular and ovarian germ cell tumors (4, 5). Four RNA isoforms, TR2-5, -7, -9, and -11, have been identified. Although TR2-11 encodes the full-length receptor, TR2-5, -7, and -9 encode truncated receptors with distinct deletions in the ligand binding domains (LBD) (1). TR2 has high homology with TR4, which places them in a unique subfamily within the nuclear hormone receptor superfamily (6). TR2 is evolutionarily conserved among species from primitive creatures to mammalians, including sea urchin, rainbow trout, axolotl, Xenopus, Drosophila, mouse, and human (1, 2, 7-11). The facts that TR2 is broadly expressed in many tissues throughout development starting at as early as mid-gestation stage (12-15) and that Drosophila with null mutations of DHR78 nuclear receptor, a homolog of human TR2, is lethal at the third-instar larval stage with severe defects in ecdysteroid-triggered metamorphosis (16), imply that the biological importance of TR2 is involved in the development process. With prominent expression throughout the active proliferating zones of the neural areas, the sensory nerve-targeted organs, and the testes during development, TR2 has been proposed to play an important role in the early development of the nervous system and the male reproductive system (12-15). Also, it has been shown that TR2 is primarily expresse...