Summary of Work: The human estrogen receptor (ER)-related orphan receptor, hERR alpha1, is an orphan member of the steroid/thyroid hormone receptor superfamily. We previously cloned the hERR alpha1 cDNA and demonstrated that it enhances ER-mediated responsiveness of the lactoferrin gene promoter in transiently transfected human endometrial carcinoma RL95-2 cells. Since the ER and hERR alpha1 binding element in the human lactoferrin gene promoter are 23 bp apart, we suggested that a physical interaction between these two receptors could stabilize ER binding to the imperfect ERE of the human lactoferrin gene promoter. To extend this study, we utilized a mammalian two hybrid system in human endometrial carcinoma cells (HEC). Using this system, we demonstrate that hERR alpha1 and ER interact with each other in a ligand-independent manner. By GST "pull-down" analysis, we show that 35S-labeled ER can be pulled-down by GST- hERR alpha1 protein in vitro. In addition, immunoprecipitation and immunoblot studies, confirm that a protein-protein interaction between these two receptors occurs. Together these results demonstrate that hERR alpha1 and ER are capable of interacting with each other and modulating each others functions. Whether heterodimerization occurs between these two receptors is yet to be established; however, the in vivo study suggests that hERR alpha1 has the potential to form homodimers and to bind to ERE. When HEC or CV1 cells were transfected with a VP16-hERR alpha1 chimera, it activates both human lactoferrin gene COUP/ ERE-CAT and 3X-vitERE-Luc reporter constructs, suggesting that hERR alpha1 can bind to the ERE and activate the reporter. In another series of experiments, we cotransfected a Ga14-BD-hERR alpha1 construct and a UAS-TATA-CAT reporter into HEC cells. An elevated CAT activity was observed. When both Ga14-BD-hERR alpha1 and Gal-AD-hERR alpha1 were cotransfected with Ga14 reporters, dramatic activation of the reporter-CAT was found. The current studies present several potential roles for hERR alpha1 in estrogen responsiveness. It could modulate ER-mediated activity positively by interacting with ER or negatively by competition of ER binding to the ERE. Because of the transactivating ability, hERR alpha1 could function as an independent receptor with unknown ligands or in cross-talk with other signaling pathways.