LXXLL-Related Motifs in Dax-1 Have Target Specificity for the Orphan Nuclear Receptors Ad4BP/SF-1 and LRH-1

Abstract

The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2). The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ERα (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL-related motifs in Dax-1 whereas HNF4 (NR2A1) and RORα (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(+1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions −2, +2, +3, and +6, respectively. Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors.

ACKNOWLEDGMENTS

We are most grateful to Ronald M. Evans, Vincent Giguère, Masato Kinoshita, G. Stanley McKnight, Ohtsura Niwa, Yoshinori Ohsumi, Keith L. Parker, Frances M. Sladek, Hitoshi Ueda, and Ruth T. Yu for providing materials and/or valuable advice; to Kaname Kawajiri, Hirofumi Mizusaki, Hidesato Ogawa, Shiho Ogawa, and Sanae Oka for constructing plasmids and/or for helpful discussions; to Ryoichiro Kageyama and Shuh Narumiya for generous support; and to the NIBB Center for Analytical Instruments for assistance in operating the laser-excited gel scanner.

This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology; Japan Science and Technology Corporation; Japan Society for Promotion of Science; and Human Frontiers Science Program.