LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1.
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, ERalpha (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 RORalpha (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.
Suzuki
Taiga
T
Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
Kasahara
Megumi
M
Yoshioka
Hidefumi
H
Morohashi
Ken-Ichirou
K
Umesono
Kazuhiko
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D047628:Estrogen Receptor alpha; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D051379:Mice; D008969:Molecular Sequence Data; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D054339:Steroidogenic Factor 1; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; C098925:Esrrb protein, mouse; D047628:Estrogen Receptor alpha; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; C539966:Nr0b1 protein, mouse; C431936:Nr5a2 protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D012097:Repressor Proteins; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D013912:Threonine; D012694:Serine