RAP80, a novel nuclear protein that interacts with the retinoid-related testis-associated receptor.
In this study, we describe the characterization of a novel nuclear protein, referred to as RAP80. The RAP80 cDNA was cloned from a human testis cDNA library and encodes a 719-amino acid protein containing two potential CX(2)CX(11)HX(3)C-type zinc finger motifs at its carboxyl-terminal region. Analysis of its genomic structure revealed that the RAP80 gene covers more than 90 kb and consists of 15 exons and 14 introns. Fluorescence in situ hybridization mapped the RAP80 gene to human chromosome 5q35. RAP80 mRNA is expressed in many human tissues, but its expression is particularly high in testis. In situ hybridization showed that RAP80 is highly expressed in germ cells of mouse testis but is not differentially regulated during spermatogenesis. Confocal microscopy showed that RAP80 is localized to the nucleus, where it is distributed in a speckled pattern. Deletion analysis showed that a bipartite nuclear localization signal at the amino terminus is important in mediating nuclear transport of RAP80. Monohybrid analysis showed that RAP80 might function as an active repressor of transcription. Mammalian two-hybrid analysis demonstrated that RAP80 was able to interact with the retinoid-related testis-associated receptor (RTR), an orphan receptor that has been implicated in the control of embryonic development and spermatogenesis. Pull-down analysis showed that RAP80 and RTR physically interact in vitro. Deletion and point mutation analyses revealed that part of the hinge domain of RTR is required for this interaction. RAP80 is able to inhibit the interaction of RTR with the co-repressor N-CoR likely by competing with N-CoR for RTR binding. Our results suggest that RAP80 may be functioning as a modulator of RTR signaling.
Yan
Zhijiang
Z
Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Kim
Yong-Sik
YS
Jetten
Anton M
AM
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D006224:Cricetinae; D004268:DNA-Binding Proteins; D015723:Gene Library; D017930:Genes, Reporter; D056488:Histone Chaperones; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D009928:Organ Specificity; D010957:Plasmids; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D017384:Sequence Deletion; D013737:Testis / Q000378:metabolism; D014162:Transfection; D016335:Zinc Fingers
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; C539950:NR6A1 protein, human; C539954:Nr6a1 protein, mouse; D009687:Nuclear Proteins; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; C466234:UIMC1 protein, human; D015500:Chloramphenicol O-Acetyltransferase