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J. Biol. Chem., Vol. 277, Issue 35, 32379-32388, August 30, 2002
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From the Cell Biology Section, Division of Intramural Research,
NIEHS, National Institutes of Health, Research Triangle Park, North
Carolina 27709
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
CX2CX11HX3C-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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF349313.
To whom correspondence should be addressed: Cell Biology Section,
Division of Intramural Research, NIEHS, National Institutes of Health,
111 T. W. Alexander Dr., Research Triangle Park, NC 27709. Tel.:
919-541-2768; Fax: 919-541-4133; E-mail: jetten@niehs.nih.gov.
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