RAP80, a Novel Nuclear Protein That Interacts with the Retinoid-related Testis-associated Receptor
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m203475200
ISSN1083-351X
AutoresZhijiang Yan, Yong Sik Kim, Anton M. Jetten,
Tópico(s)Ubiquitin and proteasome pathways
ResumoIn 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. Fluorescencein 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 interactin 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. 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. Fluorescencein 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 interactin 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. DNA binding domain retinoid-related testis-associated receptor receptor associated protein 80 activation domain fluorescence in situ hybridization enhanced green fluorescent protein upstream activating sequence nuclear co-repressor glutathione sulfotransferase chloramphenicol acetyl transferase nuclear localization signal Chinese hamster ovary high throughput human genomic sequences ligand-binding domain The nuclear receptor superfamily is composed of a large number of ligand-dependent transcription factors that include nuclear orphan receptors for which a ligand has not yet been identified (1Kumar R. Thompson E.B. Steroids. 1999; 64: 310-319Crossref PubMed Scopus (310) Google Scholar, 2Laudet V. J. Mol. Endocrinol. 1997; 19: 207-226Crossref PubMed Scopus (420) Google Scholar, 3McKenna N.J., Xu, J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 1999; 69: 3-12Crossref PubMed Scopus (362) Google Scholar, 4Willy P.J. Mangelsdorf D.J. O'Malley B.W. Hormones and Signaling. 1. Academic Press, San Diego1998: 308-358Google Scholar). Nuclear receptors share a common domain structure that includes an amino-terminal domain, a DNA-binding domain (DBD),1 hinge domain, and a ligand-binding domain (LBD). These domains are involved in the recognition of specific DNA response elements, receptor dimerization, nuclear localization, and ligand binding and contain repressor and transactivation functions. Repression and activation of transcription by nuclear receptors are mediated through interactions with co-repressor and co-activators, respectively. Nuclear receptors have been demonstrated to regulate many physiological processes, including embryonic development and cell growth and differentiation and have been implicated in a number of human diseases (5de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1192) Google Scholar, 6Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2707) Google Scholar, 7Dickson R.B. Stancel G.M. J. Natl. Cancer Inst. Monogr. 2000; 27: 135-145Crossref PubMed Scopus (156) Google Scholar, 8Jetten A.M. Kurebayashi S. Ueda E. Prog. Nucleic Acids Res. 2001; 69: 205-247Crossref PubMed Google Scholar). Less, however, is known about the role of many nuclear orphan receptors, including the retinoid-related, testis-associated receptor (RTR), also referred to as germ cell nuclear factor (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar).The orphan receptor RTR (named NR6A1 by the Nuclear Receptor Nomenclature Committee) has been cloned from several species, including mouse (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar), human (11Lei W. Hirose T. Zhang L.X. Adachi H. Spinella M.J. Dmitrovsky E. Jetten A.M. J. Mol. Endocrinol. 1997; 18: 167-176Crossref PubMed Scopus (33) Google Scholar, 12Kapelle M. Kratzschmar J. Husemann M. Schleuning W.D. Biochim. Biophys. Acta. 1997; 1352: 13-17Crossref PubMed Scopus (25) Google Scholar, 13Susens U. Borgmeyer U. Biochim. Biophys. Acta. 1996; 1309: 179-182Crossref PubMed Scopus (30) Google Scholar, 14Agoulnik I.Y. Cho Y. Niederberger C. Kieback D.G. Cooney A.J. FEBS Lett. 1998; 424: 73-78Crossref PubMed Scopus (36) Google Scholar), zebrafish (15Braat A.K. Zandbergen M.A., De Vries E. Van Der Burg B. Bogerd J. Goos H.J. Mol. Reprod. Dev. 1999; 53: 369-375Crossref PubMed Scopus (25) Google Scholar), and Xenopus laevis (16Joos T.O. David R. Dreyer C. Mech. Dev. 1996; 60: 45-57Crossref PubMed Scopus (42) Google Scholar). During embryonic development RTR is expressed in early embryonic stem cells, trophoblasts, and neuronal precursor cells while in the adult RTR expression is largely limited to testis and ovary (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 11Lei W. Hirose T. Zhang L.X. Adachi H. Spinella M.J. Dmitrovsky E. Jetten A.M. J. Mol. Endocrinol. 1997; 18: 167-176Crossref PubMed Scopus (33) Google Scholar, 17Susens U. Aguiluz J.B. Evans R.M. Borgmeyer U. Dev. Neurosci. 1997; 19: 410-420Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar, 19Mehta D.V. Kim Y.-S. Dixon D. Jetten A.M. Placenta. 2002; 23: 281-287Crossref PubMed Scopus (14) Google Scholar). In the testis, RTR is most abundant in round spermatids suggesting that RTR controls gene transcription during a specific stage of spermatogenesis (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar, 17Susens U. Aguiluz J.B. Evans R.M. Borgmeyer U. Dev. Neurosci. 1997; 19: 410-420Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar). Several studies have indicated that RTR is also important during embryonic development. Targeted disruption of the RTR gene is embryonic lethal in mice (20Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). Embryos exhibit open neural tubes and an absence of posterior structures and do not survive beyond 10.5 days post-coitus probably due to cardiovascular failure. These studies suggest that RTR is essential for the postgastrulation and neurulation stages of mouse development. A recent study identified a role for RTR in the repression of the POU-domain transcription factor Oct4 (21Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Overexpression of a dominant-negative RTR mutant in Xenopus showed that RTR has an essential function in anteroposterior differentiation during organogenesis (22David R. Joos T.O. Dreyer C. Mech. Dev. 1998; 79: 137-152Crossref PubMed Scopus (33) Google Scholar).RTR has been demonstrated to bind preferentially as a homodimer to DNA response elements consisting of the consensus sequence [AGGTCA]2 (23Yan Z.H. Medvedev A. Hirose T. Gotoh H. Jetten A.M. J. Biol. Chem. 1997; 272: 10565-10572Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 24Borgmeyer U. Eur. J. Biochem. 1997; 244: 120-127Crossref PubMed Scopus (32) Google Scholar, 25Cooney A.J. Hummelke G.C. Herman T. Chen F. Jackson K.J. Biochem. Biophys. Res. Commun. 1998; 245: 94-100Crossref PubMed Scopus (46) Google Scholar, 26Greschik H. Schule R. J. Mol. Med. 1998; 76: 800-810Crossref PubMed Scopus (27) Google Scholar). However, little is still known about the transcriptional activity of RTR. In the absence of a putative ligand, RTR has been shown to function as a transcriptional repressor (27Hummelke G.C. Cooney A.J. Front. Biosci. 2001; 6: D1186-D1191Crossref PubMed Google Scholar, 28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This repression is mediated at least in part through an interaction of RTR with the co-repressor N-CoR.In this report, we describe the cloning and sequence of the full-length coding region of a novel gene referred to as receptor-associated protein 80 (RAP80). RAP80 encodes a 79.6-kDa nuclear protein. The RAP80 protein contains two Cys-X2-Cys-X11-His-X3-Cys zinc fingers near the carboxyl terminus. RAP80 mRNA is expressed in many tissues but is most abundant in testis. In situhybridization localized RAP80 mRNA expression to the germ cells. The genomic structure of the RAP80 gene encompasses more than 90 kb and consists of 15 exons. The RAP80 gene was mapped to human chromosome 5q35. Confocal microscopy showed that RAP80 is localized largely to the nucleus. Using mammalian mono- and two-hybrid analysis, we characterized the transcriptional activity of RAP80 and its interaction with RTR. The results indicated that RAP80 is able to repress basal transcriptional activation indicating that it may function as a (co)-repressor for RTR. Deletion and point mutation analysis demonstrated that the hinge domain of RTR is important for this interaction. Previously, we reported that the hinge domain, in addition to helices 3–5 and 12, is also required for the interaction of RTR with the co-repressor N-CoR (28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We demonstrate that RAP80 is able to inhibit the interaction of the co-repressor N-CoR with RTR likely by competing with N-CoR for binding. These results suggest that RAP80 may play a role in modulating the transcriptional and biological activity of RTR.DISCUSSIONIn this study, we describe the cloning of a cDNA encoding the full-length human RAP80 protein. Examination of the RAP80 protein sequence revealed several distinctive features, two putative zinc finger motifs, three nuclear localization signals, a Glu-rich region, a PEST sequence, and a potential ubiquitin-interaction motif between Thr80 and Glu98 (37Hofmann K. Falquet L. Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The two putative zinc finger motifs at the carboxyl terminus of RAP80 exhibit the consensus sequence Cys-X2-Cys-X11-His-X3-Cys. The sequence separating the zinc finger motifs does not exhibit any homology with the consensus motif (T/S)GEKP (Y/F)X, typically found as interfinger spacer in members of the Krüppel-like zinc finger proteins (38Schuh R. Aicher W. Gaul U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Schroder C. Kemler R. Cell. 1986; 47: 1025-1032Abstract Full Text PDF PubMed Scopus (324) Google Scholar) suggesting that RAP80 does not belong to the Krüppel-like zinc finger protein superfamily. The class CCHC-type zinc finger motifs found in RAP80 has been described in only a limited number of proteins. Members of the NZF and FOG transcription factor families contain zinc finger motifs with the consensus Cys-X2-Cys-X4-His-X1–5-Cys (39Berkovits H.J. Berg J.M. Biochemistry. 1999; 38: 16826-16830Crossref PubMed Scopus (43) Google Scholar, 40Jiang Y., Yu, V.C. Buchholz F. O'Connell S. Rhodes S.J. Candeloro C. Xia Y.R. Lusis A.J. Rosenfeld M.G. J. Biol. Chem. 1996; 271: 10723-10730Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 41Matthews J.M. Kowalski K. Liew C.K. Sharpe B.K. Fox A.H. Crossley M. MacKay J.P. Eur. J. Biochem. 2000; 267: 1030-1038Crossref PubMed Scopus (53) Google Scholar). A different class of Cys-X2-Cys-X4-His-X4-Cys motifs, referred to as "zinc knuckle" fingers, are contained in DNA-binding proteins from several retroviruses (42Ramboarina S. Morellet N. Fournie-Zaluski M.C. Roques B.P. Moreller N. Biochemistry. 1999; 38: 9600-9607Crossref PubMed Scopus (40) Google Scholar). RAP80 appears to be unique and unrelated to these nuclear proteins. Zinc finger motifs can exhibit multiple functions, including a role in DNA recognition, protein-protein interactions, and nuclear localization. We are in the process of examining whether RAP80 can recognize a specific DNA sequence and whether it functions as a DNA-binding protein.Confocal microscopic analysis localized RAP80 to the nucleus, suggesting that RAP80 functions as a nuclear protein. RAP80 is distributed in a speckled pattern, suggesting that it is part of a multiprotein complex. RAP80 contains three putative nuclear localization signals: at Arg3, Arg715, and a bipartite nuclear localization signal between Lys19 and Arg35 (36Boulikas T. J. Cell. Biochem. 1994; 55: 32-58Crossref PubMed Scopus (174) Google Scholar). Deletion analysis indicated that the bipartite nuclear localization signal is important in mediating nuclear transport of RAP80.The nuclear localization and the presence of two zinc finger-like motifs suggested that RAP80 may function as a transcription factor. We therefore investigated whether it exhibited any transcriptional activity. Monohybrid analysis using different cell types showed that in several cell lines RAP80 was unable to enhance transcription of a reporter gene, suggesting that it did not function as a transcriptional activator under these conditions. Instead, Gal4(DBD)-RAP80 inhibited basal transcription, indicating that it may function as an active repressor. Deletion mutant analysis indicated that the repressor function is associated with the amino terminus of RAP80. This region has no similarity to any other known repressor domain.RAP80 exhibits a 74% identity with a partial protein sequence referred to as RIP110 encoded by one of several sequences identified by yeast two-hybrid screening using the ligand binding domain of the RXRα receptor as bait (33Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). RIP110 appears to represent the carboxyl terminus of the mouse homologue of RAP80 and has not been studied further. Because of the observed interaction of RIP110 with RXR in yeast two-hybrid analysis, we investigated the interaction of RAP80 with a number of nuclear receptors in mammalian two-hybrid analysis. This analysis included the retinoid receptors RXRα and RARα, the estrogen receptor α, the thyroid hormone receptor α, and the orphan receptors RTR and TAK1 and was carried out in either the presence or absence of their respective ligand (not shown). These analyses revealed an interaction only between RAP80 and RTR (Fig. 7). Deletion and point mutation analysis identified a region within the hinge domain of RTR that is essential for its interaction with RAP80 while the ligand binding domain of RTR was not required. Several regions within RAP80 were important for optimal interaction with RTR. These observations suggest a function for RAP80 in modulating RTR activity. RAP80 may have multiple roles in RTR signaling. It may function as a co-repressor, as a mediator to recruit other proteins to RTR or target RTR to a particular complex or compartment such as nuclear matrix or proteasome. The fact that RAP80 inhibits basal transcriptional activity may suggest that it functions as a co-repressor.Previous studies have demonstrated that RTR can function as an active repressor of transcription and is involved in the repression of transcription of the POU-domain transcriptional factor Oct4 (21Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Repression by nuclear receptors is mediated through interaction with multiprotein co-repressor complexes that contain histone deacetylase activity and affect chromatin structure. The protein N-CoR has been reported to physically interact with RTR and to function as a co-repressor for RTR. This interaction was shown to require the ligand binding domain as well as the hinge domain of RTR for optimal binding (28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because both RAP80 and N-CoR interact with RTR and require the hinge region of RTR for binding, one might predict that the interaction of RAP80 and N-CoR to RTR interfere with each other. As shown in Fig. 11, RAP80 inhibits the interaction of N-CoR with RTR likely by competing with each other for RTR binding. The hinge region is required for the interaction of N-CoR with several nuclear receptors and appears to function as a structural determinant rather than serving as a direct interface in N-CoR binding (43Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 44Nagy L. Kao H.Y. Love J.D., Li, C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (344) Google Scholar). These observations seem to indicate that RAP80 interferes with the binding of N-CoR to RTR through a mechanism of steric hindrance rather than competing for the same binding interface.RAP80 mRNA was found in many different human tissues but was most abundantly expressed in testis. In situ hybridization of sections from mouse testis indicated that RAP80 is expressed most highly in germ cells. Because no significant differences in the level or pattern of hybridization were observed between seminiferous tubules at different stages of spermatogenesis, expression of RAP80 mRNA appears not to be differentially regulated during spermatogenesis. This was supported by Northern blot analysis using RNA from different germ cell populations (not shown). Previous studies have demonstrated that RTR is differentially regulated during spermatogenesis and most highly expressed in spermatids (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar). Because RTR and RAP80 are co-expressed in spermatids and in embryonal carcinoma cells and trophoblasts (not shown) (19Mehta D.V. Kim Y.-S. Dixon D. Jetten A.M. Placenta. 2002; 23: 281-287Crossref PubMed Scopus (14) Google Scholar), RAP80 may change RTR function and signaling in these cells. What the physiological significance is of the interaction between RAP80 and RTR awaits further analysis using functional assays. Because RAP80 mRNA is also found in tissues where RTR is not expressed suggests that RAP80 has additional functions and likely interacts with other (nuclear) proteins.In summary, in this study we identify a novel nuclear protein, referred to as RAP80. This protein localizes to the nucleus and is able to repress basal transcription. We demonstrate that this protein interacts physically with the nuclear orphan receptor RTR. Deletion and point mutation analysis identified a region within the hinge domain of RTR that is essential for this interaction. This region is also required for the interaction of the co-repressor N-CoR. RAP80 and N-CoR appear to compete with each other for binding to RTR possibly through steric hindrance. Our results suggest that RAP80 may play a role in modulating the transcriptional and biological activity of RTR possibly by functioning as a co-repressor. The nuclear receptor superfamily is composed of a large number of ligand-dependent transcription factors that include nuclear orphan receptors for which a ligand has not yet been identified (1Kumar R. Thompson E.B. Steroids. 1999; 64: 310-319Crossref PubMed Scopus (310) Google Scholar, 2Laudet V. J. Mol. Endocrinol. 1997; 19: 207-226Crossref PubMed Scopus (420) Google Scholar, 3McKenna N.J., Xu, J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 1999; 69: 3-12Crossref PubMed Scopus (362) Google Scholar, 4Willy P.J. Mangelsdorf D.J. O'Malley B.W. Hormones and Signaling. 1. Academic Press, San Diego1998: 308-358Google Scholar). Nuclear receptors share a common domain structure that includes an amino-terminal domain, a DNA-binding domain (DBD),1 hinge domain, and a ligand-binding domain (LBD). These domains are involved in the recognition of specific DNA response elements, receptor dimerization, nuclear localization, and ligand binding and contain repressor and transactivation functions. Repression and activation of transcription by nuclear receptors are mediated through interactions with co-repressor and co-activators, respectively. Nuclear receptors have been demonstrated to regulate many physiological processes, including embryonic development and cell growth and differentiation and have been implicated in a number of human diseases (5de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1192) Google Scholar, 6Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2707) Google Scholar, 7Dickson R.B. Stancel G.M. J. Natl. Cancer Inst. Monogr. 2000; 27: 135-145Crossref PubMed Scopus (156) Google Scholar, 8Jetten A.M. Kurebayashi S. Ueda E. Prog. Nucleic Acids Res. 2001; 69: 205-247Crossref PubMed Google Scholar). Less, however, is known about the role of many nuclear orphan receptors, including the retinoid-related, testis-associated receptor (RTR), also referred to as germ cell nuclear factor (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar). The orphan receptor RTR (named NR6A1 by the Nuclear Receptor Nomenclature Committee) has been cloned from several species, including mouse (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar), human (11Lei W. Hirose T. Zhang L.X. Adachi H. Spinella M.J. Dmitrovsky E. Jetten A.M. J. Mol. Endocrinol. 1997; 18: 167-176Crossref PubMed Scopus (33) Google Scholar, 12Kapelle M. Kratzschmar J. Husemann M. Schleuning W.D. Biochim. Biophys. Acta. 1997; 1352: 13-17Crossref PubMed Scopus (25) Google Scholar, 13Susens U. Borgmeyer U. Biochim. Biophys. Acta. 1996; 1309: 179-182Crossref PubMed Scopus (30) Google Scholar, 14Agoulnik I.Y. Cho Y. Niederberger C. Kieback D.G. Cooney A.J. FEBS Lett. 1998; 424: 73-78Crossref PubMed Scopus (36) Google Scholar), zebrafish (15Braat A.K. Zandbergen M.A., De Vries E. Van Der Burg B. Bogerd J. Goos H.J. Mol. Reprod. Dev. 1999; 53: 369-375Crossref PubMed Scopus (25) Google Scholar), and Xenopus laevis (16Joos T.O. David R. Dreyer C. Mech. Dev. 1996; 60: 45-57Crossref PubMed Scopus (42) Google Scholar). During embryonic development RTR is expressed in early embryonic stem cells, trophoblasts, and neuronal precursor cells while in the adult RTR expression is largely limited to testis and ovary (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 11Lei W. Hirose T. Zhang L.X. Adachi H. Spinella M.J. Dmitrovsky E. Jetten A.M. J. Mol. Endocrinol. 1997; 18: 167-176Crossref PubMed Scopus (33) Google Scholar, 17Susens U. Aguiluz J.B. Evans R.M. Borgmeyer U. Dev. Neurosci. 1997; 19: 410-420Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar, 19Mehta D.V. Kim Y.-S. Dixon D. Jetten A.M. Placenta. 2002; 23: 281-287Crossref PubMed Scopus (14) Google Scholar). In the testis, RTR is most abundant in round spermatids suggesting that RTR controls gene transcription during a specific stage of spermatogenesis (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar, 17Susens U. Aguiluz J.B. Evans R.M. Borgmeyer U. Dev. Neurosci. 1997; 19: 410-420Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar). Several studies have indicated that RTR is also important during embryonic development. Targeted disruption of the RTR gene is embryonic lethal in mice (20Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). Embryos exhibit open neural tubes and an absence of posterior structures and do not survive beyond 10.5 days post-coitus probably due to cardiovascular failure. These studies suggest that RTR is essential for the postgastrulation and neurulation stages of mouse development. A recent study identified a role for RTR in the repression of the POU-domain transcription factor Oct4 (21Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Overexpression of a dominant-negative RTR mutant in Xenopus showed that RTR has an essential function in anteroposterior differentiation during organogenesis (22David R. Joos T.O. Dreyer C. Mech. Dev. 1998; 79: 137-152Crossref PubMed Scopus (33) Google Scholar). RTR has been demonstrated to bind preferentially as a homodimer to DNA response elements consisting of the consensus sequence [AGGTCA]2 (23Yan Z.H. Medvedev A. Hirose T. Gotoh H. Jetten A.M. J. Biol. Chem. 1997; 272: 10565-10572Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 24Borgmeyer U. Eur. J. Biochem. 1997; 244: 120-127Crossref PubMed Scopus (32) Google Scholar, 25Cooney A.J. Hummelke G.C. Herman T. Chen F. Jackson K.J. Biochem. Biophys. Res. Commun. 1998; 245: 94-100Crossref PubMed Scopus (46) Google Scholar, 26Greschik H. Schule R. J. Mol. Med. 1998; 76: 800-810Crossref PubMed Scopus (27) Google Scholar). However, little is still known about the transcriptional activity of RTR. In the absence of a putative ligand, RTR has been shown to function as a transcriptional repressor (27Hummelke G.C. Cooney A.J. Front. Biosci. 2001; 6: D1186-D1191Crossref PubMed Google Scholar, 28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This repression is mediated at least in part through an interaction of RTR with the co-repressor N-CoR. In this report, we describe the cloning and sequence of the full-length coding region of a novel gene referred to as receptor-associated protein 80 (RAP80). RAP80 encodes a 79.6-kDa nuclear protein. The RAP80 protein contains two Cys-X2-Cys-X11-His-X3-Cys zinc fingers near the carboxyl terminus. RAP80 mRNA is expressed in many tissues but is most abundant in testis. In situhybridization localized RAP80 mRNA expression to the germ cells. The genomic structure of the RAP80 gene encompasses more than 90 kb and consists of 15 exons. The RAP80 gene was mapped to human chromosome 5q35. Confocal microscopy showed that RAP80 is localized largely to the nucleus. Using mammalian mono- and two-hybrid analysis, we characterized the transcriptional activity of RAP80 and its interaction with RTR. The results indicated that RAP80 is able to repress basal transcriptional activation indicating that it may function as a (co)-repressor for RTR. Deletion and point mutation analysis demonstrated that the hinge domain of RTR is important for this interaction. Previously, we reported that the hinge domain, in addition to helices 3–5 and 12, is also required for the interaction of RTR with the co-repressor N-CoR (28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We demonstrate that RAP80 is able to inhibit the interaction of the co-repressor N-CoR with RTR likely by competing with N-CoR for binding. These results suggest that RAP80 may play a role in modulating the transcriptional and biological activity of RTR. DISCUSSIONIn this study, we describe the cloning of a cDNA encoding the full-length human RAP80 protein. Examination of the RAP80 protein sequence revealed several distinctive features, two putative zinc finger motifs, three nuclear localization signals, a Glu-rich region, a PEST sequence, and a potential ubiquitin-interaction motif between Thr80 and Glu98 (37Hofmann K. Falquet L. Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The two putative zinc finger motifs at the carboxyl terminus of RAP80 exhibit the consensus sequence Cys-X2-Cys-X11-His-X3-Cys. The sequence separating the zinc finger motifs does not exhibit any homology with the consensus motif (T/S)GEKP (Y/F)X, typically found as interfinger spacer in members of the Krüppel-like zinc finger proteins (38Schuh R. Aicher W. Gaul U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Schroder C. Kemler R. Cell. 1986; 47: 1025-1032Abstract Full Text PDF PubMed Scopus (324) Google Scholar) suggesting that RAP80 does not belong to the Krüppel-like zinc finger protein superfamily. The class CCHC-type zinc finger motifs found in RAP80 has been described in only a limited number of proteins. Members of the NZF and FOG transcription factor families contain zinc finger motifs with the consensus Cys-X2-Cys-X4-His-X1–5-Cys (39Berkovits H.J. Berg J.M. Biochemistry. 1999; 38: 16826-16830Crossref PubMed Scopus (43) Google Scholar, 40Jiang Y., Yu, V.C. Buchholz F. O'Connell S. Rhodes S.J. Candeloro C. Xia Y.R. Lusis A.J. Rosenfeld M.G. J. Biol. Chem. 1996; 271: 10723-10730Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 41Matthews J.M. Kowalski K. Liew C.K. Sharpe B.K. Fox A.H. Crossley M. MacKay J.P. Eur. J. Biochem. 2000; 267: 1030-1038Crossref PubMed Scopus (53) Google Scholar). A different class of Cys-X2-Cys-X4-His-X4-Cys motifs, referred to as "zinc knuckle" fingers, are contained in DNA-binding proteins from several retroviruses (42Ramboarina S. Morellet N. Fournie-Zaluski M.C. Roques B.P. Moreller N. Biochemistry. 1999; 38: 9600-9607Crossref PubMed Scopus (40) Google Scholar). RAP80 appears to be unique and unrelated to these nuclear proteins. Zinc finger motifs can exhibit multiple functions, including a role in DNA recognition, protein-protein interactions, and nuclear localization. We are in the process of examining whether RAP80 can recognize a specific DNA sequence and whether it functions as a DNA-binding protein.Confocal microscopic analysis localized RAP80 to the nucleus, suggesting that RAP80 functions as a nuclear protein. RAP80 is distributed in a speckled pattern, suggesting that it is part of a multiprotein complex. RAP80 contains three putative nuclear localization signals: at Arg3, Arg715, and a bipartite nuclear localization signal between Lys19 and Arg35 (36Boulikas T. J. Cell. Biochem. 1994; 55: 32-58Crossref PubMed Scopus (174) Google Scholar). Deletion analysis indicated that the bipartite nuclear localization signal is important in mediating nuclear transport of RAP80.The nuclear localization and the presence of two zinc finger-like motifs suggested that RAP80 may function as a transcription factor. We therefore investigated whether it exhibited any transcriptional activity. Monohybrid analysis using different cell types showed that in several cell lines RAP80 was unable to enhance transcription of a reporter gene, suggesting that it did not function as a transcriptional activator under these conditions. Instead, Gal4(DBD)-RAP80 inhibited basal transcription, indicating that it may function as an active repressor. Deletion mutant analysis indicated that the repressor function is associated with the amino terminus of RAP80. This region has no similarity to any other known repressor domain.RAP80 exhibits a 74% identity with a partial protein sequence referred to as RIP110 encoded by one of several sequences identified by yeast two-hybrid screening using the ligand binding domain of the RXRα receptor as bait (33Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). RIP110 appears to represent the carboxyl terminus of the mouse homologue of RAP80 and has not been studied further. Because of the observed interaction of RIP110 with RXR in yeast two-hybrid analysis, we investigated the interaction of RAP80 with a number of nuclear receptors in mammalian two-hybrid analysis. This analysis included the retinoid receptors RXRα and RARα, the estrogen receptor α, the thyroid hormone receptor α, and the orphan receptors RTR and TAK1 and was carried out in either the presence or absence of their respective ligand (not shown). These analyses revealed an interaction only between RAP80 and RTR (Fig. 7). Deletion and point mutation analysis identified a region within the hinge domain of RTR that is essential for its interaction with RAP80 while the ligand binding domain of RTR was not required. Several regions within RAP80 were important for optimal interaction with RTR. These observations suggest a function for RAP80 in modulating RTR activity. RAP80 may have multiple roles in RTR signaling. It may function as a co-repressor, as a mediator to recruit other proteins to RTR or target RTR to a particular complex or compartment such as nuclear matrix or proteasome. The fact that RAP80 inhibits basal transcriptional activity may suggest that it functions as a co-repressor.Previous studies have demonstrated that RTR can function as an active repressor of transcription and is involved in the repression of transcription of the POU-domain transcriptional factor Oct4 (21Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Repression by nuclear receptors is mediated through interaction with multiprotein co-repressor complexes that contain histone deacetylase activity and affect chromatin structure. The protein N-CoR has been reported to physically interact with RTR and to function as a co-repressor for RTR. This interaction was shown to require the ligand binding domain as well as the hinge domain of RTR for optimal binding (28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because both RAP80 and N-CoR interact with RTR and require the hinge region of RTR for binding, one might predict that the interaction of RAP80 and N-CoR to RTR interfere with each other. As shown in Fig. 11, RAP80 inhibits the interaction of N-CoR with RTR likely by competing with each other for RTR binding. The hinge region is required for the interaction of N-CoR with several nuclear receptors and appears to function as a structural determinant rather than serving as a direct interface in N-CoR binding (43Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 44Nagy L. Kao H.Y. Love J.D., Li, C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (344) Google Scholar). These observations seem to indicate that RAP80 interferes with the binding of N-CoR to RTR through a mechanism of steric hindrance rather than competing for the same binding interface.RAP80 mRNA was found in many different human tissues but was most abundantly expressed in testis. In situ hybridization of sections from mouse testis indicated that RAP80 is expressed most highly in germ cells. Because no significant differences in the level or pattern of hybridization were observed between seminiferous tubules at different stages of spermatogenesis, expression of RAP80 mRNA appears not to be differentially regulated during spermatogenesis. This was supported by Northern blot analysis using RNA from different germ cell populations (not shown). Previous studies have demonstrated that RTR is differentially regulated during spermatogenesis and most highly expressed in spermatids (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar). Because RTR and RAP80 are co-expressed in spermatids and in embryonal carcinoma cells and trophoblasts (not shown) (19Mehta D.V. Kim Y.-S. Dixon D. Jetten A.M. Placenta. 2002; 23: 281-287Crossref PubMed Scopus (14) Google Scholar), RAP80 may change RTR function and signaling in these cells. What the physiological significance is of the interaction between RAP80 and RTR awaits further analysis using functional assays. Because RAP80 mRNA is also found in tissues where RTR is not expressed suggests that RAP80 has additional functions and likely interacts with other (nuclear) proteins.In summary, in this study we identify a novel nuclear protein, referred to as RAP80. This protein localizes to the nucleus and is able to repress basal transcription. We demonstrate that this protein interacts physically with the nuclear orphan receptor RTR. Deletion and point mutation analysis identified a region within the hinge domain of RTR that is essential for this interaction. This region is also required for the interaction of the co-repressor N-CoR. RAP80 and N-CoR appear to compete with each other for binding to RTR possibly through steric hindrance. Our results suggest that RAP80 may play a role in modulating the transcriptional and biological activity of RTR possibly by functioning as a co-repressor. In this study, we describe the cloning of a cDNA encoding the full-length human RAP80 protein. Examination of the RAP80 protein sequence revealed several distinctive features, two putative zinc finger motifs, three nuclear localization signals, a Glu-rich region, a PEST sequence, and a potential ubiquitin-interaction motif between Thr80 and Glu98 (37Hofmann K. Falquet L. Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The two putative zinc finger motifs at the carboxyl terminus of RAP80 exhibit the consensus sequence Cys-X2-Cys-X11-His-X3-Cys. The sequence separating the zinc finger motifs does not exhibit any homology with the consensus motif (T/S)GEKP (Y/F)X, typically found as interfinger spacer in members of the Krüppel-like zinc finger proteins (38Schuh R. Aicher W. Gaul U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Schroder C. Kemler R. Cell. 1986; 47: 1025-1032Abstract Full Text PDF PubMed Scopus (324) Google Scholar) suggesting that RAP80 does not belong to the Krüppel-like zinc finger protein superfamily. The class CCHC-type zinc finger motifs found in RAP80 has been described in only a limited number of proteins. Members of the NZF and FOG transcription factor families contain zinc finger motifs with the consensus Cys-X2-Cys-X4-His-X1–5-Cys (39Berkovits H.J. Berg J.M. Biochemistry. 1999; 38: 16826-16830Crossref PubMed Scopus (43) Google Scholar, 40Jiang Y., Yu, V.C. Buchholz F. O'Connell S. Rhodes S.J. Candeloro C. Xia Y.R. Lusis A.J. Rosenfeld M.G. J. Biol. Chem. 1996; 271: 10723-10730Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 41Matthews J.M. Kowalski K. Liew C.K. Sharpe B.K. Fox A.H. Crossley M. MacKay J.P. Eur. J. Biochem. 2000; 267: 1030-1038Crossref PubMed Scopus (53) Google Scholar). A different class of Cys-X2-Cys-X4-His-X4-Cys motifs, referred to as "zinc knuckle" fingers, are contained in DNA-binding proteins from several retroviruses (42Ramboarina S. Morellet N. Fournie-Zaluski M.C. Roques B.P. Moreller N. Biochemistry. 1999; 38: 9600-9607Crossref PubMed Scopus (40) Google Scholar). RAP80 appears to be unique and unrelated to these nuclear proteins. Zinc finger motifs can exhibit multiple functions, including a role in DNA recognition, protein-protein interactions, and nuclear localization. We are in the process of examining whether RAP80 can recognize a specific DNA sequence and whether it functions as a DNA-binding protein. Confocal microscopic analysis localized RAP80 to the nucleus, suggesting that RAP80 functions as a nuclear protein. RAP80 is distributed in a speckled pattern, suggesting that it is part of a multiprotein complex. RAP80 contains three putative nuclear localization signals: at Arg3, Arg715, and a bipartite nuclear localization signal between Lys19 and Arg35 (36Boulikas T. J. Cell. Biochem. 1994; 55: 32-58Crossref PubMed Scopus (174) Google Scholar). Deletion analysis indicated that the bipartite nuclear localization signal is important in mediating nuclear transport of RAP80. The nuclear localization and the presence of two zinc finger-like motifs suggested that RAP80 may function as a transcription factor. We therefore investigated whether it exhibited any transcriptional activity. Monohybrid analysis using different cell types showed that in several cell lines RAP80 was unable to enhance transcription of a reporter gene, suggesting that it did not function as a transcriptional activator under these conditions. Instead, Gal4(DBD)-RAP80 inhibited basal transcription, indicating that it may function as an active repressor. Deletion mutant analysis indicated that the repressor function is associated with the amino terminus of RAP80. This region has no similarity to any other known repressor domain. RAP80 exhibits a 74% identity with a partial protein sequence referred to as RIP110 encoded by one of several sequences identified by yeast two-hybrid screening using the ligand binding domain of the RXRα receptor as bait (33Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). RIP110 appears to represent the carboxyl terminus of the mouse homologue of RAP80 and has not been studied further. Because of the observed interaction of RIP110 with RXR in yeast two-hybrid analysis, we investigated the interaction of RAP80 with a number of nuclear receptors in mammalian two-hybrid analysis. This analysis included the retinoid receptors RXRα and RARα, the estrogen receptor α, the thyroid hormone receptor α, and the orphan receptors RTR and TAK1 and was carried out in either the presence or absence of their respective ligand (not shown). These analyses revealed an interaction only between RAP80 and RTR (Fig. 7). Deletion and point mutation analysis identified a region within the hinge domain of RTR that is essential for its interaction with RAP80 while the ligand binding domain of RTR was not required. Several regions within RAP80 were important for optimal interaction with RTR. These observations suggest a function for RAP80 in modulating RTR activity. RAP80 may have multiple roles in RTR signaling. It may function as a co-repressor, as a mediator to recruit other proteins to RTR or target RTR to a particular complex or compartment such as nuclear matrix or proteasome. The fact that RAP80 inhibits basal transcriptional activity may suggest that it functions as a co-repressor. Previous studies have demonstrated that RTR can function as an active repressor of transcription and is involved in the repression of transcription of the POU-domain transcriptional factor Oct4 (21Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Repression by nuclear receptors is mediated through interaction with multiprotein co-repressor complexes that contain histone deacetylase activity and affect chromatin structure. The protein N-CoR has been reported to physically interact with RTR and to function as a co-repressor for RTR. This interaction was shown to require the ligand binding domain as well as the hinge domain of RTR for optimal binding (28Yan Z. Jetten A.M. J. Biol. Chem. 2000; 275: 35077-35085Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because both RAP80 and N-CoR interact with RTR and require the hinge region of RTR for binding, one might predict that the interaction of RAP80 and N-CoR to RTR interfere with each other. As shown in Fig. 11, RAP80 inhibits the interaction of N-CoR with RTR likely by competing with each other for RTR binding. The hinge region is required for the interaction of N-CoR with several nuclear receptors and appears to function as a structural determinant rather than serving as a direct interface in N-CoR binding (43Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 44Nagy L. Kao H.Y. Love J.D., Li, C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (344) Google Scholar). These observations seem to indicate that RAP80 interferes with the binding of N-CoR to RTR through a mechanism of steric hindrance rather than competing for the same binding interface. RAP80 mRNA was found in many different human tissues but was most abundantly expressed in testis. In situ hybridization of sections from mouse testis indicated that RAP80 is expressed most highly in germ cells. Because no significant differences in the level or pattern of hybridization were observed between seminiferous tubules at different stages of spermatogenesis, expression of RAP80 mRNA appears not to be differentially regulated during spermatogenesis. This was supported by Northern blot analysis using RNA from different germ cell populations (not shown). Previous studies have demonstrated that RTR is differentially regulated during spermatogenesis and most highly expressed in spermatids (9Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 10Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar, 18Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar). Because RTR and RAP80 are co-expressed in spermatids and in embryonal carcinoma cells and trophoblasts (not shown) (19Mehta D.V. Kim Y.-S. Dixon D. Jetten A.M. Placenta. 2002; 23: 281-287Crossref PubMed Scopus (14) Google Scholar), RAP80 may change RTR function and signaling in these cells. What the physiological significance is of the interaction between RAP80 and RTR awaits further analysis using functional assays. Because RAP80 mRNA is also found in tissues where RTR is not expressed suggests that RAP80 has additional functions and likely interacts with other (nuclear) proteins. In summary, in this study we identify a novel nuclear protein, referred to as RAP80. This protein localizes to the nucleus and is able to repress basal transcription. We demonstrate that this protein interacts physically with the nuclear orphan receptor RTR. Deletion and point mutation analysis identified a region within the hinge domain of RTR that is essential for this interaction. This region is also required for the interaction of the co-repressor N-CoR. RAP80 and N-CoR appear to compete with each other for binding to RTR possibly through steric hindrance. Our results suggest that RAP80 may play a role in modulating the transcriptional and biological activity of RTR possibly by functioning as a co-repressor.
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