Isolation and Characterization of a Novel Coactivator Protein, NCoA-62, Involved in Vitamin D-mediated Transcription
1998; Elsevier BV; Volume: 273; Issue: 26 Linguagem: Inglês
10.1074/jbc.273.26.16434
ISSN1083-351X
AutoresTroy A. Baudino, Dennis M. Kraichely, Stephen C. Jefcoat, Sandra K. Winchester, Nicola C. Partridge, Paul N. MacDonald,
Tópico(s)Biotin and Related Studies
ResumoThe vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR·RXR heterodimer is not well understood, but it may involve interactions of VDR·RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62,000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies andin vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1,25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways. The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR·RXR heterodimer is not well understood, but it may involve interactions of VDR·RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62,000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies andin vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1,25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways. Biological responsiveness to 1,25-dihydroxyvitamin D3(1,25-(OH)2D3) 1The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; VDRE, vitamin D response elements; SRC-1, steroid receptor coactivator 1; RAC3, receptor-associated coactivator 3; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; GRIP-1, glucocorticoid receptor-interacting protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; bp, base pair(s); WT, wild type; RXR, retinoid X receptor; RAR, retinoic acid receptor. 1The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; VDRE, vitamin D response elements; SRC-1, steroid receptor coactivator 1; RAC3, receptor-associated coactivator 3; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; GRIP-1, glucocorticoid receptor-interacting protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; bp, base pair(s); WT, wild type; RXR, retinoid X receptor; RAR, retinoic acid receptor.is mediated through an intracellular receptor termed the vitamin D receptor (VDR). VDR is a member of the superfamily of nuclear receptors for steroid hormones, and it acts as a ligand-induced transcription factor that binds to specific DNA response elements in the promoter region of vitamin D-responsive genes (1MacDonald P.N. Dowd D.R. Haussler M.R. Semin. Nephrol. 1994; 14: 101-118PubMed Google Scholar, 2Darwish H. DeLuca H.F. Crit. Rev. Eukaryot. Gene Expr. 1993; 3: 89-116PubMed Google Scholar, 3Haussler M.R. Whitfield G.K. Haussler C.A. Hsieh J.-C. Thompson P.D. Selznick S.H. Dominguez C.E. Jurutka P.W. J Bone Miner. Res. 1998; 13: 325-349Crossref PubMed Scopus (1195) Google Scholar). Vitamin D response elements (VDREs) consist of either exact or imperfect direct repeats of the hexonucleotide sequence, GGGTGA, generally separated by a three-nucleotide spacer. High affinity binding of VDR to VDREs requires an additional nuclear factor that is most likely retinoid X receptor (RXR), the nuclear receptor for 9-cis-retinoic acid (4Liao J. Ozono K. Sone T. McDonnell D.P. Pike J.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9751-9755Crossref PubMed Scopus (163) Google Scholar, 5Kliewer S.A. Umesono K. Mangelsdorf D.J. Evans R.M. Nature. 1992; 355: 446-449Crossref PubMed Scopus (1222) Google Scholar, 6MacDonald P.N. Dowd D.R. Nakajima S. Galligan M.A. Reeder M.C. Haussler C.A. Ozato K. Haussler M.R. Mol. Cell. Biol. 1993; 13: 5907-5917Crossref PubMed Scopus (230) Google Scholar). Thus, VDR and RXR heterodimerize to form a complex that binds with high affinity to VDREs, and it is the VDR·RXR heterodimer that may be the functional transcription factor in vitamin D-mediated gene expression. The mechanism that links the heterodimeric receptor complex bound at the DNA response element to the transcriptional complex is not well understood, but it is presumed to involve protein-protein interactions between the heterodimer and other transcriptional coactivator proteins. Recently, a number of putative coactivator and corepressor proteins have been described for several members of the nuclear receptor superfamily (7Horwitz K.B. Jackson T.A. Bain D.L. Richer J.K. Takimoto G.S. Tung L. Mol. Endocrinol. 1996; 10: 1167-1177Crossref PubMed Scopus (829) Google Scholar). A general property of these transcriptional cofactors is their ability to selectively interact with liganded nuclear receptors and modulate their transcriptional activity. Putative coactivators include steroid receptor coactivator 1 (SRC-1) (8Onate S.A. Tsai S.Y. Tsai M.-J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2039) Google Scholar), receptor-interacting protein 140 (9Cavailles V. Dauvois S. L'Horset F. Lopez G. Hoare S. Kushner P.J. Parker M.G. EMBO J. 1995; 14: 3741-3751Crossref PubMed Scopus (670) Google Scholar), receptor-associated coactivator 3 (RAC3) (10Li H. Gomes P.J. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (497) Google Scholar), a novel nuclear receptor coactivator (ACTR) (11Chen H. Lin R.J. Schlitz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar), CREB-binding protein (CBP) (12Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1912) Google Scholar), and glucocorticoid receptor-interacting protein (GRIP-1) (13Hong H. Kohli K. Garabedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (492) Google Scholar), also termed TIF2 (14Voegel J.J. Heine M.J.S. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (943) Google Scholar, 15LeDouarin B. Zechel C. Garnier J.-M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (572) Google Scholar). Although the precise mechanism is unclear, these coactivators are proposed to function as bridging proteins that link the receptor complex to RNA-polymerase II and the basal transcription machinery. Alternatively, the CBP and SRC-1 coactivators possess intrinsic histone acetyltransferase activity and also interact with histone acetyltransferases, suggesting that they function by altering chromatin structure within hormone-responsive promoters to affect the transcriptional response (16Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1049) Google Scholar, 17Jenster G. Spencer T.E. Burcin M.M. Tsai S.Y. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7879-7884Crossref PubMed Scopus (229) Google Scholar, 18Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2353) Google Scholar, 19Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-645Crossref PubMed Scopus (1517) Google Scholar). In the present study, we report the isolation of a cDNA that encodes a 62,000-Da protein that interacts with VDR and several other nuclear receptors. This VDR-interactive protein exhibited striking homology to a Drosophila melanogaster nuclear protein that is implicated in ecdysone-mediated transcription (20Wieland C. Mann S. von Besser H. Saumweber H. Chromosoma. 1992; 101: 517-525Crossref PubMed Scopus (28) Google Scholar). Importantly, expression of this cDNA in mammalian cells strongly augmented vitamin D-, retinoic acid-, estrogen-, and glucocorticoid-activated transcription, but it had little or no effect on basal or gal4-VP16-activated transcription. Based on its interaction with nuclear receptors and its ability to selectively augment nuclear receptor-mediated transcription, this novel protein was designated NCoA-62 (for nuclear receptor coactivator; 62,000 Da). All plasmid constructs used the pAS1 and pGAD-GH yeast expression vectors (21Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar, 22Hannon G.J. Demetrick D. Beach D. Genes Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar). A HeLa cell cDNA library (in pGAD-GH) was cotransformed with pAS1-VDR-(93–427) into the Hf7c strain of yeast as described previously (23MacDonald P.N. Sherman D.R. Dowd D.R. Jefcoat Jr., S.C. DeLisle R.K. J. Biol. Chem. 1995; 270: 4748-4752Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Transformants were plated onto media deficient in leucine, tryptophan, and histidine and containing 25 mm 3amino-1,2,4-triazole (3-AT). Colonies were assayed for β-galactosidase expression using a colony lift filter assay (21Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar). First strand cDNA was synthesized using SuperScript II reverse transcriptase, 1 μg of poly(A)+ RNA obtained from HeLa cells, and a gene-specific primer (GSP1; ATCTTGAACCTTGGAGGC), which is complementary to nucleotides 657–668 in NCoA-62. After first strand cDNA synthesis, the original mRNA template was destroyed with RNase H. Unincorporated dNTPs, GSP1, and proteins were separated from the cDNA using a GlassMAX spin cartridge. An anchor sequence was then added to the 3′-end of the cDNA using TdT and dCTP. PCR amplification was accomplished using Taq DNA polymerase, an anchor primer, and a nested gene-specific primer (GSP2; GGAACCAGGTCAGTGTAT), which is located approximately 360 base pairs into the cDNA. Following amplification, the 5′-rapid amplification of cDNA ends product was cloned into the pCR II vector (Invitrogen, CA). An insert was excised from pCR II with EcoRI andMscI restriction endonucleases. The EcoRI site was from the pCR II vector, and the Msc I site was from the 3′-end of the NCoA-62 PCR product. This fragment was then subcloned into pSG5 (Stratagene, CA) together with NCoA-62 cDNA from theMscI site to the end of the clone in order to generate the full-length NCoA-62 expression vector. The ligand binding domain of human VDR from leucine 116 to serine 427 was inserted into the pGEX-KT expression vector (24Hakes D.J. Dixon J.E. Anal. Biochem. 1992; 202: 293-298Crossref PubMed Scopus (220) Google Scholar). GST-VDR was then expressed in the DH5α strain of E. coli and purified by glutathione-agarose affinity chromatography as described previously (25Seol W. Choi H.-S. Moore D.D. Science. 1996; 272: 1336-1339Crossref PubMed Scopus (437) Google Scholar).35S-Labeled NCoA-62 was generated using the TNT-coupled transcription-translation system as described by the manufacturer (Promega, Madison, WI). GST or GST-VDR was bound to glutathione-agarose (Sigma) and equilibrated with 1× GBB (20 mm Tris, pH 7.6, 50 mm NaCl, 5 mg/ml bovine serum albumin, 1 mmdithiothreitol, 0.2% Nonidet P-40, and protease inhibitors: 0.2 mm phenylmethylsulfonyl fluoride, 4.0 μg/ml aprotinin, 2.0 μg/ml leupeptin, and 1 μg/ml pepstatin A). Equivalent volumes of [35S]methionine-labeled, NCoA-62 proteins were incubated with the immobilized GST fusion proteins in 100 μl of 1× GBB for 1 h at 4 °C. The beads were washed three times with 0.5 ml with 1× GBB and with 1 ml 50 mm Tris (pH 8.0) buffer. Bound proteins were eluted with 10 mm reduced glutathione in 50 mm Tris buffer. Eluted proteins were resolved by SDS-PAGE and visualized by autoradiography. Human NCoA-62 cDNA was subcloned into the pVL-1392 polyhedrin transfer plasmid containing a polyhistidine tag and a protein kinase A consensus site at the N terminus. Recombinant baculovirus was isolated and plaque-purified by standard methods (26Summers M. Smith G.E. A Manual of Methods for Baculovirus Vector and Insect Cell Culture Procedures. Texas Agriculture Experimental Station and Texas A & M University, College Station, TX1987Google Scholar). A 50-ml culture of Sf-9 cells (1 × 106 cells/ml) was infected for 48 h with the NCoA-62-expressing recombinant baculovirus (multiplicity of infection = 3). Whole cell extracts were prepared, and recombinant NCoA-62 was purified by nickel affinity chromatography. Whole cell extracts and Ni2+-nitrilotriacetic acid-purified, baculovirus expressed proteins from Sf9 cells or bacterial expressed GST fusion proteins were subjected to SDS-PAGE and electrophoretically transferred to a Protran BA-S 85 nitrocellulose membrane (Schleicher & Schuell). Denaturation and renaturation of the protein blot was performed as described (27Cavailles V. Dauvois S. Danielian P.S. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10009-10013Crossref PubMed Scopus (340) Google Scholar, 28Vinson C.R. LaMarco K.L. Johnson P.F. Landschulz W.H. McKnight S.L. Genes Dev. 1988; 2: 801-806Crossref PubMed Scopus (343) Google Scholar). The renatured blot was incubated in HB buffer (25 mm HEPES, pH 7.7, 25 mm NaCl, 5 mmMgCl2, 1 mm dithiothreitol) containing 5% nonfat dried milk and then in HB buffer containing 1% milk, 0.05% Nonidet P-40, and a 32P-labeled VDR probe. The VDR probe was prepared by combining purified, baculovirus-expressed His6 protein kinase A-tagged VDR (300 pmol), 12.5 μl of [γ-32P]ATP (3000 Ci/mmol), and 50 units of the catalytic subunit of protein kinase A (Sigma) in HMK buffer (20 mm Tris, pH 7.5, 100 mm NaCl, 12 mmMgCl2, 1 mm dithiothreitol) for 1 h at room temperature. The 32P-labeled VDR probe was purified on Ni2+-nitrilotriacetic acid-agarose and incubated with the membrane at approximately 500,000 cpm/ml in buffer H (20 mmHEPES, pH 7.7, 75 mm KCl, 0.1 mm EDTA, 2.5 mm MgCl2, 0.05% Nonidet P-40, 1% milk, 1 mm dithiothreitol) overnight at 4 °C. After three washes with buffer H, the protein blot was subjected to autoradiography for 3 h at room temperature. The vitamin D-responsive (VDRE)4-TK-GH growth hormone reporter plasmid contains four copies of the rat osteocalcin VDRE upstream of the viral thymidine kinase promoter. The pSG5hVDR expression plasmid was described previously (29Hsieh J.-C. Jurutka P.W. Galligan M.A. Terpening C.M. Haussler C.A. Samuels D.S. Shimizu Y. Shimizu N. Haussler M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9315-9319Crossref PubMed Scopus (185) Google Scholar). Full-length NCoA-62 cDNA was subcloned into the mammalian expression vector, pSG5 (Stratagene). All cells were cultured in Dulbecco's modified Eagle's medium containing 10% charcoal-stripped, heat-inactivated serum prior to transfection. COS-7 cells were transfected by standard calcium phosphate precipitation procedures as described previously (6MacDonald P.N. Dowd D.R. Nakajima S. Galligan M.A. Reeder M.C. Haussler C.A. Ozato K. Haussler M.R. Mol. Cell. Biol. 1993; 13: 5907-5917Crossref PubMed Scopus (230) Google Scholar). Carrier DNA (pTZ18u) was added to bring the total DNA content to 10 μg/plate. Following a 16-h incubation, the precipitate was removed with two washes of phosphate-buffered saline, and the cells were replenished with fresh media containing 10% charcoal-stripped, heat-inactivated serum. The cells were treated with ligand or vehicle for 24 h, and the amount of secreted growth hormone was determined with an immunoassay kit (Nichols Institute, San Juan Capistrano, CA). The indicated plasmid pairs were transformed into Hf7c yeast, plated on media lacking leucine and tryptophan, and incubated at 30 °C for 4 days. Triplicate colonies were harvested and cultured overnight in liquid media deficient in leucine and tryptophan. Cells were assayed for β-galactosidase activity as described previously (23MacDonald P.N. Sherman D.R. Dowd D.R. Jefcoat Jr., S.C. DeLisle R.K. J. Biol. Chem. 1995; 270: 4748-4752Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). A multiple tissue Northern blot was obtained from CLONTECHLaboratories. Each lane of the blot contained 2 μg of poly(A)+ RNA purified from several human tissues. Total RNA isolated from rat osteosarcoma cells (ROS 17/2.8) and from primary rat osteoblasts obtained from newborn calvaria (30Shalhoub V. Conlon D. Tassinari M. Quinn C. Partridge N.C. Stein G.S. Lian J.B. J. Cell Biochem. 1992; 50: 425-440Crossref PubMed Scopus (195) Google Scholar) was subjected to Northern analysis as well. A cDNA probe for NCoA-62 was labeled with [α-32P]dCTP in a random primer reaction with the Klenow fragment of DNA polymerase. The blots were hybridized with the labeled probe in ExpressHyb hybridization solution (CLONTECH Laboratories) and were processed according to the manufacturer's instructions. Autoradiographs were scanned, and the images were quantitated using ImageQuant version 3.0 software from Molecular Dynamics. We used a yeast two-hybrid system to identify cDNA clones that code for proteins that interact with the vitamin D receptor (23MacDonald P.N. Sherman D.R. Dowd D.R. Jefcoat Jr., S.C. DeLisle R.K. J. Biol. Chem. 1995; 270: 4748-4752Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The pAS1-VDR-(93–427) plasmid was used to screen a HeLa cell cDNA library that was constructed in the pGAD-GH plasmid, and positive clones were selected by growth on histidine-deficient media and for the expression of β-galactosidase activity. Specificity controls included testing each clone against pAS1-RAR, pAS1-RXR, pAS1-p53, pAS1-Gα, pAS1-VP16, and pAS1. The growth properties of one clone, designated NCoA-62, are illustrated in Fig. 1. As evidenced by growth on histidine-deficient media in the two-hybrid system, NCoA-62 interacted with the VDR hybrid protein (area 1), the RAR hybrid protein (area 3), and the RXR hybrid protein (area 5). In contrast, no interaction was detected with unrelated bait vectors including the pAS1 parent vector (area 2), AS1-p53 (area 4), or with AS1- Gα (area 6). The NCoA-62 cDNA insert was sequenced and was found to be highly similar to BX42, a nuclear protein from D. melanogaster that is involved in ecdysone-stimulated gene expression (Fig. 2) (20Wieland C. Mann S. von Besser H. Saumweber H. Chromosoma. 1992; 101: 517-525Crossref PubMed Scopus (28) Google Scholar). Other related proteins from Caenorhabditis elegans andSchizosaccharomyces pombe are also illustrated in Fig. 2. The NCoA-62 cDNA insert was 2113 bp. However, based on the BX42 sequence, the original NCoA-62 clone did not contain an obvious initiator methionine with a strong consensus Kozak sequence (31Kozak M. Cell. 1986; 44: 283-292Abstract Full Text PDF PubMed Scopus (3548) Google Scholar). Therefore, a 5′-rapid amplification of cDNA ends strategy (Life Technologies, Inc.) was used with mRNA isolated from HeLa cells, and a single product was obtained that contained an additional 30 bp of 5′ sequence, 27 of which constituted a short untranslated region followed by an in frame ATG with a perfect Kozak sequence (data not shown). Thus, the full-length NCoA-62 cDNA is 2146-bp consisting of a noncoding leader sequence of 27 bp, a 1611-bp open reading frame, and 508 bp of 3′-noncoding sequence. The size of this cDNA is consistent with Northern blot analysis of mRNA from HeLa cells, which indicated a single 2.2-kilobase pair transcript for NCoA-62 (data not shown). The tissue distribution of the NCoA-62 transcript was determined by Northern blot analysis using mRNA obtained from eight different human tissues (Fig. 3 A). NCoA-62 exhibited a wide expression pattern, being present in all of the various tissues examined. The NCoA-62 mRNA transcript was also observed in a Northern blot analysis of mRNA obtained from an osteoblast-like cell line, ROS 17/2.8, and in primary osteoblasts obtained from newborn rat calvaria (Fig. 3 B). In rat osteoblasts, the NCoA-62 transcript was a single 2.4-kilobase pair transcript that was not regulated by vitamin D treatment over a 24-h time course (Fig. 3 B). In osteoblasts obtained from newborn calvaria (Fig. 3 B, right panel), osteocalcin transcripts were up-regulated by 1,25-(OH)2D3in the differentiated and mineralizing osteoblasts as described previously (30Shalhoub V. Conlon D. Tassinari M. Quinn C. Partridge N.C. Stein G.S. Lian J.B. J. Cell Biochem. 1992; 50: 425-440Crossref PubMed Scopus (195) Google Scholar), but there was not an apparent effect on NCoA-62 transcript levels. This broad pattern of expression of NCoA-62 transcripts may reflect the wide tissue distribution observed for the VDR or, since NCoA-62 interacts with other nuclear receptors and may function in those transcriptional pathways as well, the wide expression pattern of NCoA-62 may simply reflect its role in other nuclear receptor pathways.Figure 3Tissue distribution of NCoA-62 mRNA. A, NCoA-62 transcripts in various human tissues. A blot containing approximately 2 μg of poly(A)+ RNA per lane from eight different human tissues was obtained fromCLONTECH and was hybridized with a32P-labeled NCoA-62 cDNA probe. Lanes 1–8 contain RNA from pancreas (lane 1), kidney (lane 2), skeletal muscle (lane 3), liver (lane 4), lung (lane 5), placenta (lane 6), brain (lane 7), and heart (lane 8). The blot was stripped of radioactivity and hybridized to a 32P-labeled human β-actin probe using similar conditions. Densitometric analysis revealed that the NCoA-62:actin ratios were 8.4, 1.0, 0.5, 1.0, 0.2, 2.0, 1.0, and 0.5 for pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, and heart, respectively. B, NCoA-62 transcripts in rat osteosarcoma cells and in normal osteoblasts. Cells were treated in culture with 10−8m1,25-(OH)2D3 for the indicated times (left) or for 24 h (right). A blot containing total RNA (10 μg/lane) isolated from ROS 17/2.8 cells (left) or from primary rat osteoblasts obtained from newborn calvaria in the proliferation (P), differentiation (D), and mineralization (M) stages (right) was hybridized with a 32P-labeled NCoA-62 cDNA probe. The blots were stripped and reprobed with [32P]actin cDNA (left) or32P-labeled osteocalcin cDNA. The various transcripts are indicated by arrows.View Large Image Figure ViewerDownload (PPT) The NCoA-62 cDNA was expressed as a histidine-tagged protein in a baculovirus expression system, and the expressed protein was purified (Fig. 4). The apparentM r by SDS-PAGE analysis was 66,000 (Fig. 4), while the predicted NCoA-62 translation product is 537 amino acids long with a calculated M r of 61,500. Taking into account the additional residues in the His-protein kinase A tag, there is agreement between the apparent M r by SDS-PAGE and the predicted M r of NCoA-62 based on the primary sequence. Moreover, the estimated molecular mass of the untagged NCoA-62 protein produced by in vitrotranscription/translation was approximately 62,000 daltons as assessed by SDS-PAGE analysis (see Fig. 6).Figure 6In vitrointeraction of NCoA-62 with VDR. A, far Western analysis. Soluble extracts obtained from uninfected (lane 4), WT-infected (lane 3), and NCoA-62-infected Sf9 cells (lane 2), and 2 μg of purified NCoA-62 protein (lane 1) were subjected to SDS-PAGE analysis and transferred to a nitrocellulose membrane. Following a denaturation/renaturation cycle, the blot was probed with 32P-labeled VDR and processed as described under "Experimental Procedures." B, GST pull-down analysis. Increasing amounts of in vitrotranscribed and translated 35S-labeled NCoA-62 (5, 10, or 20 μl) were incubated with either 5 μg of GST (lanes 2–4) or with 5 μg of GST-VDR (lanes 5–7). Protein-protein complexes were washed, analyzed by SDS-PAGE, and visualized by autoradiography. The input lanes represent 10% of the protein in the binding assay. C, 35S-labeled RXR (lanes 3–5) or 35S-labeled NCoA-62 (lanes 6–8) were incubated with either 5 μg of GST (lanes 3and 6) or with 5 μg of GST-VDR in the absence (lanes 5 and 8) or presence (lanes 4 and7) of 10−8m1,25-(OH)2D3. Protein-protein complexes were analyzed as described for B. D, increasing amounts of 35S-labeled NCoA-62, NCoA-62 (R438STOP), or NCoA-62 (R488STOP) were incubated with either GST (lanes 2, 7, and 12, respectively) or with GST-VDR (lanes 3–5, 8–10, and13–15). Protein-protein complexes were analyzed by SDS-PAGE and autoradiography.View Large Image Figure ViewerDownload (PPT) To identify the regions of VDR that are important for interaction with NCoA-62, several amino- and carboxyl-terminal deletion mutants of pAS1-VDR were examined in the two-hybrid system (Fig. 5 A). Nuclear receptors are characterized by an N-terminal DNA-binding domain, which targets the receptor to specific DNA response elements and a large COOH-terminal domain that binds the hydrophobic ligand. At the extreme COOH-terminal end of the ligand binding domain of these receptors is a conserved motif, designated AF-2, which is crucial for the transactivation function of many nuclear receptors. Indeed, several of the putative coactivators described thus far bind to nuclear receptors, in part, through this AF-2 domain (8Onate S.A. Tsai S.Y. Tsai M.-J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2039) Google Scholar, 9Cavailles V. Dauvois S. L'Horset F. Lopez G. Hoare S. Kushner P.J. Parker M.G. EMBO J. 1995; 14: 3741-3751Crossref PubMed Scopus (670) Google Scholar, 10Li H. Gomes P.J. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (497) Google Scholar, 32vom Baur E. Zechel C. Heery D. Heine M.J.S. Garneir J.M. Vivat V. LeDouarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: 110-124Crossref PubMed Scopus (348) Google Scholar, 33Masuyama H. Brownfield C.M. St-Arnaud R. MacDonald P.N. Mol. Endocrinol. 1997; 11: 1507-1517Crossref PubMed Scopus (127) Google Scholar). Comparison of the pAS1-VDR-(3–427) full-length construct and the pAS1-VDR-(116–427) construct clearly demonstrated that the NH2-terminal DNA binding domain of VDR is not required for VDR-NCoA-62 interactions. Furthermore, removal of the AF-2 motif located at the extreme C terminus of VDR (residues 403–427) was not detrimental to the interaction. However, removal of additional residues between 386 and 402 eliminated VDR interaction with NCoA-62. Furthermore, this COOH-terminal domain alone was sufficient to observe modest interaction with NCoA-62. For example, the pAS1-VDR-(373–427) construct, which contains only 55 amino acids from the extreme C terminus of VDR, was capable of interacting with the NCoA-62-GAD fusion in the two-hybrid system. These data strongly suggest a role for the COOH-terminal domain between amino acid residues 373 and 403 of VDR in contacting NCoA-62. A second potential interaction surface may reside between Leu116 and Ser166, since deletion of this domain also ablates VDR·NCoA-62 complex formation. However, this deletion also disrupts ligand binding, RXR interaction, and transcription factor IIB interaction as well as interaction between VDR and a number of other clones isolated in our screen. 2T. A. Baudino, D. M. Kraichely, S. C. Jefcoat, Jr., and P. N. MacDonald, unpublished data. Thus, it is also likely that this deletion alters the proper folding of the remaining ligand binding domain. Similar deletion studies of NCoA-62 are presented in Fig. 5 B. The first 220 amino acids of the N terminus of NCoA-62 appeared to be dispensable for VDR interaction, since NCoA-62-(87–536) and NCoA-62-(220–536) interacted with VDR in a manner similar to full-length NCoA-62. In fact, increased β-galactosidase activity was observed in yeast expressing these mutants compared with wild-type NCoA-62. It is possible that this amino-terminal domain
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