PNRC: A Proline-Rich Nuclear Receptor Coregulatory Protein That Modulates Transcriptional Activation of Multiple Nuclear Receptors Including Orphan Receptors SF1 (Steroidogenic Factor 1) and ERR{alpha}1 (Estrogen Related Receptor {alpha}-1)

Dujin Zhou, Keith M. Quach, Chun Yang, Stella Y. Lee, Bill Pohajdak and Shiuan Chen

Division of Immunology (D.Z., K.M.Q., C.Y., S.C.) Beckman Research Institute of the City of Hope Duarte, California 91010
Department of Biology (S.Y.L., B.P.) Dalhousie University Halifax, Nova Scotia B3H 4J1, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC’s nuclear localization was demonstrated by immunofluorescence and Western blot analyses. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERR{alpha}1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC was found to function as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor {alpha}-1), PR, and TR. By examining a series of deletion mutants of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence in the carboxy-terminal region, aa 278–300, was shown to be critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. Results from the mutagenesis study demonstrated that the two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones, including estrogens, play essential role in metabolism, sexual differentiation, and reproductive function. Considerable attention, therefore, has been directed at defining the mechanisms that control their biosynthesis. We have been studying the mechanisms that regulate the expression of the human aromatase gene in breast cancer. Aromatase catalyzes the conversion of androgens to estrogens and plays a key role in the pathogenesis of estrogen-dependent breast cancer. The control of human aromatase gene expression is complex in that several promoters direct aromatase gene expression in a tissue-specific manner (1, 2, 3, 4, 5). Research from our laboratory has identified a silencer element (6, 7) that is situated between two aromatase promoters, 1.3 (8) and II (6), which are thought to be the major promoters controlling aromatase expression in breast cancer tissue and ovary (9, 10, 11). UV cross-linking experiments (7) have found that at least four proteins bind to the silencer elements. Two orphan nuclear receptors, SF1 (steroidogenic factor 1) and ERR{alpha}1 (estrogen related receptor {alpha}-1), were shown to bind to this regulatory region (12). Cell transfection experiments have revealed that both SF1 and ERR{alpha}1 function as positive regulatory factors when they bind to the silencer element (12). To better understand the regulatory mechanism of these nuclear receptors on aromatase expression in breast, we decided to search for coregulatory proteins interacting with these proteins using bovine SF1 as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. Our screen identified a known coactivator, RIP140 (13, 14), a previously cloned protein B4–2 whose function was not known (15, 16), and four new proteins. In this study, we have performed a series of experiments to demonstrate that B4–2 is actually a nuclear receptor coregulatory protein that modulates the transcriptional activity of a number of nuclear receptors including SF1 and ERR{alpha}1. This protein is proline rich and it interacts with nuclear receptors through a carboxy- terminal SH3-binding motif.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of a Nuclear Receptor Coregulatory Protein, PNRC, by Its Interaction with SF1 in the Yeast Two-Hybrid Screening
A Gal4-based yeast two-hybrid system was used to identify proteins encoded in a human mammary gland cDNA library that interact with the bovine SF1. The coding region for the wild-type bovine SF1 was subcloned into a yeast expression vector, pGBT9, and the resulting plasmid, pGBT9-SF1, was used to transform yeast strains CG1945. The CG1945 transformants bearing pGBT9-SF1 plasmids were transformed again with a human mammary gland cDNA library (from CLONTECH Laboratories, Inc., Palo Alto, CA). Approximately 3.4 x 106 yeast transformants were screened in the absence of ligand since the ligand for SF1 is unknown. A total of 90 colonies appeared on the histidine dropout plates, 12 of which stained strongly positive when tested for expression of ß-galactosidase. To test whether SF1 was required for interaction with the products of the isolated cDNAs, all of these plasmids were retransformed into the yeast CG1945, and these transformants were used in yeast mating experiments to verify that these proteins identified from library screen can activate the reporter genes only in the presence of Gal4DNA-BD/SF1 fusion protein. All hybrid proteins were found to interact only with Gal4DB-SF1. The nucleotide sequences of these 12 cDNA inserts were generated. A database search revealed that 4 of 12 clones are identical to B4–2 protein, a proline-rich protein with unknown function, which was first cloned from a natural killer minus T cell subtractive library (15). Since we have found that this protein is a novel coregulatory protein for nuclear receptor (described in this publication), we renamed it PNRC (proline-rich nuclear receptor coregulatory protein). These four clones, clones 23, 6, 45, and 13, encode aa 141–327, aa 148–327, aa 256–327, and aa 270–327 of PNRC, respectively. Another three clones are identical to a known nuclear receptor coactivator, RIP140 (13, 14). In addition to PNRC and RIP140, we also isolated clones encoding one unknown protein with high homology to PNRC as well as three other unknown proteins.

Nuclear Localization of PNRC as Demonstrated by Immunofluorescent Staining and Western Blot Analyses
Nuclear proteins require a short peptide signal called nuclear localization signal (NLS) to guide themselves into nuclei. A putative nuclear localization sequence locates at position 94–101 of PNRC. We performed two sets of experiments to confirm PNRC as a nuclear protein. COS-1 cells that express PNRC were prepared by cDNA transfection method. As shown in Fig. 1AGo, the majority of the transfected COS-1 cells showed nuclear staining of PNRC with a low level of cytoplasmic staining, as demonstrated by immunofluorescent staining with polyclonal antiserum against PNRC (15). The few cells without the intense nuclear localization might be in a different cell cycle stage. No staining was observed for cells treated only with the secondary antibody (Cy3 antirabbit antibody) (results not shown). To further demonstrate PNRC as a nuclear protein, we performed Western blot analysis on nuclear extract and cytoplasmic fractions of PNRC-expressing Hela cells with polyclonal antiserum against PNRC. The results from Western blot analysis (Fig. 1BGo) indicate that the majority of PNRC protein is localized in nuclei although there were some PNRC retained in the cytoplasm.



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Figure 1. Nuclear Localization of PNRC

A, Immunofluorescent staining of paraformaldehyde-fixed Cos-1 cells with PNRC polyclonal antiserum diluted 1:300 as described in Materials and Methods. B, Western blot analysis of nuclear extract and cytoplasmic fraction of Hela cells transfected with pSG5-PNRC. Nuclear extract and cytoplasmic proteins were prepared from pSG5-PNRC transiently transfected Hela cells as described previously (42 ). One hundred micrograms of each nuclear extract and cytoplasmic fraction were fractionated by10% SDS-PAGE, transferred onto nitrocellulose membrane, and probed by Western blot with 1:500 dilution of polyclonal rabbit antiserum raised against recombinant PNRC. In vitro translated PNRC was included as positive control.

 
Interactions between PNRC and the Multiple Nuclear Receptors Including Orphan Receptors SF1 and ERR{alpha}1 in Yeast
The protein-protein interactions between the full-length PNRC or PNRC fragments and SF1, ERR{alpha}1, and several other nuclear receptors were analyzed by the yeast two-hybrid assays. Yeast strains Y187 expressing the Gal4DB fusion to the nuclear receptors and CG1945 expressing Gal4AD alone or Gal4 AD-PNRC fusion protein were mated by coculturing, and selected for the presence of both two-hybrid plasmids. The expression of interacting hybrid proteins in yeast diploids was analyzed for induction of HIS3 expression as shown in Fig. 2AGo and LacZ expression as shown in panels B and C. Tests in yeast two-hybrid assays indicated that PNRC interacted with orphan receptors SF1 and ERR{alpha}1 in the absence of any added activator or ligand. As shown in Fig. 2Go, A–C, PNRC also interacted specifically with all seven nuclear receptor hormone-binding domains (HBDs), including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor (TR), progesterone receptor (PR), retinoic acid receptor (RAR), and retinoid X receptor (RXR), and these interactions were completely ligand dependent. The results also showed that PNRC interacted with these nuclear receptors with different strengths. PNRC interacts strongly with HBDs for ER{alpha}, TR, PR, and RXR in the presence of cogent ligands, and the interaction between PNRC and ER{alpha}.HBD was not detected in the presence of antiestrogens such as tamoxifen (Fig. 2CGo). Only weak interactions were detected between PNRC and HBDs for GR, AR, and RAR (Fig. 2BGo). However, no interactions occurred between PNRC and an irrelevant protein such as human lamin C protein or between nuclear receptors and Gal4 activation domain alone. These results suggest that the interaction occurred only in the presence of PNRC and nuclear receptors in the forms of two-hybrid proteins, and PNRC is a nuclear receptor-interacting protein with a broad reactivity.



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Figure 2. Interaction between PNRC and Nuclear Receptors in Yeast

Yeast strains Y187, expressing the Gal4DB fusion to the nuclear receptors, and CG1945, expressing Gal4AD alone or Gal4 AD-PNRC fusion protein, were mated by coculture and selected for the presence of both two-hybrid plasmids. The expression of interacting hybrid proteins in yeast diploids was analyzed for induction of HIS3 expression (A) and LacZ expression (B and C) in the presence of ligands as described in Materials and Methods. Gal4AD was included as a control to monitor the background transcriptional activity. Relative ß-galactosidase activities in liquid cultures were expressed in Miller units as mean ± SD of three independent assays.

 
Interaction of PNRC with SF1 in Mammalian Cells
The mammalian MatchMaker two-hybrid assay system (CLONTECH Laboratories, Inc.) was used to confirm the interaction between PNRC and SF1 identified in yeast. Because the assays are performed in mammalian cells, proteins are more likely to be in their physiological environment, and the results are therefore more likely to represent biologically significant interactions. SK-BR-3 breast cancer cells were cotransfected with three expression plasmids, including pM-SF1 for Gal4 DNA-DB/SF1 fusion protein, pVP16-PNRC for VP16AD-PNRC fusion protein, and a third reporter plasmid, pG5CAT, which provides the Gal4 DNA-binding site, promoter, and the chloramphenicol acetyltransferase (CAT) reporter gene. As shown in Fig. 3Go, the CAT activities of the cells transfected with the above three plasmids is about 5- to 6-fold higher than that of the cells transfected with only CAT reporter plasmid. This interaction of PNRC is specific for SF1 since no interaction was observed between PNRC and Gal4DB or between SF1 and VP16 AD.



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Figure 3. PNRC Interacts with SF1 in Mammalian Cells

SK-BR-3 cells were transiently cotransfected with 1.5 µg reporter plasmid, pG5CAT, and 1.0 µg each of expression plasmids as indicated. The relative CAT activities were expressed as induction folds over the value obtained from the cotransfection of reporter, pM, and pVP16 vectors, which was given an arbitrary value of 1. The relative activation folds were mean ± SD of three experiments.

 
Demonstration of the Interaction of PNRC with SF1 by Glutathione-S-Transferase (GST) Pull-Down Analysis
To further confirm the interaction between PNRC and SF1 detected in both yeast and mammalian twohybrid assays, a GST pull-down binding assay was performed to study direct binding between PNRC and SF1 in vitro. Our results from yeast two-hybrid assays showed that a short 23-residue region in PNRC was sufficient for the interaction between PNRC and the nuclear receptors (explained in detail in the following section). PNRC fragments, including PNRC270-327 and PNRC278-300, were expressed as fusion protein with GST in Escherichia coli BL21, purified with glutathione Sepharose 4B beads, and tested for their ability to bind in vitro translated [35S]methionine-labeled SF1 in pull-down assays. As shown in Fig. 4Go, both GST-PNRC270-327 and GST-PNRC278-300 were found to bind SF1 (Fig. 4Go, lanes 3 and 4). The results also showed that the binding of SF1 was specific to PNRC, because GST alone retained only very small amounts of labeled SF1 (Fig. 4Go, lane 2).



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Figure 4. Interaction of PNRC with SF1 in Vitro

35S-labeled, in vitro translated SF1 was incubated with Sepharose beads containing bound GST-PNRC270–337, GST-PNRC278–300, or GST protein. The beads were washed and bound protein was eluted and analyzed by SDS-PAGE. The gel was stained with Coomasie blue (B) before being visualized by autoradiography (A). An aliquot of the in vitro translated SF1 equivalent to 10% of the sample used for the binding reactions was also analyzed (input).

 
Demonstration of the Interaction of PNRC with ER in Vivo by Coimmunoprecipitation Analysis
To examine whether PNRC associates with the nuclear receptors under more physiologically relevant conditions, both PNRC and ER were overexpressed in Hela cells that were cultured for 24 h in the presence of 100 nM 17ß-estradiol and assayed for association by coimmunoprecipitation. Because of the availability of both ER{alpha} antibody and the ligand, ER instead of SF1 was chosen as the representative of the nuclear receptors for this study. Protein complexes containing ER{alpha} were immunoprecipitated from transfected Hela cells with anti-ER{alpha} antibody (Santa Cruz Biotechnology, Inc.) and protein A agarose. The immunoprecipitated proteins were then fractionated by SDS-PAGE and hybridized separately by Western blot with antibodies against PNRC or ER{alpha}. Figure 5Go (lane 3) clearly showed that PNRC associated with ER{alpha} when both proteins were expressed in estradiol-treated Hela cells. No interaction was found when cells were cultured in the absence of the ligand estradiol.



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Figure 5. Association of PNRC with ER in Vivo

Hela cells transfected with pSG5-PNRC (10 µg) or pSG5-ER (10 µg), either separately or together as indicated above the lanes. The cells were incubated with 100 nM 17ß-estradiol for 24 h. Whole-cell extract prepared from transfected and mock transfected cells was first incubated with mouse monoclonal anti-hER{alpha} antibody (Santa Cruz Biotechnology, Inc.) and protein A agarose (Roche Molecular Biochemicals). The resulting immunoprecipitated protein complexes were then collected, washed, fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and probed by Western blot with polyclonal rabbit antiserum against PNRC (1:500 dilution). To verify the presence of ER in the immunoprecipitated complexes, elutate in an amount equal to that in lane 3 was included for the same process and probed by Western blot with anti-ER{alpha} antibody (lane 4).

 
Requirement of Functional AF2 Domain for Nuclear Receptors to Interact with PNRC
The ligand-binding domains of nuclear receptors have many functions, including dimerization and ligand-dependent transcriptional activation, that involve the AF2 domain. The transcriptional activation function of the AF2 domain is likely exerted through the interaction with coactivators that are distinct from basal transcription factors, since the binding between nuclear receptors and the basal transcription factors is unaffected by ligand binding or by mutations in the AF2 domain that abolish transcriptional activity. It has been shown that the transactivation activity of various ER AF2 mutants correlates strongly with the ability to interact with nuclear receptor coactivators GRIP1 (17) and SRC1 (18). To test whether the interaction of PNRC with mutant ER also correlates with the AF2 activity of ER, we tested the ability of PNRC to interact with a panel of single and multiple point mutations in AF2 domains of both mouse ER{alpha} and ERR3. It was previously shown that two double mutations, L543A/L544A and M547A/L548A, completely eliminated transactivation by ER{alpha}; the triple mutant, D542N/E546Q/D549N, caused substantial, but not complete, loss of function; and two single mutations, D542A and D549A, had little, if any, effect on ER transactivation activity (19). Our yeast two–hybrid assays (Fig. 6AGo) have found that PNRC interacts with these mutants in an identical manner as GRIP1 (17). These results demonstrate that the interaction between PNRC and mutants of ER correlates strongly with the AF-2 activity of ER. A functional AF-2 domain is required for ER to interact with PNRC, and the defect in transcriptional activity of AF-2-mutated ER may reflect inefficient recruitment of coregulatory proteins including PNRC. Figure 6BGo shows that PNRC interacts with ERR3 (20), whereas mutations in the AF2 region of ERR3, M432A/L433A and L428A/F429A, and a deletion of the AF2 region, -AF2, prevent its interaction with PNRC.



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Figure 6. Ability of PNRC to Interact with ER HBD or ERR3.HBD with Mutations in the AF2 Domain

Yeast strain Y187 was cotransformed with pACT2-PNRC270-327 and a yeast expression plasmid for fusion protein of Gal4 DBD and ER.HBD wild-type or various mutations in the AF2 domain. The yeast transformants bearing both plasmids were cultured in the absence or presence of 100 nM estradiol or in the presence of 10 nM tamoxifen (T). The ß-Gal activity was determined and expressed as the mean (units) ± SD of triplicate reactions from a single colony and the representative of three independent colonies (panel A). Experiments with ERR3.HBD were performed in an identical manner as those for ER.HBD, but in the absence of ligand (panel B).

 
Modulation of the Transactivation Activity of SF1, ERR{alpha}1, PR, and TR by PNRC in Mammalian Cells
PNRC has been demonstrated to specifically interact with multiple members of the nuclear receptor family in vivo and in vitro. To examine the possible biological significance of this interaction, the entire PNRC coding region was inserted into a mammalian expression vector pSG5. To study the effect of PNRC overexpression on transcriptional activation of SF1 and ERR{alpha}1, the expression plasmid pSG5-PNRC was used to transiently transfect Hela cells together with a reporter plasmid, pUMS1.3CAT-(SF1site)3, which contains three copies of extended steroid hormone half-binding site from the human aromatase gene (5'-CCAAGGTCAGAA-3'), promoter 1.3 of the human aromatase gene, and the CAT reporter gene, along with a second expression plasmid for either bovine SF1 (pSG5-SF1) or human ERR{alpha}1 (pSG5-ERR{alpha}1). As shown in Fig. 7Go, A and B, both SF1 and ERR{alpha}1 have been shown to stimulate the transcriptional function of promoter 1.3 of the human aromatase gene. Cotransfection of PNRC enhanced SF1-stimulated and ERR{alpha}1-stimulated transcription of promoter 1.3 by 1.5- and 2.2-fold, respectively. A similar effect was observed with PR- and TR-mediated transactivation function of thymidine kinase (tk) promoter (Fig. 7Go, C and D). Therefore, PNRC functions as a coactivator through interaction with the nuclear receptors. The enhancement of transactivation activity of these nuclear receptors by PNRC was not comparable to the strong interaction observed in yeast. The lack of correlation between physical interaction and coactivator activities, which was also observed for some nuclear receptors with coactivators SRC1 (21) and TIF2 (22), may be due to the endogenous levels of coactivators including PNRC in the cells. It is also possible that PNRC behaves as a weak antagonist by competing with other coactivators. At higher concentrations, PNRC was found to have an inhibitory effect on the transactivation activities of SF1, ERR{alpha}-1, PR, and TR (data not shown). The inhibitory effect of PNRC at higher concentrations was probably caused by the fact that, after saturating the binding sites on the nuclear receptors, PNRC might sequester or compete one or more other coactivators present in the cells required for transcriptional activity of these receptors. As a result of sequestering those coactivators that have higher coactivation activities than that of PNRC, the overall enhancement in the transcriptional activation by the nuclear receptors is reduced. Our preliminary results indicated that PNRC reduced the cotransactivation activity of GRIP on SF1- and ERR1-mediated transcription (data not shown).



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Figure 7. The Effect of PNRC Overexpression on Nuclear Receptor Transactivation Function

A, Effect of PNRC overexpression on SF1-stimulated transcription of promoter 1.3 of the human aromatase gene. Hela cells were transfected with 0.25 µg of p1.3aroCAT- (SF1site)3 reporter along with 5 ng of a pSG5-SF1 expression plasmid and increasing amounts of pSG5-PNRC expression plasmid as indicated below the panel. Appropriate amounts of empty vector pSG5 were included to maintain the same overall amount of total DNA in all transfections. The CAT activities in transfected cells were measured and expressed as mean ± SD of at least triplicate experiments. B, Effect of PNRC overexpression on ERR{alpha}1-stimulated transcription of promoter 1.3 of the human aromatase gene. Hela cells were transfected with p1.3CAT- (SF1site)3 reporter along with either pSG5-ERR{alpha}1 expression plasmid, pSG5-PNRC, or both, as indicated below the panel. All other features are as described in panel A. C, Effect of PNRC overexpression on PR-mediated transcription of thymidine kinase promoter. Hela cells were transfected with 1.0 µg of pRE2tkCAT reporter along with either pRSV-PR expression plasmid (0.1 µg), pSG5-PNRC expression plasmid, or both, as indicated below the panel. All other features are the same as in A. D, Effect of PNRC overexpression on TR-mediated transcription of thymidine kinase promoter. Hela cells were transfected with 0.2 µg of pDR4tkCAT reporter along with either pRSV-TR expression plasmid (0.5 µg), pSG5-PNRC expression plasmid, or both, as indicated below the panel. All other conditions are the same as in panel A.

 
Identification of the Binding Site on PNRC That Is Necessary and Sufficient for Its Interaction with Nuclear Receptors
The four PNRC clones originally isolated from the yeast two-hybrid screening encoded four C-terminal peptides, aa 141–327, aa 148–327, aa 256–327, and aa 270–327, respectively. All four of these clones showed similar ability to bind SF1. Even the shortest peptide, aa 270–327, retained the ability of PNRC to interact with SF1 (Fig. 8BGo), suggesting that the region containing residues 270–327 was responsible for the interaction with SF1. A short conserved peptide motif LxxLL (referred to as the NR box) has been identified and reported to be necessary and sufficient to mediate the binding of several coactivators to liganded nuclear receptors (23). There is one NR box-like sequence, LKTLL (aa 319–323), at the very end of the C terminus in PNRC. To determine whether this NR box-like sequence is responsible for the interaction, the PNRC fragment coding for aa 270–317 was generated by PCR, expressed as a fusion protein with Gal4 AD, and tested in the yeast two-hybrid assay for its interaction with SF1. Compared with the shortest PNRC clone isolated from the library screening, this PNRC fragment has a deletion of the last 10-amino acid segment that contains the NR box. As shown in Fig. 8BGo, the interaction intensities, as expressed by ß-Gal activity, between PNRC270-327 or PNRC270-317 and SF1 are about the same, indicating that the NR box sequence in PNRC is not necessary for the interaction. We also expressed this NR box as a fusion protein with Gal4 AD and tested its interaction with SF1 in yeast. No interaction was observed between the NR box and SF1, suggesting again that this NR box sequence is not responsible for the interaction. To directly confirm our findings, a yeast expression plasmid for a Gal4AD and PNRC301-327 fusion protein (which contains the NR box) was generated and tested in the yeast two-hybrid assay for its interaction with nuclear receptors. As shown in Fig. 8BGo, this fusion protein could not interact with SF1.



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Figure 8. Localization of the Interacting Domain within PNRC

A series of N-terminal deletion mutants of PNRC, as shown in the left schematic diagram, were generated and tested for interaction in yeast two-hybrid assays with different nuclear receptors. Gal4 AD-PNRC deletion constructs were cotransformed with Gal4DB-nuclear receptor into yeast strain Y187, and the transformants bearing both hybrid plasmids were selected and propagated in the presence of appropriate ligands. The ß-galactosidase activities in yeast, shown at the right, were determined and expressed as the average A420 of three independent assays.

 
Two additional clones were isolated from the same library screening and found to encode an unknown protein with a regional sequence homology to PNRC. Sequence comparison revealed that there is a 23-amino acid region with 100% identity between PNRC and the novel protein, and this 23-amino acid sequence is within the shortest peptide, i.e. PNRC270-327, identified in the library screening. This information suggests that the 23-amino acid region, aa 278–300, may be responsible for the interaction. To test whether the 23-amino acid region, aa 278–300, is sufficient for interaction, a yeast expression plasmid coding for Gal4AD-PNRC278-300 fusion protein was prepared and tested for interaction in both yeast and in mammalian cells. As expected, this 23-amino acid peptide was found to be able to interact with SF1 (Fig. 8BGo) and all other nuclear receptors (data not shown). In addition, PNRC278-300 was also found to retain most of the interaction of the full-length PNRC to SF1 in the mammalian two-hybrid assay (data not shown). Furthermore, the physical interaction between this short 23-residue fragment and SF1 was also demonstrated in the GST pull-down assay (Fig. 4Go, lane 4). Together, these results demonstrate that the region from aa 278 to aa 300 in PNRC is critical and sufficient for interaction with nuclear receptors.

The region from residues 270–327 is rich in proline. A proline-rich sequence has been shown to be a target for binding proteins that contain a Src-homology-3 (SH3) domain (reviewed in Ref. 24). Structural and mutagenic analysis of peptide-SH3 complexes (25) shows that the core ligand for SH3 domain appears to be a seven-residue peptide containing the consensus sequence X-P-P-X-P, where X tends to be an aliphatic residue and the two conserved prolines (P) are crucial for high-affinity binding (24). There is a putative core ligand for SH3, S-D-P-P-S-P-S (aa 286–291), in the aa 278–300 region of PNRC. The biological significance of the core ligand sequence for SH3 binding domain in PNRC was investigated by the mutagenesis experiments. Double mutations of P287A and P290A in the putative core ligand for SH3, i.e. S-D-P-P-S-P-S to S-D-A-P-S-A-S, in aa 278–300 region of PNRC almost completely abolished the interactions between PNRC278-300 and the nuclear receptors tested including SF1, PR, TR, RAR, and RXR (Fig. 9AGo). The dramatic reduction of the interaction between mutated PNRC 278-300 and the nuclear receptors was not due to the reduction of the expression level of this mutant as demonstrated by Western blot on yeast protein extract with anti-Gal4 AD antibody (Fig. 9BGo). This result strongly supports the theory that this SH3-binding motif in the region aa 278–300 is essential for PNRC to interact with the nuclear receptors. However, as shown in Fig. 6BGo, the interaction between PNRC270-327 and SF1 is stronger than the interaction between PNRC278-300 and SF1, suggesting that other residues, especially the aa 270–278 region, may also participate in the interaction with SF1. Experiments are being carried out to further determine how the aa 270–278 region interacts with the nuclear receptors.



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Figure 9. Mutational Analysis of the Core Ligand Motif for SH3 Domain in PNRC

A, ß-Galactosidase activity. The yeast expression plasmid pACT2-PNRC278-300 (mutant) was prepared as follows: two complementary oligonucleotides with the coding sequences for PNRC278–300 carrying double mutations P287A and P290A were synthesized, annealed, and cloned into pACT2 vector through EcoRI site. Yeast strain Y187 was cotransformed with pACT2-PNRC278-300 (wild type), or pACT2-PNRC278-300(mutant), along with each type of the Gal4DBD fusion protein expression plasmids for SF1, PRHBD, TRHBD, RARHBD, and RXRHBD. The Y187 transformants carrying both plasmids were cultured in YPD medium containing a proper ligand (for SF1, no ligand), and the ß-galactosidase activity in these cells was determined. B, Western blot of yeast protein extracts. Yeast protein extracts were prepared from log-phase Y187 (grown in YPD medium) and Y187 harboring pGBT9-SF1 and pACT2-PNRC278-300(wild type) or pACT2-PNRC 278-300(mutant) (grown in SD/-Leu/-Trp medium) using a trichloroacetic acid method according to the procedures provided by CLONTECH Laboratories, Inc. (PT3024–1). The protein extracts corresponding to 3 A600 units of each type of yeast cultures were fractionated by 15% SDS-PAGE, transferred to a nitrocellulose membrane, and probed by Western blot with mouse monoclonal antibody against Gal4 AD (CLONTECH Laboratories, Inc.). The immuno-protein complex was detected by a 1:2500 diluted goat antimouse horseradish peroxidase conjugate (Pierce Chemical Co.) followed by the SupeSignal Substracte (Pierce Chemical Co.).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors are transcription factors that modulate transcription of various cellular genes, either positively or negatively, by interacting with specific hormone-responsive elements located in the target gene promoters, thereby controlling diverse aspects of cell growth, development, and homeostasis. The mechanisms by which the nuclear receptors can regulate the transcription from the target gene promoters are currently under intensive investigation. Recent data show that, in addition to contacting the basal transcriptional machinery directly, nuclear receptors enhance or inhibit transcription by recruiting an array of coactivator and corepressor proteins to the transcription complex. Recently, a number of these putative coregulatory proteins for nuclear receptors have been identified and have been shown to act either as coactivators or as corepressors (reviewed in Refs. 26, 27). Among the members of a growing family of coactivators, CBP and members of the SRC-1 gene family including SRC-1/p160 (21, 28, 29), TIF2/GRIP-1 (22, 30), and CBP/p300 (31, 32) function as coactivators of nuclear receptors, and RIP140 (14), TIF1 (33), and TRIP1/SUG-1(34, 35) functions have not yet been clearly defined. Most of these cofactors of nuclear receptors have molecular masses of approximately 160 kDa and share a common motif containing a core consensus sequence LXXLL (L, leucine; X, any amino acid.), which is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. LXXLL is thus a defining feature of p160 coactivators (23).

In this study, we have identified a novel nuclear receptor coactivator, PNRC. This protein, previously named B4–2, was first isolated by differential screening of a human natural killer (NK) cell line library (15), and its rat homolog was isolated from a rat bronchiolar epithelial cell cDNA library (16). PNRC encodes a deduced 327-aa protein with a calculated molecular mass of 35 kDa. This protein exhibits interesting structural features. It is very rich in proline, from 13.4% in human PNRC to 14.4% in the rat homolog. The exact function of proline-rich regions in proteins remains unclear; however, in many proteins it appears that they mediate functionally important binding interaction (36). PNRC also contains several SPXX or TPXX sequence motifs; proteins rich in these two motifs are frequently found to be gene-regulatory proteins (37). There is a potential nuclear localization sequence located at position 94–101 of PNRC. This sequence is thought to be necessary for nuclear proteins to translocate into the nucleus (38). Based on these structural features, PNRC was postulated to be involved in gene regulation (15). In this study, the nuclear localization of PNRC was demonstrated by immunofluorescent staining as well as Western blot analyses. We isolated PNRC through its interaction with the orphan receptor SF1. The results generated from yeast and mammalian two-hybrid assays, in vitro GST pull-down assay, coimmunoprecipitation, and functional analysis have provided several lines of evidence supporting the hypothesis that PNRC is a general coactivator for the nuclear receptor superfamily. Our results indicate that PNRC interacts with nuclear receptors in a specific manner, i.e. PNRC interacts with the nuclear receptors in a ligand-dependent and AF2-dependent manner. PNRC functions as a coactivator for SF1 and ERR1 in the absence of known ligands and as a coactivator for PR and TR only in the presence of their ligands.

Unlike most of the coactivators whose interactions with the nuclear receptors depend on the LXXLL motif, the interaction between PNRC and nuclear receptors is dependent on the SH3 binding motif, S-D-P-P-S-P-S, which is in a stretch of proline-rich sequence at its carboxy terminus, aa 278–300. This domain is not found in other nuclear receptor coactivators including SRC1, GRIP1, RIP140, TIFI, TIFII, ARA70, and CBP/p300 (using the BLAST sequence homology search). This exact 23-amino acid sequence is also found to be present in another protein isolated from the same yeast two-hybrid screening. These two proteins belong to a new family of nuclear receptor coregulatory proteins that interact with nuclear receptors through a different binding mechanism. Considering the core ligand motif for SH3 in PNRC, in addition to functioning as a nuclear receptor coactivator, PNRC may also play a role in signal transduction since most of the proteins possessing SH3 domains are involved in signal transduction (24). Studies in this area are being carried out in our laboratory.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials and Reagents
The MATCHMAKER Two-Hybrid System kit including a human mammary gland MATCHMAKER cDNA library, Gal4 AD monoclonal antibody, and the yeast culture media were purchased from CLONTECH Laboratories, Inc. DNA sequencing kits were from United States Biochemical Corp. (Cleveland, OH). T4 DNA ligase and various restriction endonuclease were purchased from New England Biolabs, Inc. (Beverly, MA) and Roche Molecular Biochemicals (Indianapolis, IN). AmpiTag polymerase was obtained from Perkin Elmer Corp. (Norwalk, CT). [14C]Chloramphenicol (D-threo-[dichloroacetyl-1-14C]chloramphenicol; specific radioactivity, 55 mCi/mmol) was from Amersham Pharmacia Biotech, Inc.(Arlington Heights, IL). The CAT expression vector, pUMSVOCAT, was a gift from Dr. K. Kurachi at the University of Michigan, Ann Arbor, MI. Oligonucleotide primers were synthesized in the DNA/RNA chemistry laboratory at the City of Hope. SK-BR-3 cells from ATCC (Manassas, VA), derived from a human breast carcinoma, were maintained in McCoy’s 5A medium containing 10% FCS and glutamine. 17ß-Estradiol, progesterone, retinoic acid, deoxycorticosterone, and testosterone were purchased from Sigma (St. Louis, MO). T3 was purchased from Calbiochem (La Jolla, CA). 9-cis-Retinoic acid was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). TNT-coupled reticulocyte lysate system was purchased from Promega Corp. (Madison, WI). pGEX2TK vector for expression of GST fusion protein and glutathione Sepharose 4B affinity matrix were purchased from Pharmacia Biotech (Piscataway, NJ). Yeast transformation kit was purchased from Bio 101 (La Jolla, CA). Mouse monoclonal IgG against human ER{alpha} was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The bovine SF1 cDNA clone was kindly provided by Dr. Keith L. Parker (Duke University Medical Center, Durham, NC).

Construction of Plasmids
All recombinant DNA and plasmid constructions were prepared according to standard procedures, and the sequences and orientations of inserted DNA fragments in plasmid constructs were verified by standard DNA sequencing. A yeast expression plasmid for DBDGal4-SF1 fusion protein, named pGBT9-SF1, was made by inserting PCR-amplified cDNA fragment coding for bovine SF1 into the EcoRI site of pGBT9 vector (CLONTECH Laboratories, Inc.). pUC13-B4–2, containing the full- length coding sequence for PNRC, was generated according to Chen et al. (15). The plasmid for overexpression of PNRC in mammalian cells was made by inserting the PCR-amplified fragment with BclI at both ends into the BamHI site in pSG5 vector (Stratagene, La Jolla, CA). To construct mammalian expression plasmids for bovine SF1, human ERR{alpha}1, and human ER{alpha} (pSG5-SF1, pSG5-ERR{alpha}1, and pSG5-ER{alpha}, respectively), the coding regions for SF1, ERR{alpha}1, and ER{alpha} were amplified by PCR with EcoRI site at both ends and inserted into the EcoRI site of pSG5 vector. To construct the yeast expression plasmids for various deletion mutants of PNRC and Gal4 AD fusion proteins, the DNA fragments coding for PNRCwild type, PNRC270-327, PNRC270-318, PNRC278-300, and PNRC319-327 were generated by PCR and inserted in frame into the Gal4 activation domain vector pACT2 through the BamHI/SacI, EcoRI/SacI, EcoRI/SacI, and BamHI/SacI sites, respectively. The PCR-amplified fragments of PNRC with different restriction sites were also inserted in a proper reading frame into pGEX2TK vector (Pharmacia Biotech) through the BamHI site to express GST-PNRC wild-type and deletion mutant-fusion proteins. A set of plasmids, named pM-SF1, pM-ERR{alpha}1, pVP16-PNRC, pVP16-PNRC270-327, and pVP16-PNRC278-300, for mammalian two-hybrid assay were prepared as follows: SF1 and ERR{alpha}1 cDNA fragments were excised from pGBT9-SF1 and pGBT9-ERR{alpha}1, respectively, and inserted in frame into Gal4 DNA-binding domain vector pM (CLONTECH Laboratories, Inc.) through the EcoRI site. PCR was used to generate a PNRC full-length coding region as well as the above mentioned deleted fragments with BclI at both ends. The PCR products were digested with BclI and inserted in proper reading frame into pVP16 activation domain vector (CLONTECH Laboratories, Inc.) at the BamHI site. To construct the plasmid pGBT9-ER274-595, the coding sequence from amino acids 274–595 (ligand-binding domain) of ER{alpha} was amplified by PCR with the sense primer 5'-GCCGAATTCGGGGAGGGCAGGG-GTGAAGTG-3' and antisense primer 5'-GGCGTCGACGGATCCTCAGACTGTGGCAGGGAAACCCTC-3' and cloned into the EcoRI/SalI site of the pGBT9 yeast expression plasmid. pSG5-GRIP1, several yeast expression plasmids coding for fusion proteins of Gal4-DBD and HBDs of AR, GR, PR, TR, RAR, and RXR in pGBT9 vector, and yeast expression plasmids for fusion proteins of Gal4BD and ER.HBD or mERR3.HBD containing mutations in AF2 domain were kindly provided by Dr. Michael R. Stallcup (University of Southern California, Los Angeles, CA) (17, 20, 30, 39). The CAT reporter plasmid, pUMS1.3CAT-(SF1site)3, for mammalian transfection experiments was prepared by inserting three copies of SF1 binding site from the human aromatase gene, 5'-CCAAGGTCAGAA-3', into pUMS-64/+5CAT that contains promoter 1.3 of the human aromatase gene through the HindIII site. pRE2tkCAT, pDR4tkCAT, pRSV-PR, and pRSV-TR plasmids were kind gifts from Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX).

Yeast Two-Hybrid Screening
The yeast MATCHMAKER two-hybrid system (CLONTECH Laboratories, Inc.) was used to screen a MATCHMAKER mammary gland cDNA expression library according to the supplier’s protocol (CLONTECH Laboratories, Inc. protocol PT1030–1) using pGBT9-SF1 as the bait. Briefly, the bait plasmid, pGBT9-SF1, was transformed into yeast reporter stain CG1945 along with a human mammary gland MATCHMAKER cDNA expression library (CLONTECH Laboratories, Inc.) in the Gal4 activation domain vector (pACT2, CLONTECH Laboratories, Inc.). Transformants (3.42 x 106) were screened first for HIS3 reporter gene expression by plating them onto plates lacking histidine, leucine, and tryptophan, and His+/Leu+/Trp+ transformants were recovered and further screened for ß-galactosidase activity using the colony-lift filter assay according to the supplier’s protocol. Plasmids of both histidine-positive and ß-galactosidase-positive transformants were isolated from yeast. The nucleotide sequences of inserts in true positive hybrid plasmids were generated, and the identity of the cDNAs was determined through a homology search against known sequences in GenBank.

Immunofluorescence Microscopy
Cos-1 cells were propagated in DMEM containing 10% FBS and antibiotics at 37 C in 5% CO2. Cos-1 cells were seeded on glass coverslips the day before experiments. The cells were fixed in 4% paraformaldehyde in PBS buffer, pH 7.5, for 20 min, followed by permeabilization in 0.1 Triton X-100. PNRC polyclonal antiserum diluted 1:300 in PBS buffer containing 1% goat serum was added and incubated for 30 min at room temperature. The cells were then washed with PBS containing 0.1% Triton X-100 three times. The cells were incubated with Cy-3-conjugated antirabbit antibodies (Sigma) for 20 min at room temperature. The washing procedure was repeated after incubation. Cells were observed using a LSM confocal microscope (Carl Zeiss, Thornwood, NY).

Western Blot and Coimmunoprecipitation
Western analyses were done as previously described (40). For immunoprecipitation analysis, Hela cells in 60-mm tissue culture dishes were transfected with both pSG5-ER and pSG5-PNRC plasmid DNAs (10 µg each) or Lipofectin (BRL, Rockville, MD) alone (mock transfection). One day after transfection, the cells were cultured in MEM containing 5% charcoal-treated FBS and 100 nM of 17ß-estradiol for 24 h. The cells were then harvested for immunoprecipitation analyses using Immunoprecipitation Kit (protein A) (Roche Molecular Biochemicals) according to the manufacturer’s instructions. After being precleared by Protein A agarose, the lysate of transfected Hela cells was incubated first with anti-ER{alpha} antibody (Santa Cruz Biotechnology, Inc.) at 4 C for 4 h and then continuously incubated overnight with protein A agarose. The immunoprecipitated protein complexes were eluted from agarose beads, fractionated by 10% SDS-PAGE, and probed by Western blot with anti-PNRC antiserum or anti-ER{alpha} antibody separately.

Protein-Protein Interaction Assays
Yeast and mammalian two-hybrid assays (CLONTECH Laboratories, Inc.) were used to examine protein-protein interactions in vivo. Yeast mating approach was used in yeast two-hybrid assays to study protein-protein interactions as described in the protocol of CLONTECH Laboratories, Inc. Briefly, the yeast strain Y187 was transformed with pGBT9 (DNA-BD vector only), pGBT9/target plasmids (for SF1, ERR{alpha}1, and HBDs of other nuclear receptors), or DNA-BD/control plasmids (as negative control). The AD vector alone or wild-type PNRC and its deleted fragments containing pACT2 derivatives were used to transform yeast strain CG1945. The yeast mating was performed by picking one colony from each type and growing both colonies in liquid yeast extract-peptone-dextrose (YPD) medium with or without a proper ligand at the following concentrations: 100 nM estradiol for ER; 10 µM deoxycorticosterone for GR; 100 nM dihydrotestosterone for AR; 500 nM of progesterone for PR; 10 µM T3 for TR; 10 µM all-trans-retinoic acid for RAR; and 10 µM of 9-cis-retinoic acid for RXR. The same amount of an aliquot of the mating culture was spread on both SD/-Leu/-Trp plates to select diploid strains bearing both plasmids and SD/-Leu/-Trp/-His/+3-AT plates to score the growth of the diploid cells that express the two interacting target proteins. The His3-positive colonies were further analyzed for ß-galactosidase activity by liquid ß-galactosidase activity measurement as essentially described in the protocol. Mammalian two-hybrid assays in SK-BR-3 cells were performed according to the procedures described in the CLONTECH Laboratories, Inc. protocol (PT3002–1).

In Vitro Protein-Protein Interaction Assay (GST Pull-Down)
The wild-type bovine SF1 cDNA in pSG5 vector was translated in vitro in the presence of [35S]methionine, using the TNT Coupled Reticulocyte Lysate System (Promega Corp., Madison, WI). GST and deleted PNRC fusion proteins, GST-PNRC 270-327 and GST-PNRC278-300, were prepared using the affinity matrix glutathione Sepharose 4B (Pharmacia Biotech) according to the supplier’s instructions. Briefly, E. coli BL21 transformants carrying pGEX2TK-PNRC270-327 and pGEX2TK-PNRC278-300 were grown in LB medium to an A600 of 0.6 at 37 C, and the expression of fusion proteins was induced by addition of isopropylthiogalactosidase to a final concentration of 0.1 mM, after which incubation was continued for another 2–3 h. The cells were collected by centrifugation and lysed with B-PER Bacterial Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL). To about 100 µg total protein extract, 30 µl of 50% PBS-washed glutathione Sepharose 4B beads were added and mixed for 10 min at room temperature. The washed beads were incubated for 2 h at 4 C with 4 µl in vitro translated, [35S]methionine-labeled SF1 in a total volume of 150 µl incubation buffer (50 mM Kpi, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 10% glycerol, 0.1% Tween 20, 1.5% BSA) (41). Beads were collected by microcentrifugation and washed three times with incubation buffer without BSA. Washed beads were resuspended in 50 µl of 1xSDS sample buffer, boiled in water for 5 min, and pelleted briefly in a microfuge. Supernatant (25 µl), along with 1/10 of the input [35S]methionine-labeled SF-1, was then subjected to 10% SDS-PAGE. To control the equal loading of GST fusion proteins, gel was stained with Coomasie blue before being visualized by autoradiography. For quantification, autoradiographs were analyzed using PhosphorImager.

Mammalian Cell Transfections and CAT Assays
SK-BR-3 cells were maintained in McCoy’s 5A medium supplemented with 10% FBS, penicillin, and streptomycin at 37 C and 5% CO2. Hela cells were cultured in MEM Earle’s Salts medium supplemented with 5% charcoal dextran-treated FBS for 24 h. Cells were cultured in six-well plates for 24 h and transfected with 10 µg of Lipofectin (Life Technologies, Inc., Gaithersburg, MD) and 3.5 µg of plasmid DNA. The overall amount of total DNA in all transfections was the same by including appropriate amounts of empty vector pSG5 in addition to specific amounts of the test plasmids indicated in each experiment. After overnight incubation, media containing lipofectin and DNA were removed, and the cells were cultured in the regular growth medium. After 24 h incubation, the cells were harvested from the plates by scraping, pelleted by centrifugation, resuspended in 0.25 M Tris-HCl, pH 8.0, and disrupted by freeze-thawing four times. CAT activity in the cell extract containing an equal amount of the protein from each sample was determined by the liquid scintillation counting (LSC) method (42). Briefly, the appropriate amount of cell extracts was incubated in a reaction containing 14C-labeled chloramphenicol and n-butyryl Coenzyme A. The reaction products were extracted with a small volume of xylene. The xylene phase was mixed with scintillant and counted in a scintillation counter. Diagnostic cotransfections with a control plasmid, pSV-ß-Gal, showed the reproducibility of the transfection efficiency in our experimental conditions. For each study, the mean and SD of triplicate dishes from a single experiment and the representative of at least three independent experiments are presented.


    ACKNOWLEDGMENTS
 
We thank Dr. K. Kurachi for pUMSVOCAT vector; Dr. K. L. Parker for bovine SF1 cDNA; Dr. M. R. Stallcup for expression plasmids of Gal4-DBD fusion proteins, ER and ERR3 mutants; and Dr. M.-J. Tsai for PR and TR expression plasmids.


    FOOTNOTES
 
Address requests for reprints to: S. Chen, Division of Immunology, Beckman Coulter, Inc. Research Institute of the City of Hope, Duarte, California 91010.

This research was supported by NIH Grant CA-44735.

Received for publication October 11, 1999. Revision received March 9, 2000. Accepted for publication March 16, 2000.


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