|Molecular Vision 2005;
Received 22 March 2005 | Accepted 1 July 2005 | Published 6 July 2005
Expression and localization of leucine-rich B7 protein in human ocular tissues
Elena S. Tasheva, Ke An, Daniel L. Boyle,
Gary W. Conrad
Division of Biology, Kansas State University, Manhattan, KS
Correspondence to: Elena S. Tasheva, Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS, 66506-4901; Phone: (785) 532-6553; FAX: (785) 532-6653; email: firstname.lastname@example.org
Purpose: Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells.
Methods: Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy.
Results: RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize.
Conclusions: The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.
The gene encoding the leucine-rich B7 proteins was first described in 1996 as a part of a gene-rich cluster on human chromosome 12p13 [1,2]. Since then, the exon/intron boundaries, splice variants, and tissue expression pattern of the B7 gene have been determined mainly using various computer software programs. The B7 gene spans about 9.4 kb of continuous DNA sequence and is composed of eight exons. Two differentially spliced B7 mRNAs are transcribed from this gene. Transcript variant 1 contains all 8 exons and encodes a protein of 343 amino acids. Transcript variant 2 lacks exon 7 and encodes a protein of 312 aa. Analysis of B7 protein structure using web-based research tools  revealed that both protein isoforms contain 3 leucine-rich repeats.
The small leucine-rich proteoglycans (SLRP) are a well known family of secreted proteoglycans present in many connective tissues (reviewed in ). In the eye, they have been shown to be equally important for development and maintenance of corneal transparency and for providing the structural link between the neural retina and retinal pigment epithelium [5,6]. However, the molecular mechanisms by which the SLRPs exert their function are not well defined. To determine potential mimecan-interacting proteins, we used the search engine named Harvester  and found that B7 proteins may functionally associate with several SLRPs, including mimecan. The finding that leucine-rich B7 protein may functionally interact with SLRP family members prompted us to explore its expression in human ocular tissues. The present study is the first report on leucine-rich B7 protein expression and localization.
The Gateway Technology (Invitrogen Corporation, Carlsbad, CA) was used to generate the pcDNA-Exp40/Bovine Mimecan expression vector. Briefly, a cDNA fragment (897 bp) encoding the bovine mimecan open reading frame (ORF) was amplified using primers that contained attB sites and template that contained the bovine mimecan cDNA (AF017339) [8,9]. The amplified product was recombined with pDONR221 (Invitrogen) in an attB-attP (BP) recombination reaction to generate the pENTR221/Bovine Mimecan construct. After sequence verification, the pENTR221/Bovine Mimecan construct was recombined with pcDNA-DEST40 (Invitrogen) in an attL-attR (LR) recombination reaction to generate pcDNA-Exp40/Bovine Mimecan.
The expression vector, pReceiver.B7, containing a leucine-rich B7 ORF (NM_006992) fused to an N-terminal his tag was obtained from GeneCopoeia, Inc. (Germantown, MD). Primers were designed to amplify the B7 ORF from the pReceiver.B7 vector. An EcoRI site was incorporated into the forward primer and a BamHI site was incorporated into the reverse primer. The amplified DNA fragment containing the B7 ORF was digested with EcoRI and BamHI and ligated into the 5'-EcoRI to BamHI-3' site of the pEGFP-N2 vector (Clontech, Palo Alto, CA) to generate the pEGFP.B7 construct (Figure 1).
A polyclonal antibody against human leucine-rich B7 protein was raised by immunizing a rabbit with a synthetic peptide, whose sequence corresponded with the N-terminal region of the B7 protein (PTPLTEDMMKEGLSLLC; NP_008923), coupled to Keyhole Limpet Hemocyanin. For antibody generation, a custom service provided by Abgent, Inc. (San Diego, CA) was used. The purified antibody was shown to react with human B7 proteins by immunoblot and immunohistochemistry (IHC) analyses. The anti-V5 epitope antibody (catalog number 46-0705) was obtained from Invitrogen. Secondary antibodies, goat anti-mouse IgG Alexa Fluor 546 conjugated (catalog number A-11003), was obtained from Molecular Probes, Inc. (Eugene, OR) and goat anti-mouse IgG FITC conjugated (catalog number 1211-0231) was obtained from OrganoTeknika Corp. (West Chester, PA).
Reverse transcription-polymerase chain reaction (RT-PCR)
Whole human eyes were provided by the Missouri Lions Eye Bank (Columbia, MO). Total RNA was isolated from human eye tissues using the Totally RNA Isolation Kit (Ambion Inc., Austin, TX). Total RNA from normal human tissues was obtained from Ambion. RNA (2 μg) was reverse-transcribed using the anchor primer oligonucleotide (dT)18, and Superscript II Reverse Transcriptase (Life Technologies, Inc., Gaithersburg, MD). The single stranded cDNA products (2 μl) were used as templates in PCR amplification reactions as described [10,11]. The gene specific primers for differentially spliced B7 transcripts were designed from nucleotide sequences contained in GenBank NM_201650 and NM_006992, respectively. The primer sequences, synthesized by Integrated DNA Technologies Inc. (Coralville, IA), are shown in Table 1. One set (B7+515 [forward] and B7-944 [reverse]) spans the sequence from exons 7 and 6 while the other set (B7+515 [forward] and B7-950 [reverse]) spans the sequence from exon 8 and 6 (Figure 2). The resulting PCR products were resolved by agarose gel electrophoresis, excised from the gel, cloned into the pGEM-T vector (Promega Corp., Madison, WI), and sequence verified. A Hi-Lo DNA molecular weight marker (Minnesota Molecular, Minneapolis, MN) was used to determine fragment size. Quantum RNA18S Internal Standard (Ambion, catalog number 1717, amplifying a 324 bp fragment) was used as an endogenous standard in all PCR reactions. A 3:7 ratio of 18S primers to 18S competimers was used, as previously described [10,11].
For in vitro synthesis of B7 protein isoform 2 the TNT® Coupled Reticulocyte Lysate System (Promega Corporation, Madison, WI) with pReceiver.B7 as template was used according to the manufacturer's protocol. An aliquot of in vitro transcribed (IVT) B7 protein was diluted 1:1 in sample buffer and loaded onto a polyacrylamide gel. After electrophoresis, the proteins were transferred to a PVDF membrane. The blot was incubated with antibody B7.N, rinsed, exposed to a horseradish peroxidase conjugated secondary antibody, rinsed again, and incubated in SuperSignal West Pico Chemiluminescent reagent (catalog number 34077; Pierce Biotechnology, Inc., Rockford, IL). The blot was then exposed to blue lite autorad film (ISC BioExpress, Kaysville, UT).
Custom IHC on slides containing normal adult human eye tissues was performed by SuperBioChips Laboratories (Seoul, Korea) as previously described [10,11].
Cell culture, transient transfection and cell immunofluorescence staining
MG-63 cells were maintained in Dulbeco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics as previously described [12,13]. Transient transfection experiments were performed using FuGENE-6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol (12 μl reagent per 4 μg DNA) as described . For co-transfection experiments, 2 μg of each pEGFP.B7 and pcDNA-Exp40/BovineMimecan were used. For analysis of subcellular localization of mimecan and B7 proteins, cells were grown on coverslips. After transfection (48 h) cells were fixed in 3% paraformaldehyde in PBS for 15 min at room temperature, rinsed with PBS containing 0.2 M glycine, permeabilized with 0.1% Triton-X in PBS for 1 min at room temperature, and rinsed with PBS. For direct visualization of EGFP, fluorescence coverslips were mounted onto glass slides using VectaShield mounting medium (Vector Laboratories, Burlingame, CA) and sealed with nail polish. For indirect immunofluorescence (following fixation and permeabilization), cells were blocked with 5% (w/v) bovine serum albumin in PBS for 1 h at 20 °C and then incubated with primary antibody for 1 h at room temperature. Following incubation, the coverslips were washed 5 times in PBS and then incubated at room temperature for 1 h with the secondary antibody. Digital photomicrographs were generated using a Zeiss LSM 5 PASCAL confocal laser scanning microscope. The images were saved in JPEG format and processed with Adobe Photoshop 7.0 software.
Expression of leucine-rich B7 mRNA isoforms in human ocular and nonocular tissues
The expression of B7 mRNA transcripts was examined by RT-PCR. Primers were designed to flank introns and to detect an amplicon of 429 bp for B, isoform 1, and an amplicon of 435 bp for B7, isoform 2. As shown in Figure 2A, the forward primer contained sequences from B7 exon 4, whereas reverse primers contained sequences from exons 8 and 6 (for amplification of isoform 2) or sequences from exons 7 and 6 (for amplification of isoform 1; Figure 2A). The fidelity of PCR reactions was verified by sequencing. B7, isoform 2 was detected at moderate levels in cornea, iris, and sclera, and at a high level in retina. In contrast, B7, isoform 1 was detected at high levels in human cornea, but at very low levels in the retina (Figure 2B). Both isoforms were expressed at different levels in several nonocular human tissues, including brain, blood, lung, skeletal muscle, testis, and uterus (Figure 2B).
Expression of B7 protein in human eye
The peptide sequence that we have used for generation of an anti-human B7 antibody contained amino acid sequences from the NH2-terminal region of B7. Because the NH2-terminal region is identical in both B7 proteins (Figure 2A), our antibody would not be able to distinguish isoform 1 from isoform 2 of B7. To test whether the B7.N antibody recognized isoform 2 of B7 (the isoform that was found expressed in human eye tissues by PT-PCR in the initial experiments), western blot analysis was performed (Figure 3A). Isoform 2 of B7 was synthesized in vitro from pReceiver.B7, a plasmid that contained an ORF encoding isoform 2 of B7 (Figure 1). As a control, IVT was performed without addition of plasmid DNA template to the reaction. Indeed, on western blot analysis B7.N antibody recognized a band of about 31 kDa, the expected molecular weight of B7, isoform 2 (Figure 3A, lane 1). The smaller band (Figure 3A, lane 1) is most likely a proteolytically cleaved protein, for no protease inhibitors were added to the IVT reaction. B7.N antibody specificity was shown by a lack of recognition of any band from control IVT, performed without the addition of pReceiver.B7 (Figure 3A, lane 2). The specificity of our antibody was further confirmed by IHC analysis. As shown in Figure 3B, human spleen tissue did not show specific immunolabeling for B7, whereas skin epidermis was positive for B7. These results are consistent with gene expression data suggested by analysis of expressed sequence tag (EST) counts for the B7 gene UniGene database (NCBI, Bethesda, MD).
To determine the expression of B7 in human ocular tissues, IHC analyses on adult human eyes were performed. In the cornea, immunolabeling of B7 revealed deposits of this protein in corneal epithelial and endothelial layers (Figure 4, white arrows). Immunolabeling also was present in lens epithelial and fiber cells, and retina. In the retina, B7 immunolabeling was prominent in rod and cone layers, and was not detectable in other retinal layers (Figure 4, white arrows). Very low B7 expression was detected in iris and sclera (Figure 4). Taken together, the results presented so far establish the expression of leucine-rich B7 protein in human eye tissues.
Subcellular localization of EGFP.B7 and mimecan/V5/6xHis fusion proteins
The subcellular localization of B7 and mimecan fusion proteins was determined using transient transfections and confocal microscopy. MG-63 cells were chosen for these experiments for several reasons. First, MG-63 cells are easy to transfect. Second, in contrast to primary corneal keratocytes and other primary cell types, contact with transfection reagents does not change cellular morphology of MG-63 cells. Third, MG-63 cells are known to express both mimecan [12-14], and B7 (unpublished). The results from these studies are shown in Figure 5 and Figure 6. B7 localization was determined by direct visualization of EGFP fluorescence (Figure 5A,B). For mimecan detection, two types of secondary antibodies were used, mouse IgG, Alexa Fluor 546 conjugated and mouse IgG, FITC conjugated (Figure 5C,D). Both EGFP.B7 and mimecan/V5/6xHis fusion proteins were found to localize to the cytoplasm of MG-63 cells (Figure 5). In addition, EGFP.B7 protein also was found in the nucleus of these cells (Figure 5A,B).
Additionally, MG-63 cells transiently co-transfected with both EGFP.B7 and mimecan/V5/6xHis were analyzed by confocal microscopy (Figure 6). B7 fusion protein in cytoplasm was found to co-localize with mimecan, shown by the yellow color in the merged images (Figure 6E,F).
In this study we used a search engine named Harvester to screen the human proteome for potential mimecan/osteoglycin interacting proteins. Detailed information about Harvester and how it works can be found at the above Web site. Briefly, Harvester searches and crosslinks public bioinformatic databases and prediction servers, thereby providing fast access to protein specific bioinformatic information. With this approach we found that the leucine-rich B7 protein may functionally interact with several SLRP family members, including mimecan. Surprisingly, our attempt to obtain additional information about B7 protein by searching PubMed resulted in a total of 3 publications describing the cloning of B7 gene as a part of a gene-rich cluster on human chromosome 12p13 [1,2,15]. The absence of published studies on tissue-specific expression, cellular localization, and molecular function of leucine-rich B7 protein was the main reason to select B7 for our studies. As a prelude to conducting experiments to test the hypothesis that B7 functionally (or physically) interacts with mimecan, it was important to determine the expression pattern of B7 proteins. In this study we have used RT-PCR, IHC, and immunofluorescent cell staining to analyze the expression of leucine-rich B7 in the human eye and MG-63 cells. The results of our study showed that variant 2 of B7 mRNA is expressed in human cornea, iris, sclera, and retina, whereas variant 1 of B7 mRNA was detected at high levels in the cornea, but only at trace amounts in the retina. Both differentially spliced B7 mRNAs were detected in other human tissues, including brain, blood, and some internal organs (Figure 2). IHC analysis confirmed the results from RT-PCR and showed that B7 is present in epithelial and endothelial layers of the cornea, in epithelial and fiber cells of the lens, and in the rod and cone layer of the retina. Light immunostaining also was detectable in sclera and iris (Figure 4). The results from this study demonstrate that B7 is expressed in the human eye. Notably, the sites of expression of the B7 in the human lens and retina correlates with the sites of expression of mimecan .
The results of the current study also indicate that B7 is an intracellular protein. Confocal microscopy analyses showed that isoform 2 of B7 localized to both the nucleus and endoplasmic reticulum of MG-63 cells (Figure 5A,B). Such localization is similar to that of other proteins shown to be involved in cell signaling and transcription, such as SMADs, p38MAPK, and STATs (reviewed in [16-18]). Analysis of B7 structure using bioinformatics tools  revealed that B7 contains leucine-rich repeat (LRR) motifs, a nuclear localization signal (NLS), and leucine zipper motifs. Leucine-rich repeats are short sequence motifs found in a variety of cytoplasmic, membrane, and extracellular proteins, including proteins of the SLRP gene family . A common property of LRR proteins involves protein-protein interactions. Each of the two B7 isoforms contains 3 LRRs. The nuclear localization signal is a short amino acid region that is rich in basic residues . In both plants and animals, proteins are targeted to the nucleus by specific NLS. It is of interest to note that a consensus NLS (K/R-[X]7-RR) can be found near the carboxy-terminus of B7, isoform 2 (amino acids 245-254: KSLQYLNLRR), but not in B7 isoform 1. In B7, isoform 1 the last arginine residue of NLS is replaced with glycine residues as a result of differential splicing. The leucine zipper motif (consensus L-X-L-X-L-X-L) is present in many gene regulatory proteins, such as the Jun/AP1 family of transcription factors, and the cAMP response element (CRE) binding proteins [21-24]. The leucine zipper motif can be found near the amino-terminus of both B7 isoforms (amino acids 59-81). Considering the combination of motifs encoded within B7 the results from our confocal microscopy studies are not surprising.
Confocal microscopy analyses also revealed co-localization of B7 and mimecan in MG-63 cells transfected with pEGFP.B7 and pcDNA-Exp40/Bovine mimecan plasmids, as demonstrated by merged images shown in Figure 6E,F. The significance of such possible interactions remains to be determined.
In conclusion, to our knowledge this is the first report demonstrating the expression and localization of leucine-rich B7 mRNA and protein. Our studies should provide the basis for future analyses of B7 in health and disease. The combination of motifs and intracellular localization of B7 strongly suggests functional importance of these proteins in human eyes.
This work was supported by NIH Grant EY13395 to GWC and EST. We thank Dr. Ron Walkenbach, Tina Livesay, and Amy Giangiacomo of the Missouri Lions Eye Bank for the human eyes. We also wish to acknowledge the Kansas State University Biology Microscopy and Image Processing Facility, which has been supported, in part, by University resources and by the Kansas Agricultural Experimental Station.
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