Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein.

t(12;21) is the most frequent translocation found in pediatric B-cell acute lymphoblastic leukemias. This translocation fuses a putative repressor domain from the TEL DNA-binding protein to nearly all of the AML-1B transcription factor. Here, we demonstrate that fusion of the TEL pointed domain to the GAL4 DNA-binding domain resulted in sequence-specific transcriptional repression, indicating that the pointed domain is a portable repression motif. The TEL pointed domain functioned equally well when the GAL4 DNA-binding sites were moved 600 bp from the promoter, suggesting an active mechanism of repression. This lead us to demonstrate that wild-type TEL and the t(12;21) fusion protein bind the mSin3A corepressor. In the fusion protein, both TEL and AML-1B contribute mSin3 interaction domains. Deletion mutagenesis indicated that both the TEL and AML-1B mSin3-binding domains contribute to repression by the fusion protein. While both TEL and AML-1B associate with mSin3A, TEL/AML-1B appears to bind this corepressor much more stably than either wild-type protein, suggesting a mode of action for the t(12;21) fusion protein.

TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1.

TEL is a member of the ETS family of transcription factors that interacts with the mSin3 and SMRT corepressors to regulate transcription. TEL is biallelically disrupted in acute leukemia, and loss of heterozygosity at the TEL locus has been observed in various cancers. Here we show that expression of TEL in Ras-transformed NIH 3T3 cells inhibits cell growth in soft agar and in normal cultures. Unexpectedly, cells expressing both Ras and TEL grew as aggregates. To begin to explain the morphology of Ras-plus TEL-expressing cells, we demonstrated that the endogenous matrix metalloproteinase stromelysin-1 was repressed by TEL. TEL bound sequences in the stromelysin-1 promoter and repressed the promoter in transient-expression assays, suggesting that it is a direct target for TEL-mediated regulation. Mutants of TEL that removed a binding site for the mSin3A corepressor but retained the ETS domain failed to repress stromelysin-1. When BB-94, a matrix metalloproteinase inhibitor, was added to the culture medium of Ras-expressing cells, it caused a cell aggregation phenotype similar to that caused by TEL expression. In addition, TEL inhibited the invasiveness of Ras-transformed cells in vitro and in vivo. Our results suggest that TEL acts as a tumor suppressor, in part, by transcriptional repression of stromelysin-1.

Role of histone deacetylases in acute leukemia.

Accumulating evidence points to a connection between cancer and transcriptional control by histone acetylation and deacetylation. This is particularly true with regard to the acute leukemias, many of which are caused by fusion proteins that have been created by chromosomal translocations. Genetic rearrangements that disrupt the retinoic acid receptor-alpha and acute myeloid leukemia-1 genes create fusion proteins that block terminal differentiation of hematopoietic cells by repressing transcription. These fusion proteins interact with nuclear hormone co-repressors, which recruit histone deacetylases to promoters to repress transcription. This finding suggests that proteins within the histone deacetylase complexes may be potential targets for pharmaceutical intervention in many leukemia patients.

Glycosylation is critical for natriuretic peptide receptor-B function.

Co-transfection of a truncated natriuretic peptide receptor-B (NPR-B) with the full length receptor results in a decrease of 60-80% in wild-type receptor activity. This reduction correlates with a loss of glycosylation of the full length NPR-B. This effect is dose-dependent, and occurs with no change in the glycosylation of the truncated receptor. Co-transfection of the full length NPR-B with other receptors yields similar results. These data suggest that glycosylation may be crucial for NPR-B function. Cross-linking studies further demonstrate that only fully glycosylated NPR-B receptors are able to bind ligand. Our data therefore argue that carbohydrate modification may be critical for NPR-B receptor ligand binding.

The human 5-hydroxytryptamine 1A receptor differentially modulates phospholipase C and adenylyl cyclase activities.

In this study, we quantitate and compare the ability of the 5-hydroxytryptamine 1A (5-HT1A) receptor to modulate the activities of phospholipase C and adenylyl cyclase as a function of receptor concentration. We used a single clonal cell line permanently expressing the human 5-HT1A receptor, and progressively depleted the receptor concentration using an alkylating antagonist (N8-bromoacetyl-N1-3'-(4-indolyloxy)-2'-hydroxypropyl-Z-1,8-diamin o-p-methane, (+-) Pindobind). For serotonin-induced phospholipase C stimulation, reductions in receptor number result in dose-response curves that shift downward and rightward, reflecting both a decreasing maximal effect as well as an increasing ED50. In contrast, depletion of more than 95% of the receptors has no effect on the maximal inhibition of forskolin-stimulated adenylyl cyclase activity. Moreover, at all receptor concentrations, the amount of serotonin required to produce half-maximal phospholipase C stimulation is several-fold more than that required to produce half-maximal inhibition of adenylyl cyclase activity. We conclude that the 5-HT1A receptor modulates these two pathways differently, and that the overall response to challenge with serotonin, in terms of both phosphatidyl inositol hydrolysis and cyclic AMP production, is dependent upon receptor number.

Glycosylation of asparagine 24 of the natriuretic peptide receptor-B is crucial for the formation of a competent ligand binding domain.

UV cross-linking studies of the natriuretic peptide receptor-B (NPR-B) using radiolabeled C-type natriuretic peptide (CNP) indicate that only fully glycosylated receptors are capable of binding ligand. We therefore used site-directed mutagenesis to determine which potential glycosylation sites are occupied by carbohydrate, and the relevant mutants were characterized in order to understand the function of carbohydrate addition at those sites. Our results suggest that five of seven potential N-linked glycosylation sites are modified. In addition, mutation of asparagine 24 results in a loss of approximately 90% of receptor activity. This mutant is expressed at levels comparable to the wild-type receptor, and its activity is not significantly different from that of wild-type NPR-B in terms of EC50 for CNP. Ligand binding studies on this mutant further show that although there is no change in affinity for ligand, approximately 90% of receptor binding is lost. These data suggest that many of the mutant receptors are simply not properly folded. Our results indicate that glycosylation of asparagine 24 of NPR-B receptors may be critical for the formation of a competent ligand binding domain.

Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes.

The pathogenic human retrovirus human immunodeficiency virus type 1 (HIV-1) encodes two trans-acting nuclear proteins, tat and rev, whose functional expression is essential for viral replication in vitro. The tat protein greatly enhances the expression of both structural and regulatory genes of HIV-1 (linked to the viral long-terminal-repeat promoter element), whereas the rev gene product (previously termed art or trs) has only been shown to be required for the synthesis of structural proteins. Here, we demonstrate that rev also moderates the expression of regulatory genes of HIV-1. It decreases the expression of messenger RNAs that encode the full-length form of the viral tat gene product or the rev protein itself, and induces the synthesis of a previously unreported, truncated tat protein. These actions of rev are mediated by a dramatic shift in the ratio of spliced to unspliced cytoplasmic HIV-1 mRNA. Therefore rev not only activates the synthesis of the viral structural proteins, but also modulates the level and quality of HIV-1 regulatory gene expression.

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

Cloning and functional expression of the bovine natriuretic peptide receptor-B (natriuretic factor R1c subtype.

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED50 of 12 +/- 2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a Kd of 13 +/- 1 pM, and natriuretic peptides compete for [125I]-CNP binding with a rank order of pCNP > pBNP > rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.

Comparative binding study of rat natriuretic peptide receptor-A.

The natriuretic peptide receptor-A (NPR-A) is involved in blood pressure and body fluid regulation in order to help maintain cardiovascular homeostasis. It has been shown that these biological effects are mediated through the natriuretic peptide family of hormones, which bind NPR-A according to the rank order ANP>BNP>>CNP. Previous studies performed with rat kidney papillary tissue suggested the existence of an heterologous NPR-A population since two binding components were obtained for pBNP32, one of high affinity (pK 9.4 +/- 0.1) and the other of lower affinity (pK 7.5 +/- 0.1), while in the same preparation rANP28 binding displayed the expected affinity (pK 10.22 +/- 0.01) and was best fitted with a model involving a single class of binding sites. This apparent heterogeneity of NPR-A in rat kidney papillae could be explained by the presence of two receptor isoforms or of monomeric and oligomeric forms of the same receptor. To investigate the NPR-A binding heterogeneity, we have cloned the rat NPR-A from PC12 cells and compared its pharmacological profile with that of the papillae. Our results with rat NPR-A transfected Cos-P cells show an equivalent pharmacological profile as with the rat tissue, i.e. a high affinity for rANP28 (pK 10.4 +/- 0.1) and two distinctive affinities for pBNP32 (pK 9.74 +/- 0.05 and 7.8 +/- 0.1). Although multiple receptor glycoforms were sometimes detectable by western blotting, only one molecular form was obtained by cross-linking with 125I-rANP28. It thus appears that NPR-A alone can account for the two binding components found in the rat papillae and that a single molecular form of the protein is implicated. We therefore propose that the oligomerization state of the receptors could be responsible for the apparent binding heterogeneity of rat NPR-A.

Characterization of the phosphorylation state of natriuretic peptide receptor-C.

Many internalized receptors are known to be phosphorylated within their cytoplasmic domain. Natriuretic peptide receptor-C (NPR-C) is a covalent homodimer primarily involved in the internalization of bound ligand resulting in tissue uptake and degradation of natriuretic peptides. In this report, we have investigated the phosphorylation state of NPR-C receptors present at high level in rat aortic smooth muscle cells (RASM). 32P labeled cells, NPR-C purification and phosphoamino acid analysis clearly demonstrate that NPR-C exists as a phosphoprotein in RASM cells and that phosphorylation occurs exclusively on serine residues. Transient expression of bovine NPR-C in Cos-P cells of kidney origin confirmed that phosphorylation occurs within the cytoplasmic domain of the receptor. These results provide the first evidence for NPR-C phosphorylation as well as a model for future studies of its role in altering receptor function.

Functional analysis of the Tat trans activator of human immunodeficiency virus type 2.

The trans-activator (Tat) proteins of the related but distinct type 1 and type 2 human immunodeficiency viruses (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation of this observation, suggested by computer analysis of potential RNA secondary structures within the viral trans-activation response (TAR) elements, is that HIV-2 Tat requires the presentation of two viral RNA stem-loop sequences for full activity whereas HIV-1 Tat is maximally active upon presentation of a single stem-loop structure. Here, we demonstrate that the HIV-2 long terminal repeat indeed contains two functionally independent TAR elements. However, the second (3') TAR element of HIV-2 is significantly less active than the 5' TAR element and is functionally masked in the context of an intact HIV-2 long terminal repeat. Evidence is presented suggesting that the activities of these two HIV-2 TAR elements reflect, at least in part, their relative distances from the site of transcription initiation. Although the HIV-2 TAR element proximal to the viral mRNA cap site appears to be sufficient for effective trans activation by HIV-2 Tat in vitro, this functional redundancy may nevertheless serve to enhance HIV-2 replication in infected cells in vivo.

Functional characterization of a complex protein-DNA-binding domain located within the human immunodeficiency virus type 1 long terminal repeat leader region.

Transcriptional trans activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by the viral tat trans activator is mediated by an LTR-specific sequence located immediately 3' to the start of transcription initiation. We have used a range of molecular techniques to examine DNA-protein interactions that occur in the vicinity of this cis-acting sequence. Our results demonstrate the existence of a sequence-specific DNA-protein interaction involving the HIV-1 leader DNA and map this binding event to between -2 and +21 base pairs relative to the HIV-1 LTR transcription start site. Evidence suggesting that this interaction involves three distinct protein-DNA contact sites extending along one side of the DNA helix is presented. Mutation of these sites was found to ablate protein-DNA binding yet was observed to have no effect on either the basal or tat trans-activated level of HIV-1 LTR-specific gene expression. We therefore conclude that this DNA-protein interaction has a function distinct from the regulation of HIV-1 LTR-specific gene expression.

Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species.

The known primate lentiviruses can be divided into two subgroups consisting of the human immunodeficiency virus type 1 (HIV-1) isolates and the related HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) isolates. HIV-1 has been shown to encode a post-transcriptional trans-activator of viral structural gene expression, termed Rev, that is essential for viral replication in culture. Here, we demonstrate that HIV-2 and SIVmac also encode functional Rev proteins. As in the case of HIV-1, these Rev trans-activators are shown to induce the cytoplasmic expression of the unspliced viral transcripts that encode the viral structural proteins. Unexpectedly, the Rev proteins of HIV-2 and SIVmac proved incapable of activating the cytoplasmic expression of unspliced HIV-1 transcripts, whereas HIV-1 Rev was fully functional in the HIV-2/SIV system. This nonreciprocal complementation may imply a direct role for Rev in mediating the recognition of its viral RNA target sequence.