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1.
Mol Endocrinol ; 14(5): 605-22, 2000 May.
Article in English | MEDLINE | ID: mdl-10809226

ABSTRACT

Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.


Subject(s)
Peptides/pharmacology , Receptors, Estrogen/agonists , Receptors, Thyroid Hormone/agonists , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Amino Acid Motifs , Amino Acid Sequence , Drug Synergism , Estradiol/pharmacology , Estrogen Receptor beta , Histone Acetyltransferases , Humans , Ligands , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Receptor Coactivator 1 , Peptide Library , Peptides/chemistry , Peptides/isolation & purification , Protein Binding , Protein Structure, Tertiary , Receptors, Estrogen/chemistry , Receptors, Thyroid Hormone/chemistry , Recombinant Fusion Proteins/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Two-Hybrid System Techniques
2.
Chem Biol ; 6(11): 835-44, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10574783

ABSTRACT

BACKGROUND: Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. Here, we describe a reporter system that is based on ligand-induced DNA recombination to express the reporter gene. This system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene. RESULTS: We constructed gene fusions of Cre recombinase and mammalian promoters regulated by calcium, nuclear receptors or cyclic AMP. Reporter systems, comprising a Cre gene fusion and a loxP/reporter gene, were used to study the kinetics and dose responses to compounds that activate or inhibit the corresponding signal transduction pathway. We compared these reporters with conventional reporter systems in which the reporter gene is under the direct control of the responsive promoter. Reporter gene expression of the Cre reporters was greater than that of conventional reporters and could be measured more than a week after adding the stimulus. For all pathways studied here, the dose responses of the Cre reporters are nearly identical to those of conventional reporter systems. CONCLUSIONS: We have shown that Cre recombinase can be regulated by a variety of signal transduction pathways. It should therefore be possible to use receptor ligands to induce phenotypic conversion of mammalian cells for use in a variety of applications. One such application is high-throughput screening, and we developed loxP/luciferase reporter genes that provide an amplified and sustained luminescent response.


Subject(s)
Integrases/genetics , Nuclear Proteins , Signal Transduction , Viral Proteins , Animals , CHO Cells , Cell Division , Cricetinae , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genes, Reporter , Humans , Integrases/biosynthesis , Jurkat Cells , Kinetics , Luciferases/genetics , Mammals , Mammary Tumor Virus, Mouse/genetics , NFATC Transcription Factors , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection
3.
Cell Signal ; 11(6): 453-64, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10400318

ABSTRACT

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.


Subject(s)
Adaptor Proteins, Signal Transducing , Peptides/metabolism , Proteins/metabolism , src Homology Domains , Animals , Asparagine , Bacteriophages , COS Cells , Cloning, Molecular , GRB2 Adaptor Protein , Gene Expression , Humans , Ligands , Peptide Library , Peptides/genetics , Phosphorylation , Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis , Tyrosine
4.
Endocrinology ; 138(7): 2776-83, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9202217

ABSTRACT

Tumor necrosis factor alpha (TNF alpha) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF alpha inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARgamma and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARgamma occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF alpha-mediated inhibition of adipocyte differentiation, PPARgamma messenger RNA (mRNA) expression stays at low levels. In contrast, TNF alpha treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF alpha, basic fibroblast growth factor, transforming growth factor beta, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARgamma mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF alpha to fully differentiated adipocytes results in the rapid disappearance of PPARgamma protein expression and the rapid loss of PPARgamma DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF alpha-generated signals, whereas PPARgamma expression remains sensitive to TNF alpha at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.


Subject(s)
Adipocytes/cytology , Membrane Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/pharmacology , 3T3 Cells , Adipocytes/drug effects , Animals , Calcium/metabolism , Calcium-Binding Proteins , Cell Differentiation/drug effects , DNA/metabolism , Down-Regulation , Fibroblast Growth Factor 2/pharmacology , Intercellular Signaling Peptides and Proteins , Ionomycin/pharmacology , Ionophores/pharmacology , Mice , Phorbol Esters/pharmacology , Transforming Growth Factor beta/pharmacology
5.
Nature ; 383(6603): 837-40, 1996 Oct 31.
Article in English | MEDLINE | ID: mdl-8893011

ABSTRACT

Cells need to distinguish between transient Ca2+ signals that induce events such as muscle contraction, secretion, adhesion and synaptic transmission, and sustained Ca2+ signals that are involved in cell proliferation and differentiation. The latter class of events is blocked in lymphocytes by the immunosuppressive drugs cyclosporin A and FK506, which inhibit calcineurin, a Ca2+-activated serine/threonine phosphatase necessary for the nuclear import of NF-AT transcription factors. Here we report that sustained high concentrations of Ca2+, but not transient pulses, are required to maintain NF-AT transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation. Furthermore, overexpression and constitutive nuclear localization of NF-AT, but not Jun, Fos, NF-kappaB, Oct or Ets family members, renders the interleukin-2 enhancer in Jurkat T lymphocytes resistant to FK506 and cyclosporin A. Thus a primary effect of these immunosuppressive reagents is to control the subcellular localization of the NF-AT family of transcription factors.


Subject(s)
Calcium/metabolism , Cyclosporine/pharmacology , DNA-Binding Proteins/metabolism , Immunosuppressive Agents/pharmacology , Nuclear Proteins , Signal Transduction , T-Lymphocytes/immunology , Tacrolimus/pharmacology , Transcription Factors/metabolism , Animals , Biological Transport , Calcineurin , Calmodulin-Binding Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , Humans , Interleukin-2/genetics , Jurkat Cells , Lymphocyte Activation , Mice , NFATC Transcription Factors , Phosphoprotein Phosphatases/metabolism , T-Lymphocytes/metabolism , Transfection
6.
Mol Cell Biol ; 16(5): 2255-63, 1996 May.
Article in English | MEDLINE | ID: mdl-8628292

ABSTRACT

Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine-containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling.


Subject(s)
Cytokines/biosynthesis , Enzyme Precursors/metabolism , Lymphocyte Activation , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/immunology , src Homology Domains , Amino Acid Sequence , Antigens, Polyomavirus Transforming/biosynthesis , Cell Line, Transformed , Enzyme Precursors/biosynthesis , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Oligopeptides/pharmacology , Peptides/pharmacology , Phosphotyrosine , Protein-Tyrosine Kinases/biosynthesis , Recombinant Fusion Proteins/metabolism , Signal Transduction , Simian virus 40/genetics , Syk Kinase , Transcription, Genetic , Transfection , Tumor Cells, Cultured , ZAP-70 Protein-Tyrosine Kinase
7.
Nature ; 369(6480): 497-502, 1994 Jun 09.
Article in English | MEDLINE | ID: mdl-8202141

ABSTRACT

The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.


Subject(s)
DNA-Binding Proteins/metabolism , Lymphocyte Activation , Nuclear Proteins , T-Lymphocytes/immunology , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Calcium/metabolism , Cattle , Cell Line , Cell Nucleus/metabolism , Cytosol/metabolism , DNA, Complementary , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Gene Expression Regulation , HeLa Cells , Humans , Interleukin-2/genetics , Lymphocyte Activation/genetics , Mice , Molecular Sequence Data , NFATC Transcription Factors , Protein Kinase C/metabolism , Protein Processing, Post-Translational , RNA, Messenger/biosynthesis , Signal Transduction , T-Lymphocytes/metabolism , Thymus Gland/cytology , Thymus Gland/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/isolation & purification
8.
Genes Dev ; 7(2): 188-96, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8436291

ABSTRACT

The octamer-binding transcription factor Oct-1 is involved in a wide variety of cellular processes but appears to lack a strong transcriptional activation domain, suggesting that it functions in the context of other proteins. We demonstrated previously that Oct-1, in association with a 40-kD protein, OAP40, contributes to the induction of interleukin-2 (IL-2), an early activation gene and major growth factor for T lymphocytes. Here we report that amino acid sequences obtained from purified OAP40 are identical to regions within JunD and c-Jun. We demonstrate that each of these Jun family members can participate in a complex that includes Oct-1 and a regulatory element in the IL-2 enhancer. In transient transfections, both JunD and c-Jun can contribute to activation-specific transcription mediated by this antigen receptor response element. These studies reveal a role, distinct from AP-1 activity, for Jun family members that is controlled by a calcium-triggered, cyclosporin A-sensitive mechanism.


Subject(s)
Calcium/pharmacology , Cyclosporine/pharmacology , Interleukin-2/genetics , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , T-Lymphocytes/metabolism , Amino Acid Sequence , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic , Gene Expression Regulation , Host Cell Factor C1 , Lymphocyte Activation/drug effects , Molecular Sequence Data , Octamer Transcription Factor-1 , Phorbol Esters/pharmacology , Signal Transduction/drug effects , T-Lymphocytes/drug effects , Transcription Factors/genetics , Transcription, Genetic
9.
J Biol Chem ; 268(4): 2917-23, 1993 Feb 05.
Article in English | MEDLINE | ID: mdl-8428966

ABSTRACT

The lymphoid-specific transcription complex, NF-AT, is involved in early gene activation in T cells and is assembled from a pre-existing, T cell restricted cytoplasmic factor and an inducible ubiquitous nuclear component within 30 min after activation through the antigen receptor. Recent studies have implicated the family of AP1 factors as components of the murine NF-AT complex. Evidence is provided here that the nuclear component of human NF-AT contains the phorbol ester-inducible transcription factor AP1 (Jun/Fos). We further characterize which AP1 family members can assume this role. Antisera to Fos inhibits NF-AT DNA binding as does an oligonucleotide containing a binding site for AP1. Constitutive expression in vivo of Fos, and to a lesser extent Fra-1, eliminates the requirement for phorbol 12-myristate 13-acetate (PMA) stimulation, leaving NF-AT-directed transcription responsive to calcium ionophore alone. Overexpression of cJun or JunD, but not JunB, also eliminates the requirement for PMA, indicating that many but not all Jun- and Fos-related proteins functionally activate NF-AT-dependent transcription in the presence of the cytoplasmic component. NF-AT DNA binding can be reconstituted in vitro using semi-purified AP1 proteins mixed with cytosol from T lymphocytes. Fos proteins are not needed for this reconstitution, and although JunB is not functional, it can participate in the NF-AT DNA binding complex. Finally, we have partially purified the cytoplasmic component of NF-AT and show by elution and renaturation from SDS-polyacrylamide gel electrophoresis gels that it has a molecular mass between 94 and 116 kDa and may have multiple differentially modified forms.


Subject(s)
DNA-Binding Proteins/chemistry , Lymphocyte Activation , Nuclear Proteins/chemistry , T-Lymphocytes/chemistry , Transcription Factors/chemistry , Animals , Base Sequence , Cytoplasm/chemistry , Gene Expression Regulation , Genes, fos , Genes, jun , Humans , In Vitro Techniques , Mice , Molecular Sequence Data , Molecular Weight , NFATC Transcription Factors , Oligodeoxyribonucleotides/chemistry , Proto-Oncogene Proteins c-jun/physiology , RNA, Messenger/genetics , Rats , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic , Transcriptional Activation , Tumor Cells, Cultured
10.
J Exp Med ; 175(5): 1235-45, 1992 May 01.
Article in English | MEDLINE | ID: mdl-1569395

ABSTRACT

Glucocorticoid-dependent transcriptional enhancement is known to occur through the interaction of the glucocorticoid receptor (GR) with specific DNA response elements. In contrast, negative regulation of gene expression by this class of hormone is less well understood. Glucocorticoids are potent immunosuppressive agents acting primarily by inhibiting T lymphocyte activation and lymphokine production. Interleukin 2 (IL-2) gene expression, a critical early event during T lymphocyte activation, is inhibited in glucocorticoid-sensitive cells by hormone treatment. We have studied the mechanism of this inhibition. In transgenic mice carrying c-myc linked to the IL-2 enhancer, mitogen-induced expression of the transgene is inhibited by concurrent glucocorticoid treatment, while a similar transgene construct driven by three copies of the binding site for nuclear factor of activated T cells is not inhibited. Cotransfection experiments into glucocorticoid-insensitive jurkat cells show that the NH2 terminus of the glucocorticoid receptor is dispensable for inhibition of the IL-2 enhancer but that an intact DNA binding domain, although not necessarily binding to DNA, is required. Hybrid GRs containing the DNA binding domains of either the estrogen receptor (ER) or thyroid receptor, as well as the entire wild-type ER, all function as repressors of the IL-2 enhancer. We have localized the site of inhibition to two sequences located in the proximal half of the enhancer. These sequences bind a similar, if not identical, inducible nuclear factor that has biologic characteristics that distinguish it from AP-1. The mechanism of IL-2 inhibition likely involves direct interactions between the GR and this factor.


Subject(s)
Gene Expression Regulation , Interleukin-2/genetics , Receptors, Glucocorticoid/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , Dexamethasone/pharmacology , Enhancer Elements, Genetic , Humans , Interleukin-2/metabolism , Mice , Molecular Sequence Data , Plasmids , Transcription, Genetic , Tumor Cells, Cultured
12.
Genes Dev ; 4(10): 1823-34, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2123468

ABSTRACT

Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.


Subject(s)
Cinnamates , Receptors, Antigen, T-Cell/physiology , Signal Transduction/genetics , T-Lymphocytes/metabolism , Transcription Factors/analysis , Transcription, Genetic/physiology , Cell Cycle/physiology , Cells, Cultured , DNA-Binding Proteins/physiology , Drug Resistance/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation/physiology , Humans , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , Plasmids/genetics , Ribonucleases/antagonists & inhibitors , Transcription Factors/physiology , Transfection/genetics , beta-Galactosidase/analysis
13.
Mol Endocrinol ; 4(1): 162-70, 1990 Jan.
Article in English | MEDLINE | ID: mdl-2157974

ABSTRACT

We have isolated Chinese hamster ovary (CHO) cell lines expressing elevated levels of wild-type (W) and mutant forms of the glucocorticoid receptor (GR) using the technique of coamplification with a selectable dihydrofolate reductase (dhfr) cDNA. A prominent doublet at 90/92 kilodaltons was observed by Western blotting or labeling with [3H]-dexamethasone mesylate in extracts from cells transfected with W, the hormone binding mutant (NA), and the DNA binding mutant (NB). Quantification of receptor number by [3H]dexamethasone binding revealed the presence of approximately 10(6) receptors per cell in the W and NB-producing lines. This represents a 25- to 50-fold increase in receptor density over control CHO cells which were not transfected with GR. Comparative quantitation by Western blotting of extracts from cells expressing GR showed that cells producing NA contain a level approximately 500-fold over control CHO cells. Function of the amptified receptors was examined by transient transfection with the glucocorticoid-responsive reporter plasmid pMMTV-chloramphenicol acetyl transferase (CAT). Our results indicate that inducible CAT activity increases with the abundance of W receptor and no evidence of saturability was observed even at the highest levels of receptor. This supports previous suggestions that the concentration of the hormone-regulated transcription factor is definitely limiting with regard to maximal transcription efficiency. Interestingly, cells expressing even highly amplified levels of NA-GR or NB-GR showed no inducible response above that seen with control CHO cells. Thus these mutations are exceedingly nonleaky and are not dominant over the low endogenous activity of the CHO GR.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Gene Expression , Genetic Variation , Receptors, Glucocorticoid/genetics , Animals , Cell Line , Cricetinae , Cricetulus , DNA/biosynthesis , DNA/genetics , Dexamethasone/metabolism , Gene Amplification , Mammary Tumor Virus, Mouse/genetics , Mice , Promoter Regions, Genetic , Receptors, Glucocorticoid/metabolism , Tetrahydrofolate Dehydrogenase/genetics , Transfection
15.
Proc Natl Acad Sci U S A ; 84(21): 7413-7, 1987 Nov.
Article in English | MEDLINE | ID: mdl-2823261

ABSTRACT

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.


Subject(s)
Liposomes , Quaternary Ammonium Compounds/chemical synthesis , Simian virus 40/genetics , Transfection , Animals , Calcium Phosphates , Cell Line , DEAE-Dextran , Genetic Engineering/methods , Indicators and Reagents , L Cells/cytology
16.
Mol Endocrinol ; 1(11): 816-22, 1987 Nov.
Article in English | MEDLINE | ID: mdl-3153464

ABSTRACT

We have analyzed the domain structure of the mouse glucocorticoid receptor by expression of in vitro mutated receptor in COS-7 cells. The receptor consists of a core domain rich in Cys, Lys, and Arg amino acids which can bind specific DNA sequences (glucocorticoid response elements) and activate transcription. The activity of this centrally located domain is modulated by the activity of the other two domains. The N-terminal domain of the receptor plays a role in decreasing nonspecific DNA binding and may therefore improve the ability of the protein to discriminate between specific and nonspecific DNA binding sites. This activity maps to a small, highly acidic region of the N-terminal domain. The C-terminal domain of the receptor contains the glucocorticoid binding site and in addition represses the transcriptional activity of the receptor in the absence of hormone. Hormone binding relieves the repression allowing transcription activation. The C-terminal domain contains a short sequence conserved among steroid receptors; its deletion yields a receptor that activates transcription in the absence of hormone.


Subject(s)
DNA-Binding Proteins/physiology , Receptors, Glucocorticoid/physiology , Transcription, Genetic/physiology , Amino Acid Sequence , Animals , Cell Line, Transformed , Cellulose/metabolism , Gene Expression Regulation/physiology , Molecular Sequence Data , Plasmids/genetics , Transfection/genetics
17.
EMBO J ; 5(10): 2513-22, 1986 Oct.
Article in English | MEDLINE | ID: mdl-3780669

ABSTRACT

We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor.


Subject(s)
Cloning, Molecular , DNA/metabolism , Mutation , Receptors, Glucocorticoid/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Lymphoma, Non-Hodgkin/metabolism , Mice , Plasmids , Receptors, Glucocorticoid/metabolism , Transcription, Genetic
18.
J Biol Chem ; 261(24): 11064-70, 1986 Aug 25.
Article in English | MEDLINE | ID: mdl-3015953

ABSTRACT

We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein.


Subject(s)
DNA/analysis , Receptors, Glucocorticoid/genetics , Alleles , Animals , Cell Line , Cloning, Molecular , DNA Restriction Enzymes/metabolism , Liver Neoplasms, Experimental/genetics , Lymphoma/genetics , Mice , Molecular Weight , Phenotype , RNA, Messenger/analysis , Rats , Thymoma/genetics , Thymus Neoplasms/genetics , Transcription, Genetic
19.
J Biol Chem ; 260(10): 6398-403, 1985 May 25.
Article in English | MEDLINE | ID: mdl-3997828

ABSTRACT

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.


Subject(s)
Liver Neoplasms, Experimental/metabolism , Lymphoma/metabolism , Receptors, Glucocorticoid/genetics , Receptors, Steroid/genetics , Affinity Labels/metabolism , Animals , Antibodies, Monoclonal , Clone Cells/metabolism , Liver Neoplasms, Experimental/genetics , Lymphoma/genetics , Mice , Mutation , Rats , Receptors, Glucocorticoid/immunology , Receptors, Glucocorticoid/metabolism
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