Biomedical subjects

M R Stallcup

Publications and source records attributed to M R Stallcup.

At least 55 records · Page 3Linked to original sources

PubMed1994

Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity.

Site-directed mutagenesis was employed to make two single amino acid substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid receptor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.

Amino Acid Sequence↗

PubMed1993

Relationship between defective mouse mammary tumor virus envelope glycoprotein synthesis and GRP78 synthesis in glucocorticoid-treated mouse lymphoma cells. Evidence for translational control of GRP78 synthesis.

We have found that synthesis of the 78-kDa glucose-regulated protein, GRP78, was increased following glucocorticoid treatment of S49 and W7MG1 mouse lymphoma cell lines. These lines synthesized a 74-kDa mouse mammary tumor virus envelope glycoprotein precursor, Pr74, both constitutively and in response to glucocorticoid treatment. In these cell lines, nascent Pr74 was not processed normally but was retained within the endoplasmic reticulum where it was bound stably by GRP78. GRP78 synthesis was not increased following glucocorticoid treatment of the W7.2 mouse lymphoma cell line, which does not synthesize Pr74, suggesting that the increase in GRP78 synthesis following glucocorticoid treatment of S49 and W7MG1 cells was secondary to the glucocorticoid-induced increase in Pr74 synthesis. Consistent with this hypothesis, the glucocorticoid-induced increase in Pr74 synthesis preceded the increase in GRP78 synthesis. Also, there was a direct correlation between the level of GRP78 synthesis and the level of Pr74 synthesis among multiple subclones of the W7-ENV/N line. This line was derived from the W7.2 line by retroviral mediated transfer of a constitutively expressed sequence encoding a defective form of Pr74 that was bound stably by GRP78 within the endoplasmic reticulum. An elevation in the steady state level of GRP78 mRNA was not detected either in glucocorticoid-treated S49 cells and W7MG1 cells or in subclones of the W7-ENV/N line that have an increased level of GRP78 synthesis. Therefore, it appears that the binding of Pr74 to GRP78 induces an increase in GRP78 synthesis that is regulated at a translational level, rather than at a transcriptional level.

Animals↗

PubMed1992

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

Maturation of mouse mammary tumor virus envelope protein is blocked by a specific point mutation.

Expression of a mouse mammary tumor virus (MMTV) envelope gene, ENV2, in COS-7 cells resulted in the synthesis of a precursor protein Pr74 that underwent complete proteolytic processing to the mature envelope species gp52 and gp33. In contrast, expression of a second MMTV envelope gene, ENV1, in COS-7 cells resulted in the synthesis of a precursor protein Pr74 that failed to undergo proteolytic processing to the mature products. Expression and sequencing of various chimeric ENV1/ENV2 genes determined that a single glycine-to-glutamic acid mutation at position 54 was responsible for the nonprocessable phenotype of ENV1-encoded Pr74. The significance of this point mutation in relation to the requirements for precursor protein folding and maturation are discussed.

Animals↗

PubMed1992

The effect of glucocorticoid on the subcellular localization, oligomerization, and processing of mouse mammary tumor virus envelope protein precursor Pr74.

Mouse lymphoma cell line W7MG1 is stably infected with mouse mammary tumor virus and produces the viral envelope glycoprotein precursor Pr74, but the mature envelope proteins gp52 and gp33, which are derived from Pr74 by posttranslational processing, are produced only when the cells are cultured with a glucocorticoid agonist. The current study demonstrated that even when W7MG1 cells are grown with hormone, the conversion of Pr74 to gp52 and gp33 is an inefficient process in this cell line. At least 2 h of exposure to glucocorticoid were required to induce the appearance of gp52 and gp33; furthermore, Pr74 labeled in the absence of hormone was not converted to gp52 and gp33 upon subsequent addition of hormone. RNA synthesis inhibitors blocked the hormonal induction of gp52 and gp33, indicating that the hormone acts by promoting the expression of a new gene(s) required for the production of gp52 and gp33, rather than by inhibiting the expression of a gene(s) that prevents processing of Pr74. Subcellular fractionation studies demonstrated that Pr74 produced in either the presence or absence of hormone was associated primarily with the ER, whereas gp52 and gp33 were found in the Golgi and plasma membrane fractions. The Pr74 molecules from W7MG1 cells grown either with or without glucocorticoid coimmunoprecipitated with BiP/GRP78 and sedimented as aggregates of heterogeneous size. In contrast, Pr74 from virus-producing GR3A mouse mammary tumor cells, which process Pr74 more efficiently, sedimented as apparent monomers, dimers, and trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells.

In the mouse mammary tumor virus (MMTV)-infected mouse T-lymphoma cell line W7MG1, glucocorticoid hormone regulates two aspects of MMTV gene expression: hormone stimulates MMTV gene transcription and increases the ratio of mature envelope proteins to envelope precursor protein produced. To separate these two effects and determine the mechanism by which hormone regulates the conversion of the envelope precursor Pr74 to the mature cleaved products gp52 and gp33, we constructed expression vectors in which the envelope gene is constitutively transcribed. Surprisingly, the envelope precursor protein Pr74 encoded by two independently isolated, allelic envelope genes behaved differently. Pr74-P (encoded by the ENV/P gene) was processed efficiently to the mature products gp52 and gp33, independently of the level of expression, hormonal induction of cellular genes, or the presence of other MMTV proteins. In contrast, under the same conditions, Pr74-N (encoded by the ENV/N gene) was not processed further despite being relatively stable. In sucrose gradient analyses, Pr74-P sedimented as monomers, whereas Pr74-N was found in high mol wt aggregates of heterogeneous size. Coimmunoprecipitation analysis determined that Pr74-N associated with BiP, whereas Pr74-P did not. This is indicative of improper folding of Pr74-N in the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Two point mutations in the hormone-binding domain of the mouse glucocorticoid receptor that dramatically reduce its function.

Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional.

Amino Acid Sequence↗

PubMed1990

The order of processing events in mouse mammary tumor virus envelope protein maturation: implications for the location of the glucocorticoid-regulated step.

Treatment of the W7MG1 mouse T lymphoma cell line with glucocorticoid stimulates directly or indirectly two observable steps in the processing of mouse mammary tumor virus (MMTV) envelope glycoprotein precursor Pr74: cleavage of Pr74 to yield the mature glycoprotein products gp52 and gp33, and processing of the N-linked oligosaccharides to endoglycosidase H (endo H)-resistant forms found on the mature products but not on the precursor. Therefore, the primary hormone-regulated event in this pathway must occur at or before the point where MMTV envelope proteins become endo H resistant. Pulse-chase analyses identified a novel endo H-resistant 80-kDa species (designated gp80) as a processing intermediate. Therefore, in contrast to conclusions drawn for the envelope proteins of several other retroviruses, proteolytic cleavage of MMTV envelope proteins occurs after acquisition of endo H resistance. Also, proteolytic cleavage cannot be the primary hormone-regulated step. Second, inhibition of mannosidase II by the drug swainsonine did not prevent Pr74 from being proteolytically processed, thus demonstrating that conversion of oligosaccharide chains from endo H-sensitive to -resistant forms was not a prerequisite for proteolytic cleavage. Therefore, the requisite hormone-regulated event in MMTV glycoprotein processing must precede both acquisition of endo H resistance and proteolytic cleavage. This places the regulated event in the endoplasmic reticulum or early Golgi.

Alkaloids↗

PubMed1990

Glucocorticoid-dependent maturation of mouse mammary tumor virus glycoproteins in mouse lymphoma cells: isolation of variants with constitutive viral protein maturation and normal glucocorticoid receptor function.

The posttranslational maturation and cell surface localization of mouse mammary tumor virus (MMTV) envelope glycoproteins is regulated by glucocorticoid hormone in mouse T-lymphoma cell line W7MG1. Only when the cells are cultured with glucocorticoid is the MMTV envelope precursor, Pr74, converted efficiently to the two mature proteolytic products, gp52 and gp33. By immunological selection we have isolated protein-processing variants that express the mature viral proteins constitutively on the cell surface. The rate of synthesis of Pr74 is indistinguishable in variant and wild-type cells, but the variants efficiently convert Pr74 to gp52 and gp33 even when grown without the hormone. The variant phenotype persists when the variant cells are fused with uninfected wild-type cells to form somatic cell hybrids, indicating that the variant phenotype resulted from expression of a new or altered function that is not expressed in wild-type cells grown without glucocorticoid. Although the specific gene whose structure or regulation is altered in the variant has not yet been determined, some possibilities have been eliminated. First, the number and function of the glucocorticoid receptors in the variant cells was normal, suggesting that alterations in this protein were not responsible for the variant phenotype. Second, comparison by two-dimensional gel electrophoresis of gp52 produced in variant and wild-type cells revealed no differences in size or charge, indicating no gross differences in the processing of the viral proteins in the variant and wild-type cells.

Animals↗

PubMed1990

Characterization of oligosaccharide chains on mouse mammary tumor virus envelope proteins and the implications for the mechanism of their glucocorticoid regulated processing.

Glucocorticoid hormone is required for complete posttranslational processing of the glycosylated mouse mammary tumor virus envelope precursor, Pr74env in the murine T-lymphosarcoma cell line, W7MG1. Metabolic labeling studies with [35S]methionine, [3H]galactose, and [3H]mannose, combined with enzymatic digestion analyses with a variety of endoglycosidases, demonstrated that both proteolytic processing and N-linked oligosaccharide maturation depended, either directly or indirectly, on glucocorticoid action. Pr74 is found in both control and hormone-treated cells. In both cases Pr74 molecules carry high mannose and/or hybrid, but not complex, oligosaccharide chains with very little or no sialic acid. When cells are grown with glucocorticoid, Pr74 is converted to gp52 and gp33 with greatly increased efficiency, and these mature glycoproteins carry complex oligosaccharides containing sialic acid. No O-linked carbohydrate was detected on any of these species. According to this evidence, the glucocorticoid-regulated step in this pathway must occur at or before the final mannose trimming step in the Golgi that is required for formation of complex carbohydrate chains.

Animals↗

PubMed1988

A role for divalent cations in specifying the start site for transcription from chromatin templates in vitro.

An assay that employs nucleoside 5'-O-(2-thiotriphosphates) was used to detect initiation of mouse mammary tumor virus (MMTV) RNA chains in preparations of isolated nuclei from cultured rat hepatoma cells containing stably integrated proviruses. RNA chains initiated with adenosine 5'-O-(2-thiotriphosphate), guanosine 5'-O-(2-thiotriphosphate), or uridine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated RNA chains with a T1 nuclease protection assay. Combined use of the thionucleotide transcription reaction with the T1 nuclease assay allowed precise localization of the transcription start sites. The majority of MMTV RNA chains were initiated with guanosine 5'-O-(2-thiotriphosphate) at a template site 133 nucleotides upstream from a PvuII site that coincides with the right end of the long terminal repeat. However, some RNA chains were also initiated with adenosine 5'-O-(2-thiotriphosphate) and uridine 5'-O-(2-thiotriphosphate) at template sites within three nucleotides of the primary guanosine start site. When Mn2+ was substituted for Mg2+ in the transcription reaction, MMTV RNA chains were initiated with approximately the same efficiency, but the start site was shifted to a position approximately 40 nucleotides downstream from the physiological start site; in the presence of Mn2+, MMTV RNA chains were initiated only with guanosine 5'-O-(2-thiotriphosphate). When the nuclei were exposed to both Mn2+ and Mg2+, transcription initiated at the manganese-dependent site. Mn2+ also caused the transcription start site for 45 S pre-rRNA to shift about 10 nucleotides upstream from the physiologically correct start site.

Animals↗

PubMed1988

Optimized reaction conditions and specific inhibitors for initiation of transcription by RNA polymerase II in nuclei from cultured mammalian cells.

An assay that employs guanosine 5'-O-(2-thiotriphosphate) was used to measure correct initiation of RNA chains in isolated cell nuclei, where chromatin structure is relatively undisturbed. RNA chains initiated with guanosine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated mouse mammary tumor virus RNA with a T1 nuclease protection assay. The monovalent cation concentration dependence for initiation in isolated nuclei was similar to that previously observed for initiation from naked DNA templates (optimum near 90 mM) but different from that for elongation of nascent RNA chains. However, in contrast to the systems that employ naked DNA templates, initiation efficiency in the nuclear system was relatively unaffected by moderate changes in pH (6.7-8.3), temperature (25-37 degrees C), and magnesium ion concentration (1-9 mM). The optimized assay was used to assess the inhibitory activity of several compounds that have been reported to be specific inhibitors of transcription initiation on naked DNA templates. Both Sarkosyl and heparin were effective inhibitors of specific initiation by RNA polymerases I and II in isolated nuclei without inhibiting elongation of nascent chains, but 5,6-dichlorobenzimidazole riboside was relatively ineffective as a specific inhibitor of initiation by RNA polymerase II.

Animals↗

PubMed1988

Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.

Animals↗

PubMed1987

Glucocorticoid-dependent maturation of viral proteins in mouse lymphoma cells: isolation of defective and hormone-independent cell variants.

Maturation of mouse mammary tumor virus proteins is dependent on glucocorticoid hormones in W7MG1, a stably infected mouse T-lymphosarcoma cell line derived from WEH17. We used an immunological procedure to select variant cell lines with altered levels of viral glycoproteins on the cell surface. One variant, W7M329a, expressed lower-than-normal levels of the major viral glycoprotein, gp52env, on the cell surface before and after exposure to hormone. Two other variants, W7M302b and W7M326.4, expressed elevated levels of gp52env on the cell surface even in the absence of hormone. Analysis of the levels and/or rates of synthesis of viral RNA and glycoproteins before and after hormone treatment indicated that the variant phenotypes resulted from changes in posttranslational steps of viral gene expression. The hormone-independent maturation of MMTV proteins is a novel variant phenotype that has not previously been reported.

Animals↗

PubMed1987

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.

Animals↗

PubMed1987

Mouse lymphoma cell variants with genetically dominant alterations in the maturation of viral glycoproteins.

The appearance of Mouse Mammary Tumor Virus (MMTV) glycoproteins on the surface of infected mouse lymphoma cells is regulated by glucocorticoid hormones. The hormone stimulates transcription of the proviral genes and is also required for maturation of viral proteins. We have reported earlier the isolation of variant cell lines with altered patterns of cell-surface expression of the MMTV antigens. One type of variant expresses high levels of viral glycoproteins on the cell surface in a constitutive or hormone-independent manner. Another type of variant expresses reduced levels of viral glycoproteins on the cell surface compared with wild-type cells, both in the presence and in the absence of glucocorticoids. In this report pulse-chase studies demonstrate that the variant phenotypes are caused by changes in the efficiency of processing the envelope polyprotein precursor into the mature glycoproteins. In addition, both variant phenotypes are dominant over the wild-type phenotype in stable hybrid cell lines produced by cell fusion.

Animals↗

PubMed1984

Hormonal regulation of transcription of rDNA: use of nucleoside thiotriphosphates to measure initiation in isolated nuclei.

Nuclei isolated from cultured mouse and rat cell lines initiated pre-rRNA chains at the correct site and with the correct nucleotide specificity (A for mouse, G for rat). Nucleic acid filter hybridization and S1 nuclease mapping were used to analyze the RNA products initiated with nucleoside (beta-S)triphosphates. Initiation of pre-rRNA was completely resistant to alpha-amanitin but was inhibited by either actinomycin D or heparin. Experiments with P1798 mouse lymphoma cells indicated that the antiproliferative effects of glucocorticoids on lymphoid cells includes a reduction in the ability of nuclei to initiate pre-rRNA chains.

Adenosine Triphosphate↗

PubMed1984

Asymmetric transcription of mouse mammary tumor virus genes in vivo and in vitro.

Mouse mammary tumor virus (MMTV) DNA fragments were cloned into M13 bacteriophage, and the single-stranded recombinant phage DNAs were used as strand-specific nucleic acid hybridization probes to measure synthesis of plus (genomic) and minus strands of MMTV RNA in cultured cell lines and in cell-free preparations of nuclei. Pulse-labeling studies showed that synthesis of MMTV RNA in three different cell lines was highly asymmetric. In nuclear preparations from a cloned line of MMTV-infected rat hepatoma cells, elongation of nascent MMTV RNA chains and initiation of new MMTV RNA chains with nucleoside (beta-S)triphosphates were also highly asymmetric.

Animals↗