Dense Amino Acid Labeling Research Articles

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit.

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.

Measurement of 1,25-dihydroxyvitamin D3 receptor turnover by dense amino acid labeling: changes during receptor up-regulation by vitamin D metabolites.

We have previously demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors are up-regulated by exposure to 24,25-(OH)2D3 and other vitamin D metabolites in several mammalian cell cultures systems as well as in vivo in rats. The goal of the current study was to determine how changes in receptor turnover could give rise to increased receptor levels after treatment with 24,25-(OH)2D3, whether by an increased rate of synthesis or a decreased rate of degradation. Receptor turnover was studied by the dense amino acid shift technique in cultured pig kidney cells (LLC-PK1). In control cells, the half-life of the 1,25-(OH)2D3 receptor was estimated to be 4.3 +/- 0.4 h, with a degradation rate constant (kD) of 0.16 +/- 0.01 h-1. In parallel experiments, cells were treated with 24,25-(OH)2D3 for about 48 h to reach a new up-regulated steady state before the measurement of receptor turnover. In up-regulated cells, the receptor half-life was prolonged to 8.9 +/- 1.0 h and the kD was 0.07 +/- 0.01 h-1. Assuming a steady state, the values obtained for total receptor levels and the degradation rate allow calculation of the synthesis rate (kS) at equilibrium. Control cells synthesized receptors at the rate of 3.4 +/- 0.4 vs. up-regulated cells at 4.9 +/- 0.5 fmol mg protein-1 h-1. In conclusion, the data indicate that up-regulation of receptors after exposure of cells to vitamin D metabolites results predominantly from a prolongation of receptor half-life in addition to a relatively smaller increase in the rate of receptor synthesis.

Glucocorticoid induction of beta-adrenergic receptors in the DDT1 MF-2 smooth muscle cell line involves synthesis of new receptor.

We have shown that glucocorticoids induce the appearance of beta 2-adrenergic receptors in membranes of the ductus deferens smooth muscle cell line (DDT1 MF-2). A concomitant increase in isoproterenol stimulated adenylate cyclase activity in the absence of exogenously applied GTP was observed as was a significantly increased (p less than 0.05) sensitivity of the adenylate cyclase system to exogenously applied GTP. However, no significant difference in the maximal velocity of adenylate cyclase between control and steroid treatment was measurable in the presence of sodium fluoride. Induction of beta 2-adrenergic receptors in DDT1 MF-2 cells is correlated with the presence of steroid receptors (androgen and glucocorticoid) in the cells since estrogens and progesterones had no effect on receptor levels. Finally, utilizing dense amino acid labeling of cells to measure old versus newly synthesized receptor sites by a density shift method, we have documented that glucocorticoid induction of beta 2-adrenergic receptors involves synthesis of new receptor protein.

The rate of appearance of thyroid hormone nuclear receptor is increased during deoxyribose nucleic acid synthesis in GC cells: analysis of thymidine-treated GC cells using dense amino acid labeling.

The growth rate of GH-producing rat pituitary tumor cells (GC cells) is dependent on thyroid hormone. Previous studies have shown that GC cells can be partially synchronized in the DNA synthesis phase (S-phase) of the cell cycle by a 25-h incubation with 2 mM thymidine. Measurements in GC cells partially synchronized in S-phase showed a significant increase in cellular thyroid hormone nuclear receptor appearance and disappearance in thymidine-treated GC cells using dense amino acid labeling and subsequent sucrose gradient ultracentrifugation. In comparison with GC cells in asynchronous culture, thymidine treatment causes a steady state increase in thyroid hormone nuclear receptors which results from an increase in the receptor appearance rate without an effect on the disappearance rate. Thus, position in the cell cycle appears to be another factor that influences the appearance rate of nuclear thyroid hormone receptors.

Antiestrogen action in breast cancer cells: modulation of proliferation and protein synthesis, and interaction with estrogen receptors and additional antiestrogen binding sites.

Antiestrogens have proven to be effective in controlling the growth of hormone-responsive breast cancers. At the concentrations of antiestrogens achieved in the blood of breast cancer patients taking antiestrogens (up to 2 X 10(-6) M), antiestrogens selectively inhibit the proliferation of estrogen receptor-containing breast cancer cells, and this inhibition is reversible by estradiol. Antiestrogens also inhibit estrogen-stimulation of several specific protein synthetic activities in breast cancer cells, including increases in plasminogen activator activity, progesterone receptor levels and production of several secreted glycoproteins and intracellular proteins. Antiestrogens bind with high affinity to the estrogen receptor and to additional microsomal binding sites to which estrogens do not bind. These latter sites, called antiestrogen binding sites (AEBS), are present in equal concentrations in estrogen receptor-positive and -negative breast cancer cells and are present in a wide variety of tissues, with highest concentrations being found in the liver. The antiestrogenic and growth suppressive potencies of a variety of antiestrogens correlate best with their affinity for estrogen receptor and not with affinity for AEBS. Antiestrogens undergo bioactivation and metabolism in vivo and hydroxylated forms of the antiestrogen have markedly enhanced affinities for the estrogen receptor. Detailed studies with high affinity radiolabelled antiestrogens indicate that antiestrogens induce important conformational changes in receptor that are reflected in the enhanced maintenance of a 5 S form of the estrogen receptor complex; reduced interaction with DNA; and altered activation and dissociation kinetics of the antiestrogen-estrogen receptor complex. These conformational changes effected by antiestrogens likely result in different interactions with chromatin, causing altered cell proliferation and protein synthesis. Analyses of the rates of synthesis and turnover of the estrogen receptor through pulse-chase experiments utilizing the covalently attaching antiestrogen, tamoxifen aziridine, and studies employing dense amino acid labeling of estrogen receptor reveal that the antiestrogen-occupied receptor is degraded at a rate (t 1/2 = 4 h) similar to that of the control unoccupied receptor. Hence, antiestrogens do not prevent estrogen receptor synthesis and they do not either accelerate or block estrogen receptor degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the subunit nature of nuclear estrogen receptors by chemical cross-linking and dense amino acid labeling.

We have used chemical cross-linking and dense amino acid labeling of estrogen receptors to characterize the subunit nature and rate of turnover of nuclear 5S estrogen-receptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbe ne (CI628M) or (Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl] 1,2-diphenyl-1-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KCl and then chemically cross-linked with the cross-linker 2-iminothiolane, the cross-linked receptor complexes sediment as a 5.4S species on 3 M urea-containing sucrose gradients, while the noncross-linked species are 4S. Sodium dodecyl sulfate-polyacrylamide gel analyses of these cross-linked nuclear receptor complexes labeled with the covalently attaching ligand [3H]tamoxifen aziridine reveal a species of about 130,000 mol wt, while the noncross-linked or the cross-linked but mercaptan-cleaved receptor is 65,000 mol wt. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rates, cells exposed for different time periods to medium containing dense (15N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [1-[4-(2-dimethylaminoethoxy)phenyl]1-[4-hydroxyphenyl] 2-phenylbut-1-(2)ene (trans-hydroxytamoxifen) or CI628M] or [3H]estradiol, and salt-extracted nuclear estrogen receptors were analyzed on sucrose gradients. The normal density 5S form shifted to a broader, more dense peak at 2 and 4 h and finally, by 8-10 h, to a more dense, sharply sedimenting species. The time course of this shift is the same as that seen for the 4S urea-dissociated nuclear receptor form (t1/2 approximately 4h), suggesting that the 5.4S nuclear receptor is composed of two species which turn over at the same rate. We conclude from these cross-linking and density shift experiments that the nuclear 5S receptor complex consists of two similarly sized units, which turn over with similar half-lives. These data provide strong evidence that the 5S nuclear receptor complex is a homodimer of two 4S, 65,000 mol wt monomers.

Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form.

Glucocorticoid-receptor activation in GH1 cells results from the conversion of a 10 S oligomeric cytosolic form to a 4-5 S nuclear-binding species (Raaka, B. M., and Samuels, H. H. (1983) J. Biol. Chem. 258, 417-425). In this study, we report that triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide) elicits a time- and dose-dependent reduction of total-cell (nuclear + cytoplasmic) receptor. The mechanism of receptor regulation was studied by dense amino acid labeling of receptor using media containing 2H, 13C, and 15N-labeled amino acids. Total cell receptor was extracted with 0.4 M KCl and newly synthesized dense receptor was separated from pre-existing receptor of normal density by centrifugation in gradients of 15-30% sucrose (w/v) in D2O. Receptor levels in cells grown without [3H]triamcinolone acetonide was 260 +/- 19 fmol/100 micrograms of DNA (16,000 molecules/cell), and, with 10 nM [3H]triamcinolone acetonide, this decreased to 130 +/- 14 fmol/100 micrograms of DNA after 30 h. Receptor half-life was 19 +/- 1.9 h in the absence and 9.5 +/- 0.3 h in the presence of triamcinolone acetonide and accounted for the decrease in steady-state receptor levels. Receptor synthesis was 9.7 +/- 0.3 fmol/100 micrograms of DNA/h (580 molecules/cell/h) both in the presence and absence of 10 nM [3H]triamcinolone acetonide. Triamcinolone acetonide reduced the half-life in proportion to the extent of receptor occupancy and activation. During the approach to steady-state conditions, 10 nM [3H]triamcinolone acetonide shortened receptor half-life almost immediately to the value in cells grown with [3H]triamcinolone acetonide for 24 h or longer. Cycloheximide did not prevent the triamcinolone acetonide-mediated decrease in receptor half-life and the shortening of receptor half-life is rapidly reversed by removal of hormone. These studies support a model of receptor regulation in which triamcinolone acetonide converts the unactivated 10 S receptor to the activated 4-5 S form which is degraded at an increased rate by the cell.

Photoaffinity labeling of thyroid hormone nuclear receptors. Influence of n-butyrate and analysis of the half-lives of the 57,000 and 47,000 molecular weight receptor forms.

The thyroid hormone receptor is a nuclear-associated protein which appears to mediate the actions of 3,5,3'-triiodo-L-thyronine (L-T3) and 3,5,3',5'-tetraiodo-L-thyronine (L-T4) in mammalian cells. In a previous study we reported that N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodo-L-thyronine (L-T3-PAL) serves as an effective photoaffinity label probe of the receptor in GH1 cells, a growth hormone producting rat pituitary cell line. Irradiation of cells at 254 nm covalently cross-links L-[125I]T3-PAL to two molecular weight (Mr) nuclear receptor forms, an abundant 47,000 Mr component and a less abundant 57,000 Mr species (Pascual, A., Casanova, J., and Samuels, H. H. (1982) J. Biol. Chem. 257, 9640-9647). In this study we have explored a number of possible interrelationships of the different Mr receptor forms. Denaturing gel electrophoresis and autoradiography indicates that the 57,000 Mr form is a doublet species which differ in Mr by 1,000 to 2,000. The various receptor forms are not an artifact of the L-[125I]T3-PAL probe, and identical forms can be labeled at 310 nm using underivatized L-[125I]T4 with a 15-fold lower coupling efficiency. The 57,000 and 47,000 Mr receptor forms are not generated by indiscriminate proteolysis, UV peptide cleavage, or zero length protein-protein cross-linking by irradiation at 254 nm. Micrococcal nuclease excises both the 57,000 and 47,000 Mr forms, and receptor is not identified in the residual nuclear matrix fraction. Receptor is also not detected in the cytoplasmic fraction. By coupling dense amino acid labeling and photoaffinity labeling of receptor we determined a half-life of 2.4 h for the 57,000 Mr species and 5.6 h for the 47,000 Mr form while both species have similar relative synthetic rates. n-Butyrate has been previously shown to decrease receptor levels in GH1 cells. We demonstrate that n-butyrate decreases receptor levels primarily by shortening the half-life of the 47,000 Mr form.

The estrogen receptor in MCF-7 cells: evidence from dense amino acid labeling for rapid turnover and a dimeric model of activated nuclear receptor.

Upon treatment of MCF-7 cells with estradiol at 37 C, practically all of the estrogen receptor is found in the nuclear compartment. This nuclear estrogen receptor form sediments close to 5S on high salt sucrose density gradients, similar to receptor activated in vitro by gentle heating. Upon extraction of the nuclear receptor, this 5S form is not stable at 0-4 C under our buffer conditions and is converted to a 4S form in 10-14 h. Incubation of cells in the presence of dense amino acids for various amounts of time in the absence of estradiol, followed by a 20- to 30-min labeling period with 3 nM [3H]estradiol at 37 C, permits separation of the newly synthesized estrogen receptor on sucrose gradients by means of its different sedimentation properties. With the fresh nuclear extract, we observe a progressive shift to a heavier form on sucrose gradients in proportion to the time of incubation in dense amino acids. The 5S nuclear receptor gradually becomes denser as the time of incubation of cells in dense medium increases and the peak moves from light to heavy without resolving into discrete heavy and light forms at intermediary time points. In contrast, when the nuclear extract, prepared after varying periods of incubation of the cells with dense amino acids and brief treatment with [3H]estradiol, is left standing at 0-4 C for a period of 10-14 h, we observe discrete peaks of heavy and light receptors. The resolution into discrete peaks of heavy and light forms at each time point permits a calculation of the turnover rate for this receptor (t 1/2 = approximately 2.25 h). These findings are consistent with a model in which the 5S nuclear receptor is an oligomer of randomly associated heavy and light monomers, whereas the aged 4S nuclear receptor is a monomer. We conclude from these data that estrogen receptor synthesis is extremely rapid in log growth phase MCF-7 cells; and the activated nuclear receptor appears to be an oligomer of at least two monomers closely related in size and metabolized at a similar rate. The random association between different monomers (heavy and light) results in a broad peak of nuclear receptor and is at least consistent with a dimerization reaction occurring as part of receptor activation.

The glucocorticoid receptor in GH1 cells. Evidence from dense amino acid labeling and whole cell studies for an equilibrium model explaining the influence of hormone on the intracellular distribution of receptor.

The glucocorticoid receptor in GH1 cells cultured without glucocorticoids and harvested in hypotonic buffer is primarily a cytosolic protein with a sedimentation coefficient of 10 S in low salt gradients. In gradients containing 0.4 M KCl, the receptor sediments at 3.5 S. Following incubation of cells for various times in culture medium containing dense amino acids, discrete peaks of newly synthesized dense receptor and normal density 3.5 S receptor are resolved in salt-containing gradients. The half-life of the 3.5 S receptor is 9.5 h. In contrast, the 10 S receptor gradually becomes more dense as the time of incubation of cells with dense medium increases and discrete normal and dense receptor peaks are not resolved in sucrose gradients. These results suggest that the 10 S receptor is an oligomer and is in a rapidly exchanging equilibrium in intact cells at 37 degrees C with its 3.5 S hormone-binding subunit. When GH1 cells are incubated for 1 h at 37 degrees C with various concentrations of [3H]9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione, 16,17-acetonide ([3H]triamcinolone acetonide), receptor is found both in the cytosol and the nucleus. Sedimentation of the cytosol in low salt gradients reveals that a 4 S receptor form is present along with 10 S receptor. As the concentration of [3H]triamcinolone acetonide is increased, the amounts of both 4 S cytosolic receptor and nuclear receptor increase and the amount of 10 S cytosolic receptor decreases. Following removal of [3H]triamcinolone acetonide from cells, the amounts of both 4 S cytosolic receptor and nuclear receptor decrease and the amount of 10 S cytosolic receptor increases. Density labeling indicates that this increase in 10 S receptor is not due to new receptor synthesis. The 4 S receptor form, but not the 10 S form, binds to purified DNA. Resedimentation studies suggest that the 4 S receptor found in low salt gradients is identical with or derived from the 3.5 S form found in salt-containing gradients. A model for hormone-mediated receptor translocation to the nucleus is proposed in which binding of hormone shifts the equilibrium between the oligomeric 10 S receptor and its 4 S subunit toward the 4 S form. The 4 S form is in equilibrium with 4 S receptor bound to chromatin. The model provides a molecular explanation for many experimental findings concerning glucocorticoid and other steroid receptors.

Regulation of thyroid hormone nuclear receptor levels in GH1 cells by 3,5,3'-triiodo-L-thyronine. Use of dense amino acid labeling to determine the influence of hormone on the receptor half-life and the rate of appearance of newly synthesized receptor.

Regulation of thyroid hormone nuclear receptor levels in GH1 cells by 3,5,3'-triiodo-L-thyronine. Use of dense amino acid labeling to determine the influence of hormone on the receptor half-life and the rate of appearance of newly synthesized receptor.