Dense Amino Acids Research Articles

Protein kinase-C activation stimulates synthesis of gonadotropin-releasing hormone (GnRH) receptors, but does not mediate GnRH-stimulated receptor synthesis.

Gonadotropes respond to GnRH with LH synthesis and release, desensitization, changes in GnRH receptor number, and GnRH receptor synthesis. Activation of protein kinase-C (PKC) appears to be involved in LH beta gene expression, but is not required for acute LH release, desensitization, or receptor down-regulation. The present studies were conducted to determine whether PKC mediates GnRH-stimulated receptor synthesis. We have adapted the density shift technique to measure the synthesis of GnRH receptors in pituitary culture. Pituitary cells from female weanling rats were exposed to medium containing treatments, dense amino acids (greater than 95% 13C, 15N, and 2H), dialyzed horse serum (10%, vol/vol), and fetal calf serum (2.5%, vol/vol). Treatments consisted of medium alone, phorbol myristate acetate (PMA), phorbol dibutyrate (PdBu), or GnRH. To deplete cells of PKC, cultures were exposed for 8-16 h to 1 microM PMA. Short term treatment with PKC activators (PMA or PdBu, 1 microM) or GnRH (0.1 nM) was given for 30 min. After treatment, GnRH receptors were covalently linked to [125I]Tyr5-azidobenzoyl-D-Lys6-GnRH and solubilized. Newly synthesized (densely labeled) GnRH receptors were separated from normal receptors by velocity sedimentation (156,000 X g; 24 h; 0-20% sucrose) and quantified by gamma-spectroscopy. Treatment with GnRH significantly stimulated the synthesis of GnRH receptors. Treatment of pituitary cell cultures with PMA (8-16 h) also stimulated the synthesis of GnRH receptors, although to a lesser extent than that observed after GnRH treatment. The synthesis of GnRH receptors in response to 0.1 nM GnRH was not different in cells with a normal complement of PKC compared to those depleted of PKC activity. This indicates that the ability of GnRH to stimulate the synthesis of its own receptor is not mediated by PKC. Short term treatment of cell cultures with 1 microM PMA or PdBu (30 min) stimulated GnRH receptor synthesis similar to treatment with 0.1 nM GnRH. When PMA and GnRH were administered simultaneously, GnRH receptor synthesis was stimulated to a greater extent than with either agent alone, suggesting differing mechanisms of action. These results indicate that although activators of PKC can stimulate the synthesis of GnRH receptors, PKC does not mediate the effects of GnRH on homologous receptor synthesis.

Regulation of the Synthetic Rate of Gonadotropin- Releasing Hormone Receptors in Rat Pituitary Cell Cultures by Inhibin*

Regulation of steady state levels of plasma membrane receptors for GnRH is the arithmetic result of processes that contribute to the appearance of receptors (synthesis, recycling, and unmasking) less those that contribute to the loss of receptors (degradation, internalization, and inactivation). We have adapted the density shift technique to evaluate specifically the rate of synthesis of GnRH receptors in rat pituitary cell cultures. Recently, it has been shown that inhibin can decrease the steady state levels of GnRH receptors in rat pituitary cell cultures and can block homologous up-regulation of GnRH receptors. In the present study we have evaluated the ability of purified inhibin to affect the synthesis rate of GnRH receptors under basal conditions and after exposure of cultured gonadotropes (from female weanling rats) to GnRH. Cells were exposed to inhibin alone (4 or 12 ng/ml) or to GnRH (10(-10) M) plus inhibin (0.4, 4, or 12 ng/ml) in the presence of densely labeled amino acids. GnRH was administered as a 20-min pulse, but inhibin treatment was continued for up to 2 days. After these treatments, GnRH receptors were covalently linked to a radio-labeled photoaffinity probe (125I- Tyr5-[azido-benzoyl-D-Lys6] GnRH) and solubilized with 1% sodium dodecyl sulfate. Newly synthesized GnRH receptors (those that had incorporated the dense amino acids) were separated from previously synthesized receptors (those containing normal amino acids) by velocity sedimentation through sucrose gradients (O-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After velocity sedimentation, gradients were fractionated, and the radioactivity in each fraction was quantified. Treatment with inhibin alone had no effect on the synthesis rate of GnRH receptors compared to that of control cultures (t1/2, 23.5 +/- 0.3 vs. 23.3 +/- 0.3 vs. 22.9 +/- 0.9 h for control, 4 ng/ml inhibin, and 12 ng/ml inhibin, respectively). In contrast, inhibin blocked the stimulation of homologous receptor synthesis by GnRH in a dose-dependent manner (t1/2, 12.2 +/- 0.7 vs. 14.0 +/- 0.7 vs. 19.2 +/- 1.5 vs. 20.0 +/- 2.9 h for GnRH alone and GnRH plus 0.4, 4, or 12 ng/ml inhibin, respectively). These data indicate that in rat pituitary cell cultures, inhibin does not decrease basal levels of GnRH receptors by affecting the synthesis rate of receptors, but prevents up-regulation of GnRH receptors by blocking stimulation of GnRH receptor synthesis by homologous hormone.

Altered Rate of Synthesis of Gonadotropin-Releasing Hormone Receptors: Effects of Homologous Hormone Appear Independent of Extracellular Calcium*

Steady state levels of plasma membrane receptors (measured in radioligand assays) result from processes that contribute to the production of receptors (synthesis, recycling, and unmasking) as well as those that contribute to the loss of receptors (degradation, internalization, and inactivation). Accordingly, we have previously adapted the density shift technique to determine the contribution due to synthesis of GnRH receptors. In the present study we have evaluated the ability of homologous hormone to affect the rate of synthesis of these receptors. Additionally, because of its role in other actions of the releasing hormone, the requirement for extracellular Ca2+ to mediate the effects of GnRH was assessed. Cultures of pituitary cells, prepared from female weanling rats, were used for all studies. After treatment with GnRH, a GnRH antagonist, or the calcium ionophore A23187, with or without EGTA (a calcium chelator), cells were further cultured for up to 24 h in medium containing either dense or normal amino acids. After this treatment, receptors for GnRH were covalently linked to a radiolabeled photoaffinity probe [( 125I]Tyr5-[azido-benzoyl-D-Lys6-GnRH]) then solubilized in 1% sodium dodecyl sulfate. Receptors that had incorporated the dense amino acids (i.e. newly synthesized receptors) were separated from those that had been synthesized before the addition of dense amino acids by velocity sedimentation in sucrose gradients (0-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After centrifugation, gradients were fractionated, and the radioactivity in each fraction was quantified. GnRH treatment (10 or 0.1 nM) increased the rate at which dense amino acids were incorporated into GnRH receptors (t 1/2 = 13 +/- 2, 15 +/- 1, and 25 +/- 2 h for 0.1 nM GnRH, 10 nM GnRH, and control values, respectively). GnRH antagonist alone did not change the rate of GnRH receptor synthesis (t 1/2 = 22 +/- 3 h) compared to the control value (t 1/2 = 25 +/- 2 h) and was able to block the effects of GnRH. The effects of GnRH were not antagonized by inclusion of 3 mM EGTA during treatment (t 1/2 = 15 +/- 1 h vs. 13 +/- 2 h for 0.1 nM GnRH in the presence and absence of 3 mM EGTA, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

Conservative Segregation of Tetrameric Units of H3 and H4 Histones during Nuclesome Replication

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.

Synthesis of Gonadotropin-Releasing Hormone Receptors by Gonadotrope Cell Cultures: Both Preexisting Receptors and Those Unmasked by Protein Kinase-C Activators Show a Similar Synthetic Rate*

Responsiveness of gonadotropes to GnRH depends, in part, on the number of plasma membrane GnRH receptors. Since the steady state level of these plasma membrane receptors is a function of the rates of both receptor generation (synthesis, unmasking, and recycling) and loss (internalization, degradation, and inactivation) we have sought to quantify the rate of synthesis of GnRH receptors in pituitary cell cultures. Further, since the protein kinase-C activator phorbol 12-myristate 13-acetate (PMA) has been shown to unmask a class of GnRH receptors that appear to be uncoupled from phosphoinositide turnover, we have measured the rate of synthesis of this second receptor population. The present studies use the density shift technique; incorporation of densely labeled amino acids confers a higher density to newly synthesized proteins and allows their separation by physical means. Cultures of pituitary cells were prepared from female weanling rats. After cells had attached to the culture dishes, medium was replaced at 12-h intervals with medium containing either densely labeled or normal amino acids. After the incubation, GnRH receptors were covalently linked to a photoaffinity receptor agonist [( 125I]Tyr5-[azido-benzoyl-D-Lys6-GnRH]), then solubilized with 1+ sodium dodecyl sulfate. In some cultures PMA (50 nM) was included during the photoaffinity agonist-binding step. Newly synthesized (dense) receptors were separated from previously synthesized receptors by velocity sedimentation (0-20% sucrose in 1% sodium dodecyl sulfate-10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). Gradients were fractionated, and the radioactivity contained in each fraction was quantified. Newly synthesized GnRH receptors exhibited a higher density, as evidenced by further migration into the gradient, than did normal GnRH receptors. There was a delay of approximately 6 h between exposure to dense amino acids and the appearance of densely labeled GnRH receptors at the plasma membrane. Equilibrium for incorporation of dense amino acids into GnRH receptors was 48 h of exposure to dense amino acids. The time required for synthesis of half the entire population of GnRH receptors was 28 +/- 2 h (mean +/- SEM; n = 4). Scatchard analysis and the pattern of GnRH-stimulated LH release from densely labeled cells indicated that they bound the photoaffinity label (Kd = 0.4 nM; approximately 1 fmol receptor/microgram DNA) and secreted gonadotropin normally. Additionally, treatment with PMA caused a significant increase (181 +/- 24%) in photoaffinity agonist binding, consistent with previous observations.

Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands.

Density labeling procedures have been utilized to study the dynamics of histone-histone interactions in vivo. Cells were labeled for 60 min with dense amino acids, and the label was chased for up to 22 h (two replication events for these cells). Nuclei were isolated and treated with formaldehyde to stabilize the histone-histone interactions with a covalent cross-link that produces an octameric complex of two each of H3, H2B, H2A, and H4. This complex was then extracted from the DNA and analyzed on density gradients. The results indicate that new H3,H4 deposits as a tetramer and does not dissociate in the subsequent chases. New H2A,H2B deposited as a dimer and also does not dissociate in subsequent chases. These new histones form hybrid octamers with old histones. On the basis of the new:old ratio in the hybrid octamers, we propose that additional old H2A,H2B from elsewhere in the genome interacts with tetramers of new H3,H4 to form the newly synthesized nucleosomes. It is also observed that 5% of the cross-linked complexes produced by formaldehyde are octamer-octamer (dioctamer). Upon analysis of the density of the dioctamer, the hybrid octamers were found adjacent to octamers that were homogeneous with respect to containing normal density histones. Control experiments are presented to demonstrate that the octamer-octamer cross-links are a product of intrastrand and not interstrand interactions between nucleosomes. These same control experiments also indicate that these procedures do not induce histone exchange during the preparative procedure prior to density gradient analysis. The significance of these results with regard to the dynamics of histone-histone interactions at the replication fork and the potential role in the maintenance of differentiation is discussed.

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4.

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.

Dynamics of estrogen receptor turnover in uterine cells in vitro and in uteri in vivo.

The rate at which the estrogen receptor (ER) is synthesized and degraded (turns over) in target cells is a major factor regulating the levels of ER and cell sensitivity to estrogen. We have used the ability of tamoxifen aziridine [(TAZ)(Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-1-buten e] to affinity label the ER in intact cells to study the dynamics of ER turnover in uterine cells in vitro and in intact rat uteri in vivo. In primary cultures of rat uterine cells (from 21-day-old rats), ER is degraded with a half-life of 3-4 h, as determined by pulse-chase experiments in which ER in cells are covalently labeled with [3H]TAZ (20 nM) and then exposed to a chase of 10(-6) M estradiol. Density shift experiments, in which uterine cells are exposed for different time periods to medium containing dense (15N,13C,2H) amino acids and the shift of receptor from a normal density to a more dense species is analyzed on sucrose gradients, also confirm this rapid turnover for the uterine ER. A similar half-life is obtained for ER in rat uteri in vivo, after intralumenal installation of [3H]TAZ to label effectively ER covalently. Analysis of nuclear ER on sodium dodecyl sulfate-polyacrylamide gels during the chase period reveals the loss of the 65,000 mol wt [3H]TAZ-labeled receptor species; no lower mol wt [3H]TAZ-labeled fragments are observed. Likewise, immunoblot analyses of receptor with the ER monoclonal antibody H222Sp gamma reveal only the 65,000 mol wt receptor species, with no evidence of any smaller receptor forms in cells. Interestingly, incubation of uterine cells with 10(-5) M cycloheximide (which inhibits 98% of protein synthesis) completely arrests ER turnover, suggesting that ER turnover, which is rapid, is probably dependent upon the synthesis of other rapidly turning over proteins. Hence, we conclude that ER turnover is rapid in uterine cells, that turnover of uterine ER occurs at a rate similar to that previously reported for ER turnover in MCF-7 human breast cancer cells, and that ER turnover is similar in uterine cells under in vitro and in vivo conditions. These findings indicate that the receptor is a dynamic and rapidly turning over protein whose level could be adjusted quickly by alterations in the rate of synthesis and/or degradation.

Hormonal regulation of estrogen and progestin receptors in decidual cells.

Total estrogen receptor (Re) and total progestin receptor (Rp) were measured in the cytosol and nuclear fractions from hamster deciduomal tissue and decidual cell cultures. Correlation of serum steroid (estradiol and progesterone) and deciduomal receptor profiles revealed a significant loss of Re during the first four days of decidualization that was not attributable to changes in serum steroid levels. A decidual cell-tissue culture system was used to study the receptor's recovery response to progesterone withdrawal. Decidual cells were plated and grown in Ham's F12/Dulbecco's modified Eagle's medium with 5% horse serum supplemented with insulin, transferrin, selenium and progesterone (10 ng/ml). Within 48 h of culture large, multinucleate decidual cells were observed by phase microscopy. At 72 h of culture in medium containing progesterone, only Rp was detectable in decidual cells. Re was not detectable (less than 200 fmol/mg DNA) in either cytosol or nuclei from cells maintained in the presence of progesterone. However, when progesterone was deleted from the medium, cytosol Re recovered progressively from 8 h to 16 h of culture. Progesterone withdrawal also caused parallel increases in cytosol and nuclear Rp, and estradiol treatment (2 ng/ml) in combination with progesterone withdrawal further enhanced Rp levels in decidual cell cultures. These results with cultured decidual cells demonstrate that progesterone down-regulates Re and Rp, Re recovers rapidly upon progesterone withdrawal, and the Re system is competent to respond to estrogen action in terms of Rp induction. We used the density-shift method to determine that progestin increases the turnover of nuclear Re in hamster decidual cells within 3 h. Hamster decidual cells were isolated from the endometrium and cultured in progesterone-free medium containing normal amino acids (1H, 12C, 14N) for 2 days. Confluent monolayers of cells were exposed to 1 nM estradiol (E2) for 1 h to maximize the amount of occupied Re in the nuclear fraction. Then, at time 0, cells were transferred to medium supplemented with dense (2H, 13C, 15N) amino acids and either 1 nM E2 or E2 plus 100 nM progesterone. After Re was labeled with dense amino acids for 1, 3, 6 and 9 h, nuclear Re was extracted with 10 mM pyridoxal -5' phosphate and labeled with 125I-iodoestradiol (5 nM). Two radioactive peaks representing preexisting and newly synthesized Re were separated by sucrose density-gradient centrifugation. The halflife of nuclear Re in decidual cells was 3.7 h when cells were treated with E2 alone.(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.

Mechanism of androgen-receptor augmentation. Analysis of receptor synthesis and degradation by the density-shift technique.

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) contains receptors for, and is stimulated by, androgens. Cells cultured in the absence of androgens maintain a basal level of androgen receptors. Following incubation with various concentrations of the synthetic androgen methyltrienolone (R1881) for 1-6 h, the concentration of these receptors increased from 6.0 to 12.2 fmol/micrograms of DNA, while the equilibrium dissociation constant (Kd) of 0.5 nM for this steroid remained unchanged. The steroid-induced increase in androgen receptor levels was specific for androgens and dependent upon protein synthesis. The mechanism of receptor augmentation was examined by utilization of isotopically dense amino acids to determine rates of receptor appearance and degradation in the presence or absence of [3H]R1881. In the absence of androgens, the half-life of the androgen receptor was 3.1 h, with a rate constant (kD) of 0.22/h. In the presence of 1 nM [3H]R1881, however, the half-life was 6.6 h, with kD = 0.11/h. The rate constant for receptor synthesis (ks) in the absence or presence of [3H]R1881 was calculated to be 1.35 and 2.23 fmol/micrograms of DNA/h, respectively. Thus, androgen-induced androgen-receptor augmentation is explained by an increase both in receptor half-life and in rates of receptor synthesis.

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.

Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique.

The level of estrogen receptor (ER) is a major factor regulating cell sensitivity to estrogen. We have determined the rates of ER synthesis and turnover in MCF-7 breast cancer cells by incubating cells in medium supplemented with 13C15N2H-amino acids (dense amino acids) and monitoring the shift from "old-light" (preexisting) to "new-dense" (newly synthesized) receptors by velocity sedimentation on 0.4-M KCl, 5-20% sucrose gradients prepared in buffered deuterium oxide. Cytosol or unoccupied nuclear ER prepared from control cells grown in the presence of 12C14N1H-amino acids (normal amino acids) and labeled in vitro with [125I]iodoestradiol sediments as a single, normal density peak. When cells were incubated in medium supplemented with dense amino acids for various periods of time, the [125I]iodoestradiol-labeled receptor showed a progressive shift with time to a denser species; by 3 h, a more rapidly sedimenting shoulder was observed on the normal density peak; by 6 h, 40% of the receptor sedimented at the normal density rate, with 60% at a more rapid rate; by 8 h, the predominant peak (70%) was at the more dense position, and at 15 h, all of the sites sedimented as the denser form. Thus, the magnitude of the receptor peak of normal density as a function of time in dense medium indicates that the receptor in control cells has a half-life of 4.0-4.5 h. To determine whether estradiol or the antiestrogens nafoxidine (U11,100A; (1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy] ethyl)pyrrolidine hydrochloride) or CI628 affected the turnover rate of the receptor, measurements were performed on cells grown in the continuous presence of estradiol or antiestrogen (steady state conditions). Exposure of cells to 10 nM estradiol was found to increase the turnover rate of the nuclear receptor, while exposure to 200 nM nafoxidine or CI628 (alpha-(p-[2-(1-pyrrolidino)ethoxy]phenyl)4-methoxy-alpha'- nitrostillbene) did not substantially alter the turnover rate of the nuclear receptor. The half-lives of receptor were found to be 4.02 +/- 0.23 and 4.47 +/- 0.26 h for control unoccupied cytosol and nuclear receptors, 3.00 +/- 0.38 h for estradiol-occupied nuclear receptors, 4.90 +/- 0.66 h for CI628-occupied nuclear receptors, and 3.43 +/- 0.37 h for nafoxidine-occupied nuclear receptors. These results indicate that ER turns over rapidly, with a half-life of 3-5 h, in the presence or absence of estradiol or antiestrogen, and that receptor synthesis is also rapid, with the rate of appearance of newly synthesized receptor being 0.3-0.5 pmol/mg DNA . h. These rates provide the cell with the capacity for dynamic and rapid regulation of its ER levels.

Differential effects of androgens and glucocorticoids on regulation of androgen receptor concentrations and cell growth

This report describes androgen induced up-regulation of intracellular androgen receptor concentrations in the DDT 1MF-2 cell line derived from the hamster ductus deferens and the R3327H-G8-A1 line derived from the Dunning prostate adenocarcinoma. Within 6 h of exposure to androgens the receptor concentration is increased approximately 2-fold. Incorporation of dense aminoacids and subsequent analysis by sucrose density gradient centrifugation indicates that the observed increase in receptors is possibly due to de novo androgen receptor synthesis. In both cell lines this increase is inhibited by 30–50% within 6 h and completely during a subsequent 18 h period by the potent glucocorticoid triamcinolone acetonide (TA). TA also inhibits the growth of both cell lines and antagonizes the stimulatory effect of androgens. Flow cell cytometry studies indicate that TA blocks the cells in the G1 phase of the cell cycle. This event may be associated with regulation of androgen receptor concentrations.

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.

Assembly of new histones into nucleosomes and their distribution in replicating chromatin.

We studied the assembly of new histones into nucleosomes and their distribution in replicating chromatin in growing P815 mouse cells. New histones and new DNA were density-labeled with 13C, 15N, 2H-substituted amino acids together with [3H]arginine or with 5-iododeoxyuridine and [3H]thymidine, respectively, for 1 hr (approximately 20% of S phase). Mono- di-, tri-, tetra- and larger oligonucleosomes were isolated by sucrose gradient centrifugation of micrococcal nuclease-digested chromatin, and their density distribution was analyzed, without fixation, in metrizamide/triethanolamine density gradients [Russev, G. and Tsanev, R. (1976) Nucleic Acids Res. 3, 697-707] in which mono- and oligonucleosomes containing dense amino acids or 5-iododeoxyuridine separate from the corresponding normal nucleosomes. Under these conditions, approximately 74% of the new histones are found in nucleosomes on newly replicated DNA, and the remainder are on unreplicated DNA. The majority of new histones form entirely new nucleosomes; a minor fraction may form hybrid nucleosomes that also contain preexisting histones. New nucleosomes are distributed to both new daughter DNA molecules with approximately equal probability, and our evidence suggests, but does not prove, that they are distributed in a random manner along new DNA.

Synthesis, insertion into the plasma membrane, and turnover of alpha-bungarotoxin receptors in chick sympathetic neurons.

alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.

Conservative assembly and segregation of nucleosomal histories

The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3–4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2–3 generations.