Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.     

ATP-dependent activation of L cell glucocorticoid receptors to the steroid binding form.

The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.     

Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells

1. A competitive binding assay was adapted for determination of the specific binding of glucocorticoids to cytoplasmic receptors from rat thymus cells. The steroid–receptor complexes prepared by incubation of a cytoplasmic fraction from rat thymus cells with [1,2-³H₂]cortisol or with [1,2,4-³H₃]triamcinolone acetonide had rates of dissociation at 37°C similar to those from intact cells. 2. The cytoplasmic receptor was unstable at 3°C, but the rate of inactivation was decreased in the presence of 2.5mm-EDTA. The steroid–receptor complex was stable. 3. Rate constants for association and for dissociation, and association constants, were determined for the interactions of cortisol, cortexolone, dexamethasone and triamcinolone acetonide with the cytoplasmic receptor at 3°C. Differences in the association constants for different steroids could largely be accounted for by the differences in the rate constants for dissociation, but the rate constants for association did not vary greatly; the implications of these findings for the nature of the steroid-binding site are discussed. 4. A cytoplasmic fraction prepared from cells which had been incubated at 37°C under anaerobic conditions bound much less [1,2-³H₂]cortisol than did a fraction from aerobic cells, but the binding capacity was restored after exposure of the anaerobic cells to O₂. 5. The specific binding of [1,2-³H₂]-cortisol to intact thymus cells incubated aerobically was not affected by the presence of 0.1mm-cycloheximide, nor did this concentration of cycloheximide inhibit the recovery of specific binding observed when anaerobic cells were transferred to an aerobic atmosphere. 6. The energy dependence of specific binding of cortisol to the receptor is discussed with reference to possible mechanisms.     

Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60°C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cellls transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.     

Effect of contraceptive treatment on thymic glucocorticoid receptors and hepatic microsomal enzyme system of adult rats.

Contraceptive steroid treatment accounted for about a 30 per cent decrease in the number of thymic glucocorticoid receptors of adult rats. Neonatal allylestrenol treatment had no influence on that treatment. The activity of the hepatic microsomal (PSMO) enzyme system was not changed by the contraceptive treatment. It appears that contraceptive treatment may account for overlaps on receptors in adulthood.     

ATP-dependent activation of glucocorticoid receptor from rat liver cytosol.

The glucocorticoid--receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5--10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an 'activated' transformed form, which readily bound to nuclei, ATP--Sepharose, phosphocellulose and DNA--cellulose. This transformation was comparable with the activation achieved by warming the steroid--receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid--receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.     

Monoclonal antibodies against human DNA polymerase-alpha do not cross-react with glucocorticoid receptors

None of 16 independent monoclonal antibodies against human (KB cell) DNA polymerase-alpha recognizes epitopes on cytoplasmic glucocorticoid receptors prepared from the same cells. Consistently negative results are obtained with separate assays that measure antibody binding to uncharged receptors or to charged receptor complexes that have been preloaded with a specific steroid ligand. These results must qualify the interpretation of possible immunological relations between polymerase-alpha and glucocorticoid receptors that were inferred from studies with polyclonal antisera of poorly defined specificity.     

Structural determinants underlying constitutive dimerization of unoccupied human follitropin receptors

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr¹¹⁰ in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269–277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr¹¹⁰ to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr¹¹⁰ of the ECD.     

Apparent nuclear localization of unoccupied receptors for 1,25-dihydroxyvitamin D3

Previous studies have demonstrated that unoccupied 1,25-dihydroxyvitamin D₃ receptors are associated with crude chromatin under hypotonic conditions $\text{in}\text{vitro}$. The data presented herein show that unoccupied 1,25-dihydroxyvitamin D₃ receptors appear to be associated with chromatin prior to solubilization by dilution/homogenization in both high and low salt buffers. Additionally the unoccupied receptors are recovered nearly quantitatively from purified nuclei. These results suggest that unoccupied 1,25-dihydroxyitamin D₃ receptors may be localized within nuclei $\text{in}\text{vivo}$.     

Heteromeric nature of glucocorticoid receptors

The wild-type and a mutant receptor of S49.1 lymphoma cells have been shown by photoaffinity labelling to contain steroid-binding polypeptides of Mr 94 000 and 40 000, respectively. We investigated the molybdate-stabilized forms of these receptors and obtained Mr 325 000 and 285 000, respectively, by gel filtration and sedimentation analysis. Mild chymotrypsin treatment of the large wild-type receptor resulted in a form of about Mr 290 000 which contained a steroid-binding polypeptide of Mr 40 000. The data suggest that the high -Mr forms of glucocorticoid receptors are heteromeric in nature and contain one steroid-binding polypeptide per complex.     

Intracellular location of unoccupied 1,25-dihydroxyvitamin D receptors: a nuclear-cytoplasmic equilibrium

In order to investigate the subcellular distribution of unoccupied 1,25-dihydroxyvitamin D3 receptors, highly purified cytoplasts and nucleoplasts were prepared from two kidney cell lines (PK1 and MDBK). This was accomplished utilizing the technique of enucleation by cytochalasin B and density gradient centrifugation. Unoccupied 1,25-dihydroxyvitamin D3 receptors were found in both the nuclear and cytosolic compartments, with approximately 70% of the receptors localized in the cytoplasm. When cells were pretreated with 1,25-[3H]dihydroxyvitamin D, prior to enucleation, it was found that 90% of the receptor-hormone complex was associated with nucleoplasts, thus demonstrating that cytochalasin B treatment does not alter the high-affinity association of the receptor-hormone complex with the nucleus. The ratio of unoccupied receptor/protein was found to be the same in whole cells, cytoplasts, and nucleoplasts for both cell types. The ratio of unoccupied receptor/DNA was highest in cytoplasts and lowest in nucleoplasts. Taken together, these data indicate that the unoccupied 1,25-dihydroxyvitamin D receptor is generally associated with cell proteins and not specifically associated with cell DNA. We therefore propose, at least for these cells, that the unoccupied 1,25-dihydroxyvitamin D receptor exists in equilibrium between the nuclear and cytosolic compartments of the whole cell, and receptor-hormone binding shifts this equilibrium to favor nuclear localization.     

Chemical cross-linking of heteromeric glucocorticoid receptors

Glucocorticoid receptors of wild-type and nti ("increased nuclear transfer") mutant S49.1 mouse lymphoma cells exist in extracts under low-salt conditions predominantly as high molecular weight species (Mr greater than or equal to 300,000). These receptor-hormone complexes are unable to bind to DNA. High salt (300 mM KCl) produces dissociated receptors of Mr 116,000 and 60-A Stokes radius (wild type) and Mr 60,000 and 38-A Stokes radius (nti mutant), both of which bind to DNA. We used reaction with bifunctional N-hydroxysuccinimide esters as well as oxidation with Cu2+/o-phenanthroline to stabilize the high molecular weight structures. These cross-linked complexes do not interact with DNA, but reductive cleavage again produces the dissociable receptor forms and restores their ability to bind to DNA. The protein modifying reagents iodoacetamide and diethyl pyrocarbonate also produce stabilized high molecular weight receptor complexes. Cross-linking of the high molecular weight receptor forms can also be achieved in intact cells. Immunochemical techniques were used to prove that the complexes cross-linked either in vivo or in cell extracts do contain the heat shock protein of Mr 90,000 as a common constituent. The data show that the high molecular weight receptor complexes are preexisting in intact cells and that dissociation generates DNA binding ability.     

Persistent influence of neonatal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment on glucocorticoid receptors and on the microsomal enzyme system.

Neonatal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) accounted for a considerable decrease in the number of thymic glucocorticoid receptors in both male and female rats, as assessed at 6 weeks of age. TCDD also gave rise to a marked and prolonged increase in microsomal enzyme activity in the female rats but had practically no such effect on the males. These experimental observations attract attention on to the lasting microsomal inducer effect of the herbicide contaminant dioxin which damages foreign receptors and substantiate the chemical imprinting potential of aromatic hydrocarbons.     

Effect of C-reactive protein on platelet-activating factor-induced platelet aggregation and membrane stabilization

C-reactive protein (CRP) is an acute phase reactant which humoral concentration rises drastically following tissue injury or inflammation. CRP of all species binds to phosphorylcholine residues. In the present studies CRP was found to inhibit platelet-activating factor-induced platelet aggregation, and to stabilize platelet membranes against the lytic effect of lysophosphatidylcholine. Inhibition of platelet aggregation by CRP is accompanied by an inhibition of arachidonic acid release from both phosphatidylcholine and phosphatidylinositol. This suggests that phospholipases are inhibited. Hydrolysis of multilamellar dipalmitoylphosphatidylcholine liposomes by purified phospholipase A2, was also inhibited by CRP. These results suggest that CRP can stabilize membranes from the detergent-like effects of lysolipids and from potentially toxic materials such as platelet-activating factor. By inhibition of phospholipases, production of inflammatory mediators would be blocked. CRP might thus act as an early protective recognition mechanism in acute inflammatory states.     

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.     

Inactivation of hepatic glucocorticoid receptors by heparin

Heparin dramatically enhanced the rate of unbound glucocorticoid receptor inactivation in vitro in a concentration, time and temperature-dependent manner. Control specific binding decreased only about 25% after incubation for 6 h at 4°C. However in the presence of heparin (40 μg per ml cytosol) receptor binding decreased about 75%. At 25°C liver receptor specific binding was found to have a half0life of about 60 min in control cytosol. However, in the presence of heparin (40 μg per ml cytosol) the glucocorticoid receptor had a half-life of only 15 min at 25°C. Interestingly, 10 mM molybdate (with or without 5 mM dithiothreitol) greatly inhibited heparin-dependent receptor inactivation at 4°C. Dithiothreitol (alone) significantly stabilized receptor binding in control samples at 4°C, but provided no protection from heparin-dependent receptor inactivation. Heparin had no apparent inactivating effect on prebound glucocorticoid receptor complexes at 4°C. Interestingly however, heparin altered the sedimentation coefficient of prebound hepatic glucococorticoid-receptor complexes in low salt gradients from 7–8 S to about 3–4 S. When molybdate plus dithiothreitol were added with heparin, the sedimentation coefficient was found to be approx. 6—7 S. These results demonstrate that heparin, which is often used pharmacologically and which occurs naturally in animal tissues, has significant effects on liver glucocorticoid receptors in vitro.     

Tumor necrosis factor-induced downregulation of its receptors in HeLa cells

Tumor necrosis factor (TNF) induced loss of TNF receptors in HeLa cells was studied using acid elution technique, which could distinguish surface occupancy and real loss of receptors. Exposure of HeLa cells to TNF resulted in a rapid reduction in the number of TNF receptors without affecting the apparent binding affinity. The binding of transferrin after treatment with unlabeled TNF was unaffected. The TNF-mediated decrease in receptor number on the cells was reversible. Following removal of TNF from growth medium, binding activity was restored within 3 h. Cycloheximide prevented the restoration of TNF receptors, suggesting that de novo synthesis of receptors was required to restore the binding activity.