Import and export of nuclear proteins: Focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg²⁺ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.     

Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: Identification of the proteins involved

An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.     

A nuclear transforming factor that converts the goat uterine nonactivated estrogen receptor to nuclear estrogen receptor II

A 62-kDa nuclear protein that transforms the goat uterine nonactivated estrogen receptor (naER) to nuclear estrogen receptor II (nER II) has been isolated and purified. This is being identified as the naER-transforming factor (naER-TF). The transformation is achieved through deglycosylation of the naER. It is observed that the naER-TF action on the naER introduces significant changes in the structural and functional features of the naER. The capacity of the naER to bind estradiol increases 8- to 10-fold, while its hormone binding affinity reduces to a considerable extent following its exposure to naER TF. There is a critical ratio in the concentration of the two proteins, the TF and the naER, that would ensure an optimum transformation process. The transformed naER is incapable of dimerization with the estrogen receptor activation factor (E-RAF).     

Cholesterol inhibits the nuclear entry of estrogen receptor activation factor (E-RAF) and its dimerization with the nonactivated estrogen receptor (naER) in goat uterus

An alternative form of estrogen receptor isolated from goat uterus, the nonactivated estrogen receptor (naER), has no DNA-binding function, although it is closely similar to the classical estrogen receptor (ER) in its hormone binding affinity and specificity. The naER dimerizes with a DNA binding protein, estrogen receptor activation factor (E-RAF). The heterodimer binds to the DNA. Assays carried out during the purification of E-RAF showed that an endogenous inhibitor that is heat stable and dialyzable bound to the E-RAF and prevented the formation of the heterodimer. The inhibitor has been isolated and purified. GC-MS analysis identifies this molecule to be cholesterol. Circular dichroism measurement has shown that the high-affinity binding of cholesterol to E-RAF results in subtle changes in the secondary and the tertiary structure of the protein. The E-RAF with altered conformation fails to dimerize with the naER. Instead of facilitating E-RAF entry into the nucleus, dimerization with the naER prevents it. Similarly, cholesterol binding blocks the nuclear entry of the protein, showing that E-RAF with altered conformation is incapable of interaction with the nuclear pore complex/membrane proteins. The naER-E-RAF heterodimer remains at the nuclear periphery, incapable of further transport. These results indicate the possibility that the dimerization between naER and the E-RAF takes place only within the nuclear compartment. The observation that cholesterol binding prevents nuclear entry of the E-RAF reflects the similarity of E-RAF with the sterol regulatory element (SRE) binding protein that enters the nucleus and binds to SRE only when the intracellular level of cholesterol remains low.     

Nuclear estrogen receptor II (nER-II) is involved in the estrogen-dependent ribonucleoprotein transport in the goat uterus I. Localization of nER-II in snRNP.

Exposure of goat uterine nuclei to estradiol in vitro results in an immediate exit of ribonucleoproteins (RNP) from the nuclei to the medium. This RNP exit appears to be mediated by an estrogen receptor localized in small nuclear ribonucleoproteins containing U1 and U2 snRNA. Available evidence indicates that the estrogen receptor involved is not the ERalpha, but an alternative form, which is also a 66 kDa protein. This is the nuclear estrogen receptor II (nER-II) that has no DNA-binding capacity. The transport is estrogen-specific since non-estrogenic steroids do not stimulate the transport of the RNP where the receptor is localized.     

Proteins which mediate the nuclear entry of goat uterine non activated estrogen receptor (naER) following naER internalization from the plasma membrane

The nuclear transport of the internalised naER is influenced by a 58 kDa protein, p58, that appears to recognize the nuclear localization signals on the naER. At the nuclear pore complex the naER-p58 complex binds to a 62 kDa protein, p62; p58 recognizes p62 in this interaction. It is further observed that p62 gets 'docked' at a 66 kDa nuclear pore complex protein, npcp66. The nuclear entry of naER is an ATP-dependent process. An ATP-dependent biphasic nuclear entry of naER, has been observed. It is possible that the docking of p58-naER complex at the nuclear pore complex and the eventual nuclear entry of naER following its dissociation from the p58 are influenced by two different ranges in the concentration of ATP. In this process, it appears that, the nuclear entry requires an additional quantum of energy, provided by the hydrolysed ATP, in contrast to the energy requirement associated with, the nuclear 'docking' event.     

Localization of estrogen receptors in uterine cells. An appraisal of translocation.

The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The ‘nuclei’ which are obtained after lysis have a median diameter of 1μm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a model in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0 °C, provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressively accumulated in the nucleus; afterwards, estrogen receptor was progressively lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction, which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.     

Nuclear estrogen receptor II (nER-II) is involved in the estrogen-dependent ribonucleoprotein transport in the goat uterus: II. Isolation and characterization of three small nuclear ribonucleoprotein proteins which bind to nER-II.

Three proteins of a goat uterine small nuclear ribonucleoprotein (snRNP) fraction, which bind to nuclear estrogen receptor-II (nER-II) have been isolated and purified. These are the p32, p55, and p60 of which p32 is the major nER-II binding protein. Indirect evidence reveals that p32 binds to the nuclear export signal (NES) on the nER-II. nER-II is a snRNA binding protein while p32 does not bind to the RNA. nER-II along with p32 and p55 form an effective Mg(++)ATPase complex, the activation of which appears to be the immediate reason behind the RNP exit from the nuclei following estradiol exposure. The three nER-II binding proteins bind to the nuclear pore complex; nER-II does not possess this property.     

Estradiol dependent anchoring of the goat uterine estrogen receptor activation factor (E-RAF) at the endoplasmic reticulum by a 55 kDa anchor protein (ap55)

The primary intracellular site of localization of the estrogen receptor activation factor (E-RAF) is shown here to be the endoplasmic reticulum where the protein remains anchored through an estrogen dependent mechanism. The retention of E-RAF by the endoplasmic reticulum is facilitated by two proteins: (1) a 55 kDa anchor protein (ap55) which is an integral membrane protein of the endoplasmic reticulum. ap55 is a high affinity estrogen binding protein. A conformational change induced by estrogen binding is thought to favor the anchoring process. (2) The anchoring of E-RAF by ap55 is mediated by yet another protein. This is the 66 kDa transport protein (tp66) which recognizes ap55 on the one hand and E-RAF on the other. The presence of estradiol that saturates the hormone binding sites on ap55 appears to favor the anchoring of tp66-E-RAF complex to ap55. This interaction appears to be weakened by levels of estradiol below 7 nM concentration leading to the dissociation of the tp66-E-RAF complex from ap55. The tp66-E-RAF complex moves towards the nucleus.     

Progesterone abbreviates the nuclear retention of estrogen receptor in the rat oviduct and counteracts estrogen action on egg transport

Progesterone has synergistic or antagonistic effects on several estrogenic actions. The effects of progesterone on estrogen-induced accelerated ovum transport and on the dynamics of estrogen receptors in the rat oviduct were examined. The involvement of the progesterone receptors in these phenomena was assessed. On Day 1 of pregnancy, rats were treated with estradiol, estradiol plus progesterone, or either one plus the progesterone receptor-blocking agent RU486. Control animals received the oil vehicle alone. The number of eggs remaining in the oviduct was assessed 24 h after treatment. Cytoplasmic and nuclear estrogen receptor levels in the oviduct, as well as plasma concentrations of estradiol and progesterone, were measured at various intervals--up to 11 h and 24 h after treatment, respectively. Accelerated oviductal egg transport induced by estrogen was blocked by the concomitant administration of progesterone. This effect of progesterone was not associated with changes in estrogen circulating levels and was preceded by a reduction in the total amount of estrogen receptors and by a shortened retention of estrogen receptors in the nucleus. The effects of progesterone on egg transport and on the levels of estrogen receptors were reversed by blocking the progesterone receptor with RU486, suggesting that both effects were receptor-mediated. These findings demonstrate that progesterone antagonizes the effect of estrogen on oviductal egg transport in the rat, and suggest that this antagonism is mediated by a reduction both in the amount of estrogen receptors and in their retention time in the nucleus.     

The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay. While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery.     

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M_r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA.The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes.The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.     

Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.     

The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins

The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo- cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin alpha/beta-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.     

The retention of core ribonucleoproteins in the nuclear matrix isolated from nuclei treated with the phenantroline-copper complex

The nuclear matrix isolated from rat liver nuclei whose protein sulfhydryl groups were oxidised with the o-phenantroline-copper (OP-Cu) complex was enriched with a set of 32-44 kd polypeptides identified as core proteins of ribonucleoprotein particles (RNP). The most conspicuous protein in the nuclear matrix was a 36 kd protein present as a disulfide-linked homodimer. The propensity of protein 36 to be oxidised and form intermolecular associations suggests that it may contribute to the interaction of RNP particles with the nuclear matrix and thus to their spatial distribution in the nucleus.     

Melatonin blocks the activation of estrogen receptor for DNA binding.

The present study shows that melatonin prevents, within the first cell cycle, the estradiol-induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol-estrogen receptor complexes to estrogen-responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol-estrogen receptor complex to the estrogen responsive element.