Receptors In HeLa Cells Research Articles

Potential in vitro therapeutic effects of targeting SP/NK1R system in cervical cancer.

Cervical cancer, an aggressive gynecological cancer, seriously threatens women's health worldwide. It is recently reported that neuropeptide substance P (SP) regulates many tumor-associated processes through neurokinin-1 receptor (NK1R). Therefore, we used cervical cancer cell line (HeLa) to investigate the functional relevance of the SP/NK1R system in cervical cancer pathogenesis. Cellular proliferation and cytotoxicity were analyzed by colorimetric MTT assay. Quantitative real-time PCR (qRT-PCR) was used to measure mRNA expression levels of desired genes. Cell cycle distribution and apoptosis were evaluated by flow cytometry. A wound-healing assay was employed to assess migration ability. We found that the truncated isoform of NK1R(NK1R-Tr) is the dominantly expressed form of the receptor in Hela cells. We also indicated that that SP increased HeLa cell proliferation while treatment with NK1R antagonist, aprepitant, inhibited HeLa cell viability in a dose and time-dependent manner. SP also alters the levels of cell cycle regulators (up-regulation of cyclin B1 along with downregulation of p21) and apoptosis-related genes (up-regulation of Bcl-2 along with downregulation of Bax) while aprepitant reversed these effects. Aprepitant also induced arrest within the G2 phase of the cell cycle and subsequent apoptosis. Furthermore, SP promoted the migrative phenotype of HeLa cells and increased MMP-2 and MMP-9 expression while aprepitant exposure significantly reversed these effects. Collectively, our results indicate the importance of the SP / NK1R system in promoting both proliferative and migrative phenotypes of cervical cancer cells and suggest that aprepitant may be developed as a novel treatment for combating cervical cancer.

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER.

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

Conjugated anisotropic gold nanoparticles through pterin derivatives for a selective plasmonic photothermal therapy: in vitro studies in HeLa and normal human endocervical cells

This article reports a simple one-step method for anisotropic gold nanoparticle synthesis for plasmonic photothermal therapy medical purposes, using 1,4-bis[(2-ethylhexyl) oxy]-1,4-dioxo-2-butanesulfonic acid-sodium (AOT) reverse micelles as nanoreactor, where under specific condition, AOT acts as both a reducing and stabilizing agent. Obtained AuNPs were functionalized by attaching compounds derived from 2-aminopteridin-4(3H)-ona (pterin family) such as (2S)-2-[(4-{[(2-amino-4-hydroxypteridin-6-yl) methyl] amino} phenyl) formamido] pentanedioic acid (folic acid/FA) and 2-amino-4-hydroxypteridine-6-carboxylic acid (PCA) in order to evaluate its effect as targets for folate receptor-mediated cellular uptake in HeLa and normal human endocervical cells. The nanoconjugates were characterized through transmission electron microscopy (TEM), ultraviolet-visible, fluorescence, and Fourier transform infrared spectroscopies. Results showed an effective photothermal response of AuNPs in solution under NIR exposure with concentration dependence and none effect of the conjugation. In vitro studies in HeLa cells showed a concentration-dependent cytotoxicity of AuNPs; thus, conjugation to biomolecules such as FA or PCA has provided a biocompatible coating onto AuNPs and made them highly cytocompatible. Results demonstrated despite AF and PCA are analogue molecules, the folate receptors in HeLa cells are specific, and the different chemical groups available on the AuNPs surface have drastically different cell membrane penetration properties. The specific cell uptake through folate receptor (FR) was observed for short treatment time, while for a long treatment time, other mechanisms as penetration or adhesion were shown involved. In the particular case, of AF@AuNPs, the cell uptake through FR-mediated endocytosis was evidenced to have been decreasing cell viability in 24% after 2 h of treatment and 5 min under NIR exposure. This was confirmed by morphological changes in cells, as well the selective uptake of the FA@AuNPs by HeLa cells compared to normal cells, due folate receptor overexpression in HeLa cells. The findings from this study will have implications in the chemical design of nanostructures for plasmonic photothermal therapy. The obtained results provide evidences at in vitro level to support the fact that AF@AuNP nanoconjugate will accumulate in the affected tissue preferentially through the EPR (enhanced permeability and retention) effect by folate-targeting mechanism which will significantly enhance the efficacy of NIR-induced local photothermal effects.

Microtubules and Actin Filaments Facilitate Fusions and Separations of Vesicules on the Endocytic Pathway of EGF-Receptor Complexes

The effect of microtubules disassembly with nocodazole and depolymerization of the actin cytoskeleton with cytochalasin D, as well as their combined effect on endocytosis dynamics of complexes of epidermal growth factor and its receptor in HeLa cells, was studied. Using confocal microscopy, it was shown that in cells fixed at different intervals after endocytosis stimulation, there is a high degree of colocalization of the receptor with the marker of early endosomes EEA1, which correlates with enlargement of endosomes as a result of fusion at early stages, and translocation of endosomes to the juxtanuclear area (JNA) at later stages. Translocation towards the JNA depends on microtubules. Nocodazole does not interfere with the formation of endosomes, but blocks their fusions and translocations. At the same time, depolymerization of actin microfilaments, not affecting the process of EEA1-dependent fusions and transport of endosomes into the JNA, leads to enlargement of endosomes, preventing the separation of early endosomes and mature multivesicular endosomes. With the destruction of both types of cytoskeleton, the newly formed receptor-containing endocytic vesicles do not enlarge, do not mature, and do not move into the JNA. Live cell imaging of endosomes with EGF associated with quantum dots confirmed the data obtained on fixed cells and made it possible to analyze the nature of an endosome motility. It turned out that rare periods of a fast linear run are changed for prolonged chaotic oscillations within a small area, while the directions of linear runs can be different, and not only towards the MTOC. Our data suggest that the role of microtubules is not only to ensure the translocations of endosomes, but also to provide a platform for their fusion. This assumption can also explain the seemingly chaotic movements of endosomes, which make it possible to increase the probability of their contacts and, consequently, fusions.

PhotonSABER: new tool shedding light on endocytosis and learning mechanisms in vivo

ABSTRACTIn the central nervous system, activity-dependent endocytosis of postsynaptic AMPA-type glutamate receptors (AMPA receptors) is thought to mediate long-term depression (LTD), which is a synaptic plasticity model in various neuronal circuits. However, whether and how AMPA receptor endocytosis and LTD at specific synapses are causally linked to learning and memory in vivo remains unclear. Recently, we developed a new optogenetic tool, PhotonSABER, which could control AMPA receptor endocytosis in temporal, spatial, and cell-type-specific manners at activated synapses. Using PhotonSABER, we found that AMPA receptor endocytosis and LTD at synapses between parallel fibers and Purkinje cells in the cerebellum mediate oculomotor learning. We also found that PhotonSABER could inhibit endocytosis of epidermal growth factor receptors in HeLa cells upon light stimulation. These results demonstrate that PhotonSABER is a powerful tool for analyzing the physiological functions of endocytosis in non-neuronal cells, as well as the roles of LTD in various brain regions.

Multimodal highly fluorescent-magnetic nanoplatform to target transferrin receptors in cancer cells

BackgroundSite-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). MethodsThe Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. ResultsThe FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. ConclusionsThe prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. General significanceThis study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.

A cationic conjugated polymer coupled with exonuclease I: application to the fluorometric determination of protein and cell imaging.

A strategy is described for the detection of protein by using a cationic fluorescent conjugated polymer coupled with exonuclease I (Exo I). Taking streptavidin (SA) as model protein, it is observed that Exo I can digest single-stranded DNA conjugated with biotin and carboxyfluorescein (P1) if SA is absent. This leads to the formation of small nucleotide fragments and to weak fluorescence resonance energy transfer (FRET) from the polymer to P1. If, however, SA is present, the high affinity of SA and biotin prevents the digestion of P1 by Exo I. This results in the sorption of P1 on the surface of the polymer through strong electrostatic interaction. Hence, efficient FRET occurs from the fluorescent polymer to the fluorescent label of P1. Fluorescence is measured at an excitation wavelength of 370nm, and emission is measured at two wavelengths (530 and 425nm). The ratio of the two intensities (I530/I425) is directly related to the concentration of SA. Under the optimal conditions, the assay has a detection limit of 1.3ng·mL-1. The method was also applied to image the folate receptor in HeLa cells, thus demonstrating the versatility of this strategy. Graphical abstract A fluorometric strategy is described for protein detection and cell imaging based on a cationic conjugated polymer (PFP) coupled with exonuclease I (Exo I) trigged fluorescence resonance energy transfer (FRET).

Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF).

Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother’s breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N’ terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

A novel fluorescent histamine H1 receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands

Fluorescent ligands facilitate the study of ligand-receptor interactions at the level of single cells and individual receptors. Here, we describe a novel fluorescent histamine H(1) receptor antagonist (mepyramine-BODIPY630-650) and use it to monitor the membrane diffusion of the histamine H(1) receptor. The human histamine H(1) receptor fused to yellow fluorescent protein (YFP) was transiently expressed in CHO-K1 cells. The time course of binding of mepyramine-BODIPY630-650 to the H(1) receptor was determined by confocal microscopy. Additionally, fluorescence correlation spectroscopy (FCS) was used to characterize the diffusion coefficient of the H(1) receptor in cell membranes both directly (YFP fluorescence) and in its antagonist-bound state (with mepyramine-BODIPY630-650). Mepyramine-BODIPY630-650 was a high-affinity antagonist at the histamine H(1) receptor. Specific membrane binding, in addition to significant intracellular uptake of the fluorescent ligand, was detected by confocal microscopy. However, FCS was able to quantify the receptor-specific binding in the membrane, as well as the diffusion coefficient of the antagonist-H(1) receptor-YFP complexes, which was significantly slower than when determined directly using YFP. FCS also detected specific binding of mepyramine-BODIPY630-650 to the endogenous H(1) receptor in HeLa cells. Mepyramine-BODIPY630-650 is a useful tool for localizing the H(1) receptor using confocal microscopy. However, its use in conjunction with FCS allows quantification of ligand binding at the membrane, as well as determining receptor diffusion in the absence of significant bleaching effects. Finally, these methods can be successfully extended to endogenously expressed untagged receptors in HeLa cells.

S100A1: a physiological modulator of RYR1, Ca2+ release, and contractility in skeletal muscle. Focus on “S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle”

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

Depletion of the lipid raft constituents, sphingomyelin and ganglioside, decreases serotonin binding at human 5‐HT7(a) receptors in HeLa cells

The localization and function of several G protein-coupled receptors, including beta-adrenergic receptors and NK 1 receptors, are regulated via lipid rafts in the plasma membrane. These domains are enriched in cholesterol, gangliosides and sphingolipids, and play an important role in regulating signal transduction in most cell types. Serotonin (5-hydroxytryptamine, 5-HT), acting via 14 different receptors, regulates as diverse effects as mood, metabolism and smooth muscle contraction. 5-HT(7) receptors are involved in the regulation of depression, circadian rhythms, thermoregulation and vasodilatation. Ligand binding and signalling via the 5-HT(7) receptor are regulated by membranous cholesterol. Here we investigated the role of sphingomyelin and gangliosides on binding of 5-HT to 5-HT(7) receptors to further examine the role of lipid raft constituents on 5-HT(7) receptor function. HeLa cells stably transfected with the human 5-HT(7) receptor were treated with Fumonisin B(1) or (+/-)-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) to reduce sphingomyelin or ganglioside levels, respectively. The effects of these treatments were investigated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) viability assay, cholesterol analysis and [(3)H]5-HT binding studies on intact cells. Treatments with 20 mum Fumonisin B(1) for 24 h or with 10 mum PDMP for 48 h had no effects of total levels if 5-HT(7) receptors, but caused significant decreases in maximum [(3)H]5-HT binding to 5-HT(7) receptors. The effects were cholesterol-independent as levels of cholesterol remained unaffected by either treatment. These data demonstrate a role for sphingomyelin and gangliosides in regulating binding of [(3)H]5-HT to 5-HT(7) receptors. These observations further strengthen that actions of 5-HT via 5-HT(7) receptors are dependent upon lipid raft integrity.

The interactome of human EGF/ErbB receptors

Mol Syst Biol. 2: 2006.0006 When you search PubMed for ‘EGF receptor’ (EGFR), the database returns more than 5000 hits. This is not entirely surprising given the receptor's eminent role in cancer biology. However, it is truly surprising that two recent papers published by the groups of Gavin MacBeath and Matthias Mann add a whole new dimension to the problem of EGFR signaling. They do that by using protein microarray and quantitative proteomics technologies to comprehensively and quantitatively identify proteins associated with the cytosolic domains of the human EGFR family (Jones et al , 2005; Schulze et al , 2005). This family of membrane receptors consists of four different proteins called EGFR/ErbB1/HER1, ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. Under normal physiological conditions, the ErbB receptors play crucial roles in propagating signals regulating cell proliferation, differentiation, motility, and apoptosis (Holbro and Hynes, 2004). They are activated by ligand binding, which leads to homo‐ or heterodimerization followed by trans‐phosphorylation of specific tyrosine residues. These phosphorylated tyrosines, in turn, provide recognition sites for cytoplasmic proteins, which link ErbB receptors to downstream signaling transduction cascades such as the MAP kinase pathway. Among all ErbB family members, EGFR and ErbB4 are fully functional receptor tyrosine kinases, whereas ErbB2 does not …

Phospholipase D2 Is Required for Efficient Endocytic Recycling of Transferrin Receptors

RNA interference-mediated depletion of phospholipase D2 (PLD2), but not PLD1, inhibited recycling of transferrin receptors in HeLa cells, whereas the internalization rate was unaffected by depletion of either PLD. Although reduction of both PLD isoforms inhibits PLD activity stimulated by phorbol 12-myristic 13-acetate, only depletion of PLD2 decreased nonstimulated activity. Cells with reduced PLD2 accumulated a greater fraction of transferrin receptors in a perinuclear compartment that was positive for Rab11, a marker of recycling endosomes. EFA6, an exchange factor for Arf6, has been proposed to stimulate the recycling of transferrin receptors. Thus, one consequence of EFA6 overexpression would be a reduction of the internal pool of receptors. We confirmed this observation in control HeLa cells; however, overexpression of EFA6 failed to decrease the internal pool of transferrin receptors that accumulate in cells previously depleted of PLD2. These observations suggest that either PLD2 is required for a constitutive Arf6-mediated recycling pathway or in the absence of PLD2 transferrin receptors accumulate in recycling endosomes that are not responsive to overexpression of EFA6.

Targeting quantum dots to surface proteins in living cells with biotin ligase

Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.

Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids.

Ligand-dependent down-regulation of the glucocorticoid receptor (GR) has been shown to limit hormone responsiveness, but the mechanisms involved in this process are poorly understood. The glucocorticoid receptor is a phosphoprotein that upon ligand binding becomes hyperphosphorylated, and recent evidence indicates that phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover. Because phosphorylation is a key signal for ubiquitination and proteasomal catabolism of many proteins, we evaluated whether the ubiquitin-proteasomal pathway had a role in glucocorticoid receptor down-regulation and the subsequent transcriptional response to glucocorticoids. Pretreatment of COS-1 cells expressing mouse glucocorticoid receptor with the proteasome inhibitor MG-132 effectively blocks glucocorticoid receptor protein down-regulation by the glucocorticoid dexamethasone. Interestingly, both MG-132 and a second proteasome inhibitor beta-lactone significantly enhanced hormone response of transfected mouse glucocorticoid receptor toward transcriptional activation of glucocorticoid receptor-mediated reporter gene expression. The transcriptional activity of the endogenous human glucocorticoid receptor in HeLa cells was also enhanced by MG-132. Direct evidence for ubiquitination of the glucocorticoid receptor was obtained by immunoprecipitation of cellular extracts from proteasome-impaired cells. Examination of the primary sequence of mouse, human, and rat glucocorticoid receptor has identified a candidate PEST degradation motif. Mutation of Lys-426 within this PEST element both abrogated ligand-dependent down-regulation of glucocorticoid receptor protein and simultaneously enhanced glucocorticoid receptor-induced transcriptional activation of gene expression. Unlike wild type GR, proteasomal inhibition failed to enhance significantly transcriptional activity of K426A mutant GR. Together these findings suggest a major role of the ubiquitin-proteasome pathway in regulating glucocorticoid receptor protein turnover, thereby providing a mechanism to terminate glucocorticoid responses.

Quantity of partial agonist activity for antiglucocorticoids complexed with mutant glucocorticoid receptors is constant in two different transactivation assays but not predictable from steroid structure

An unsolved question in steroid hormone action is why the amount of agonist activity displayed by antisteroids is not constant but varies with the assay conditions. Receptor mutations have provided insight into hormone action, presumably due to changes in the tertiary structure of the receptor that alter its interaction surfaces with the transcriptional machinery or/and co-factors. We have now employed two mechanistically different induction assays to determine whether disparate transactivation processes are similarly altered by receptor mutations. The two activation assays studied were (i) the standard induction of GREtkLUC in transiently transfected CV-1 cells and (ii) a novel modulation of endogenous receptor activity by transiently transfected receptors in HeLa cells. Five different mutations in the ligand binding and DNA binding domains of the rat glucocorticoid receptor (CS1, CS1/CD, 451/9, C656G, and R732Q) and seven steroids of varied structures (five antagonists and two agonists) were selected for use. The results in both induction assays were the same. However, no generalizations regarding steroid structure and activity emerged. Neither of two potent glucocorticoids were active with GR-CS1, or GR-CS1/CD, while RU 486 was the only antisteroid with appreciable agonist activity. With the GR-451/9 mutant, three antagonists afforded partial agonist activity. We confirmed that the C656G mutant is both “super-sensitive” and “super-selective” for transactivation. In contrast, the R732Q mutation caused significant decreases in activity with both antagonists and subsaturating concentrations of agonists. This inability to generalize about the behavior of any class of steroids with mutant receptors may reflect an induced fit for each receptor–steroid complex. Nevertheless, the activity of a given steroid appeared to be constant in two different transactivation assays for a given mutant receptor. Thus, disparate transactivation processes may utilize identical receptor surfaces, even in the expression of partial agonist activity for specific antiglucocorticoids.

Vacuolation induced by cytotoxin from Helicobacter pylori is mediated by the EGF receptor in HeLa cells

The bacterial toxin VacA produced by H. pylori induces large vacuoles in several types of cultured cells such as HeLa cells or gastric cells. To determine the mechanism of vacuolation induced by this toxin we employed several inhibitors of membrane trafficking and endocytosis. The development of vacuolation induced by VacA in HeLa cells were prevented by bafilomycin A1 and low temperature conditions that inhibited vesicle transport or endocytosis. Formation of large vacuoles was also inhibited by an antibody against EGF receptor, which was previously shown to be internalized by endocytosis, but not by an anti-transferrin receptor antibody. Moreover, proteins of 58 and 37 kDa, corresponding to fragments of VacA, were recognized by an anti- H. pylori antibody after immunoprecipitation with anti-EGF receptor of cell extracts from HeLa cells treated with VacA, but not from untreated HeLa cells. We suggest that VacA may enter cells by endocytosis mediated by the EGF receptor. These are the first data indicating that the EGF receptor may be significant in the development of vacuolation caused by VacA.

Cloning and functional characterization of a sigma receptor from rat brain.

We have cloned a sigma receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain sigma receptor is highly homologous to that of the sigma receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain sigma receptor cDNA leads to a two- to threefold increase in haloperidol binding, and this cDNA-induced binding is sensitive to inhibition by several sigma receptor-specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain sigma receptor interacts with haloperidol with an apparent dissociation constant (K(D)) of 3 nM. Functional expression of the cloned rat brain sigma receptor in HeLa cells also leads to an increase in the binding of two other sigma ligands, namely, (+)-pentazocine and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain sigma receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1 ,3-di(2-tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF-7 cells, which do not express type 1 sigma receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol-sensitive binding of (+)-pentazocine, a selective type 1 sigma receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 sigma receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the sigma receptor-specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.