DEAE-dextran Method Research Articles

The Effect of Resveratrol on the Werner Syndrome RecQ Helicase Gene and Telomerase Activity

Various protein factors, including telomerase and WRN helicase, are involved in telomere maintenance. Resveratrol (Rsv), a polyphenol that extends the lifespan of diverse species is an activator of SIRT1, a NAD+ dependent deacetylating enzyme in mammalian cells. Here, we examined the changes in gene expressions and promoter activities of WRN helicase and telomerase after Rsv treatment. This treatment increased the amount of WRN transcript and protein product by activating its promoter and telomerase promoter activity and gene expression. However cell proliferation was not changed. This suggests that Rsv induces telomere maintenance factors like WRN helicase without affecting cell proliferation. Keywords: BLM, Resveratrol, Telomerase, Sp1, TERT, WRN, Werner Syndrome, deacetylating enzyme, cell proliferation, eukaryotic chromosomes, fibroblasts, Reactive oxygen species (ROS), mitochondrial respiration, caloric restriction (CR), superoxide dismutases (SODs), Human cervical carcinoma (HeLa S3), Dulbecco's modified Eagle's (DME) medium, MTS assay, Luciferase, plasmids, DEAE-dextran method, Reverse Transcriptase Polymerase Chain Reaction (RTPCR), acrylamide gels, ethidium bromide, Real time PCR analysis, GAPDH, plasmid-transfected cells, anti-actin, anti-Sp1 antibodies, DNA-damage responses, DNA-repair synthesis, insulin signaling, epigenetic, sirtuin (SIRT1)-mediated deacetylation

Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

DEAE-Dextran Transfection of Lymphocyte Cell Lines

INTRODUCTIONIn this procedure, negatively charged DNA binds to the positively charged diethylaminoethanol (DEAE) moieties of a DEAE-dextran polymer. The soluble complex is added to the cells, which take it up by nonspecific endocytosis. The method described here uses an initial incubation with a DNA/DEAE-dextran mixture, followed by a second incubation in growth medium supplemented with chloroquine. Chloroquine is thought to enhance transfection efficiencies by neutralizing lysosomal hydrolases, which can degrade the DNA. This protocol describes transfection of lymphocyte cell lines, but it can be used for transient transfection of both adherent and nonadherent cells. However, it rarely is used for stable transfection experiments because toxicity prevents long-term maintenance of the transfected cells. One notable advantage of the DEAE-dextran method is that a relatively small amount of DNA is needed for each experiment.

RIBEYE Recruits Munc119, a Mammalian Ortholog of the Caenorhabditis elegans Protein unc119, to Synaptic Ribbons of Photoreceptor Synapses

Munc119 (also denoted as RG4) is a mammalian ortholog of the Caenorhabditis elegans protein unc119 and is essential for vision and synaptic transmission at photoreceptor ribbon synapses by unknown molecular mechanisms. Munc119/RG4 is related to the prenyl-binding protein PrBP/delta and expressed at high levels in photoreceptor ribbon synapses. Synaptic ribbons are presynaptic specializations in the active zone of these tonically active synapses and contain RIBEYE as a unique and major component. In the present study, we identified Munc119 as a RIBEYE-interacting protein at photoreceptor ribbon synapses using five independent approaches. The PrBP/delta homology domain of Munc119 is essential for the interaction with the NADH binding region of RIBEYE(B) domain. But RIBEYE-Munc119 interaction does not depend on NADH binding. A RIBEYE point mutant (RE(B)E844Q) that no longer interacted with Munc119 still bound NADH, arguing that binding of Munc119 and NADH to RIBEYE are independent from each other. Our data indicate that Munc119 is a synaptic ribbon-associated component. We show that Munc119 can be recruited to synaptic ribbons via its interaction with RIBEYE. Our data suggest that the RIBEYE-Munc119 interaction is essential for synaptic transmission at the photoreceptor ribbon synapse.

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

Pax-6 and c-Maf Functionally Interact with the α-Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression

Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of alpha-cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in alpha-cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Evidence for Actin Cytoskeleton-dependent and -independent Pathways for RelA/p65 Nuclear Translocation in Endothelial Cells

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

Membrane Potential-regulated Transcription of the Resting K+ Conductance TASK-3 via the Calcineurin Pathway

The 2P domain K(+) channel TASK-3 is highly expressed in cerebellar granule neurons where it has been proposed to underlie the K(+) leak conductance, IKso. In a previous work we showed that expression of TASK-3 increases in cerebellar granule neurons as they mature in culture. Here we show that within the cerebellum, levels of TASK-3 mRNA increase as granule neurons migrate to their adult positions and receive excitatory mossy fiber input. To understand the mechanism of this increase in TASK-3 expression we used an in vitro model culturing the neurons in either depolarizing conditions mimicking neuronal activity (25K, 25 mm KCl) or in conditions which approach deafferentation (5K, 5 mm KCl). An important increase in TASK-3 mRNA is uniquely observed in 25K and is specific since other background K(+) channel levels remain unchanged or decrease. The rise in TASK-3 mRNA leads to an increase in TASK-3 protein and the IKso conductance resulting in hyperpolarization. Blocking L-type calcium channels or their downstream effector calcineurin, abrogates TASK-3 expression and IKso, leading to hyperexcitability. This is the first study demonstrating that depolarization-induced Ca(2+) entry can directly regulate cellular excitability by dynamically regulating the transcription of a resting K(+) conductance. The appearance of this conductance may play an important role in the transition of depolarized immature neurons to their mature hyperpolarized state during neuronal development.

Telmisartan downregulates angiotensin II type 1 receptor through activation of peroxisome proliferator-activated receptor γ

Telmisartan, an angiotensin II type 1 receptor (AT1R) antagonist, was found to have a unique property: it is a partial agonist of peroxisome proliferator-activated receptor gamma (PPARgamma). Since previous studies have demonstrated that PPARgamma activators suppressed AT1R expression, we examined whether telmisartan affects AT1R expression in vascular smooth muscle cells. Vascular smooth muscle cells were derived from the thoracic aorta of Wistar-Kyoto rat. Northern and Western blotting analysis were used to examine AT1R mRNA and protein expression, respectively. The DEAE-dextran method was used for transfection, and the promoter activity of AT1R was examined by luciferase assay. Telmisartan decreased the expression of AT1R at the mRNA and protein levels in a dose- and time-dependent manner. Decreased AT1R promoter activity with unchanged mRNA stability suggested that telmisartan suppressed AT1R gene expression at the transcriptional level. However, the expression of AT1R was not suppressed by other AT1R antagonists such as candesartan or olmesartan. Since the suppression of AT1R expression was prevented by pretreatment with GW9662, a PPARgamma antagonist, PPARgamma should have participated in the process. The deletion and mutation analysis of the AT1R gene promoter indicated that a GC box located in the proximal promoter region is responsible for the telmisartan-induced downregulation. Our data provides a novel insight into an effect of telmisartan: telmisartan inhibits AT1R gene expression through PPARgamma activation. The dual inhibition of angiotensin II function by telmisartan - AT1R blockade and downregulation - would contribute to more complete inhibition of the renin-angiotensin system.

Epitope Mapping of Monoclonal Antibody to Integrin αL β2 Hybrid Domain Suggests Different Requirements of Affinity States for Intercellular Adhesion Molecules (ICAM)-1 and ICAM-3 Binding

Integrin undergoes different activation states by changing its quaternary conformation. The integrin beta hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin alpha(L) beta2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the beta2 hybrid domain. Integrin alpha(L) beta2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to alpha(L) beta2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated alpha(L)beta2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, alpha(L) beta2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that alpha(L)beta2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by alpha(L)beta2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation.

Regulation of the Expression of Caspase-9 by the Transcription Factor Activator Protein-4 in Glucocorticoid-induced Apoptosis

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at -1.55 and -1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases -1140 and -944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Paired Activation of Two Components within Muscarinic M3 Receptor Dimers Is Required for Recruitment of β-Arrestin-1 to the Plasma Membrane

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

Microsomal Triglyceride Transfer Protein Promotes the Secretion of Xenopus laevis Vitellogenin A1

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Tumor Necrosis Factor α-induced Desumoylation and Cytoplasmic Translocation of Homeodomain-interacting Protein Kinase 1 Are Critical for Apoptosis Signal-regulating Kinase 1-JNK/p38 Activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.

A Genome Wide Screening Approach for Membrane-targeted Proteins

Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.

Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface.

Collagen XVII/BP180, an epithelial adhesion molecule, belongs to the group of collagenous transmembrane proteins, which are characterized by ectodomain shedding. We recently showed that ADAMs can cleave collagen XVII, but also that furin participates in this process (Franzke, C. W., Tasanen, K., Schäcke, H., Zhou, Z., Tryggvason, K., Mauch, C., Zigrino, P., Sunnarborg, S., Lee, D. C., Fahrenholz, F., and Bruckner-Tuderman, L. (2002) EMBO J. 21, 5026-5035). To define the cleavage region in the juxtamembranous NC16A linker domain and assess its structure and requirements for shedding, we constructed deletion mutants of the NC16A domain, expressed them in COS-7 cells, and analyzed their structural integrity and shedding behavior. A mutant lacking the furin consensus sequence was shed in a normal manner, demonstrating that furin does not cleave collagen XVII but rather activates ADAMs (a disintegrin and metalloproteinase). Large deletions of the NC16A domain prevented shedding, and analysis of defined smaller deletions pointed to the stretch of amino acid residues 528-547 as important for sheddase recognition and cleavage. Secondary protein structure predictions showed that deletion of this stretch resulted in an NC16A domain with a positive net charge and an amphipathic alpha-helix, which can cause conformational changes in the collagen XVII homotrimer. Assessment of triple-helix folding of the mutants revealed a lower thermal stability of all non-shed variants than of wild-type collagen XVII or the shed mutants. In contrast, deletion of the putative nucleation site for triple-helix folding of collagenous transmembrane proteins did not affect folding of collagen XVII. The data indicate that the conformation of the NC16A domain and steric availability of the cleavage site influence shedding and is important for folding of collagen XVII.

Detection of ERK1/2 activities using affinity reagents.

The three major mammalian mitogen-activated protein kinase (MAPK) subfamilies include the extracellular signal-regulated kinases (ERK1/2), the c-Jun N-terminal kinases (JNKs), and the p38 kinases. These kinases are differentially activated in response to extracellular stimuli and show differential specificity toward their substrates, which is provided, in part, by the existence on the different substrates of specific kinase docking sites (reviewed in ref. ). Specific substrates of JNKs and p38s include the transcription factors c-Jun and ATF2, respectively, and GST-c-Jun and GST-ATF2 fusion proteins are widely used as suitable substrates to measure in vitro the kinase activity of these enzymes (, , , ). On the other hand, although a wide number of cytosolic, membrane-bound, and nuclear proteins are substrates of ERK1/2 (reviewed in refs. and ), measurement of ERK1/2 kinase activity in vitro is routinely performed using the nonphysiologic substrate myelin basic protein (MBP) (,). ERK1/2 and p38α bind to the protein tyrosine phosphatase PTP-SL through a kinase interaction motif (KIM) located outside of the protein tyrosine phosphatase (PTP) catalytic domain; on binding, PTP-SL is phosphorylated by these MAPKs, mainly at the Thr253 residue (,). Phosphorylation by active ERK1/2 and p38α of a GST-PTP-SL fusion protein containing the KIM and the MAPKs phosphorylation site (GST-PTP-SL 147–288) is illustrated in Fig. 1, in comparison with the phosphorylation of MBP. Open image in new window Fig. 1. Phosphorylation of GST-PTP-SL 147–288 and MBP by ERK1/2 and p38α. (A) Rat 1 cells were plated at semiconfluency and left for 6 h in the absence of fetal calf serum (FCS). Then, cells were stimulated with 10% FCS for 10 min (for ERK1/2; lanes 1 and 2) or with 0.5 M sorbitol for 20 min (for p38α, lanes 3 and 4) and lysed in lysis buffer. For each point, ERK1/2 and p38α were immunoprecipitated from cell lysates (0.75 mg of protein for ERK1/2, and 2.25 mg of protein for p38α) using specific anti-ERK1/2 or anti-p38α antibodies and protein A-Sepharose and subjected to immune complex in vitro kinase assays in the presence of equimolar amounts of GST-PTP-SL 147–288 (2 βg; lanes 1 and 3) or MBP (0.85 μg; lanes 2 and 4). (B) COS-7 cells were transfected with pCDNA3-HA-ERK2 or pECE-HA-MAPK (HA-p38α) in six-well plates using the DEAE-dextran method (1 μg of DNA/well). Cells were activated with 50 ng/mL of EGF for 5 min (for HA-ERK2; lanes 1 and 2) or with 0.5 M sorbitol for 20 min (for HA-p38α; lanes 3 and 4) and lysed. For each point, HA-ERK2 and HA-p38α were immunoprecipitated from cell lysates (from one well from a six-well plate for HA-ERK2, and from six wells from a six-well plate for HA-p38α) with the 12CA5 anti-HA monoclonal antibodies and protein A-Sepharose and subjected to in vitro kinase assays as in (A). For ERK1/2 and HA-ERK2, 2 μCi of [γ-32P]ATP/sample were used. For p38α and HA-p38α, 6 μCi of [γ-32P]ATP/sample were used. Note that autophosphorylation of HA-p38α is observed in these assays (asterisk with an arrow in [B], lane 4), which could mask the phosphorylation of GST-PTP-SL 147–288. No autophosphorylation of HA-ERK2 is observed. Autoradiographs of representative experiments are shown. Since the in vitro specific activity of ERK1/2 is much higher than that of p38α, different amounts of cell lysates and [γ-32P]ATP (see above), and different exposure times of the autoradiographs, were used for the assays with these two MAPKs. The exposure times were as follows: For (A), lanes 1 and 2 = 30 min; lanes 3 and 4=6 h. For (B), lanes 1 and 2 = 6 h; lanes 3 and 4 = 16 h. The relative expression of endogenous ERK1/2 and p38α in Rat 1 cells, and of recombinant HA-ERK2 and HA-p38α in COS-7 cells, was similar (not shown). All samples were analyzed by 15% SDS-PAGE under reducing conditions.

Identification of CD226 ligand on colo205 cell surface.

To confirm the existence of CD226 ligand and its distribution, which is a novel molecule cloned in 1996. The mRNA was extracted from TPA activated Jurkat cells and used as a template for reverse-transcription. After PCR amplification, the fragment including CD226 extracellular region and the splice donor sequence "ACTTACCTGT" was obtained and cloned into fusion expression vector pIG. The recombinant vector pCD226/Ig was transfected in COS-7 cells by DEAE-Dextran method, the secreting fusion protein was identified by Sandwich ELISA, and was purified by anti-CD226 affinity chromatography. This fusion protein was used as a probe in the investigation of CD226 ligand by immunohistochemistry. Existence of CD226 ligand was further identified by adhesion experiment. Expression of a secreting fusion protein was identified by sandwich ELISA,indicating that both CD226 extracellular domain and IgGFc domain could be recognized respectively by anti-CD226 and anti-hIgFc mAb. About 130g CD226/Ig fusion protein could be obtained from 100mL COS-7 culture supernatants by anti-CD226 affinity chromatography purification. SDS-PAGE showed that this fusion protein has a molecular mass of 83ku. It was confirmed by immunohistochemistry that CD226 ligand expressed on the Colo205 cells, but not on Jurkat cell, U937 cell and mixed lymphocyte culture cells. In adhesive assay, resting Jurkat cells did not have significant adhesion to Colo205 cells. In contrast, activated Jurkat cells could bind to colon carcinoma Colo205 cells and this adhesive reaction could be blocked by CD226/Ig fusion protein or anti-CD226 mAb. Immunochemical experiment showed that Colo205 cells could be specifically stained by CD226/Ig, indicating that CD226 ligand exists on the surface of Colo205 cells. Existence of CD226 ligand on the surface of Colo205 cells was identified by immunohistochemistry and adhesion blocking experiment. In addition, the secreting CD226/Ig fusion protein prepared in this study will be a potential tool for further investigation of CD226 ligand.