Cellular Ligands Research Articles

NKp44L expression on CD4+ T cells is associated with impaired immunological recovery in HIV-infected patients under highly active antiretroviral therapy

HIV-infected immunological nonresponders (InRs) patients fail to show satisfactory CD4+ T-cell recovery despite virologically effective HAART. We propose that NKp44L, the cellular ligand of an activating natural killer (NK) receptor, expressed only on uninfected bystander CD4+ T cells from HIV-1 infected patients, could play a major role in this phenomenon by sensitizing these cells to NK killing. Phenotype and multifunctional status of CD4+ T cells, especially the subsets expressing and not expressing NKp44L, were characterized for HIV-infected patients receiving HAART for at least 2 years, during which their viral load remained less than 40 copies/ml; 53 were InRs (CD4 cell count always <350 cells/µl), and 82 immunological responders (CD4 cell count always ≥350 cells/µl). Flow cytometry determined NKp44L expression in association with specific markers of proliferation, maturation, activation, homeostasis, and intracellular cytokine production. Degranulation of NKp44+ determined the functional capacity of NK cells. InRs exhibited high levels of NKp44L+CD4+ T cells. Compared with NKp44L negative cells, the frequency of naive CD45RA+CCR7+ T cells expressing NKp44L fell (P < 0.001) and their proliferative capacity grew. Moreover, apoptosis and a unique ability to produce multiple cytokines (IL-2, IFN-γ, and TNF-α) without or after phytohemagglutinin or anti-CD3/CD28 stimulation distinguished NKp44L+ T cells. InR status is associated to a significant expansion of highly differentiated, multifunctional and apoptotic CD4+ T cells expressing NKp44L. This could explain a rapid CD4+ T-cell turnover in InR preventing immune recovery. These data suggest a new target for developing therapeutic strategies to prevent NKp44L expression and then stimulating immune recovery in InRs.

Human Cytomegalovirus (HCMV) Glycoprotein gB Promotes Virus Entry In Trans Acting as the Viral Fusion Protein Rather than as a Receptor-Binding Protein

ABSTRACTHuman cytomegalovirus (HCMV) glycoproteins gB and gH/gL are both necessary and sufficient for cell-cell fusion. However, it is not clear what roles these glycoproteins play in virus entry, whether acting directly in membrane fusion or in binding receptors. With other herpesviruses, it appears that gB is the fusion protein and is triggered by gH/gL, which, in some cases, binds receptors. However, for HCMV, there is published evidence that gB binds cellular ligands necessary to promote virus entry into or signaling of cells. Most mechanistic information on herpesvirus fusion proteins involves cell-cell fusion assays, which do not allow a determination of whether gB or gH/gL in the virion envelope must be oriented toward cellular membranes that contain receptors. Here, we showed that HCMV virions lacking gB were unable to enter normal cells but entered cells that expressed gB. Analyses of gB mutants lacking the cytoplasmic domain or with substitutions in putative “fusion loops” provided evidence that gB fusion activity was required for this “entry in trans.” In gB-mediated entry in trans, gB is oriented toward the virion envelope that apparently lacks receptors, arguing against an essential role for gB in binding receptors or signaling molecules. In contrast, particles lacking gH/gL did not enter cells expressing gH/gL, apparently because gH/gL must be oriented toward cellular membranes (which have receptors). Coupled with our previous interference studies, in which gH/gL expressed in cells blocked HCMV entry, our findings here support the hypothesis that HCMV gH/gL binds cellular receptors before triggering gB, which acts as the fusion protein.

Abstract 1838: nc886, a putative tumor suppressive non-coding RNA in gastric cancer.

Abstract nc886, a novel type of non-coding RNA, was originally recognized as a microRNA precursor (pre-miRNA) or a vault RNA (vtRNA), but it has been shown to be barely processed into mature microRNAs and be mostly dissociated from the vault complex. So far, nc886’s best studied role is as a cellular RNA ligand and regulator of PKR (Protein Kinase RNA-activated). When nc886 is suppressed, PKR is activated and this activation leads to apoptosis through the canonical PKR-eIF2α cell death pathway. Given that the expression of nc886 was found to be suppressed in a variety of cancer cells, nc886/PKR's such role is thought to be critical in eliminating pre-cancerous cells during certain stages of tumorigenesis. Thus, nc886 plays a sentinel role against tumorigenesis by controlling PKR activity in some cells. We have found that nc886 is decreased also in gastric cancer. Our measurement of nc886 in 88 specimens from gastric cancer patients indicates that nc886 tends to be decreased in tumors relative to the adjacent normal tissues. Also, DNA methylation of the nc886 locus exhibits an anti-correlation to nc886 level, and therefore epigenetic silencing is likely to be the mechanism for the decrease of nc886 in gastric cancer. Moreover, hypermethylation of the nc886 promoter region is associated with poor survival of gastric cancer patients. All our data indicates a putative tumor suppressive role for nc886 and the possibility of a clinical application for nc886 as a prognostic marker for gastric cancer. Citation Format: Seon-Young Kim, Jong Lyul Park, Yong Sung Kim, Kwang Soo Lee, Sung Ho Jeon, Yong Sun Lee. nc886, a putative tumor suppressive non-coding RNA in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1838. doi:10.1158/1538-7445.AM2013-1838

Systems level immune response analysis and personalized medicine

The immune system is an anatomically structured, orchestrated interaction of different cell types that communicate via a large number of receptors recognizing both soluble and cellular ligands. Recent technological advances now allow large-scale measurements for better appreciation of this complexity. Despite these advances, only a few immunological parameters are routinely measured in clinical practice. The authors believe that these measurements are insufficient to describe the immune function of individual patients and thus cannot be used to evaluate immune-mediated diseases or response to therapy. Our current knowledge of immunology comes largely from work in murine model systems where the immune system has been characterized in great detail. This impressive volume of knowledge has proven to be difficult to translate into novel therapies in humans; one reason for this is the lack of large-scale immune monitoring allowing for systems-wide analysis of the human immune system. The authors propose a systems approach to immunology, where the focus is moved from analysis of individual cell types towards more integrated studies of the entire immune system. Exercising ‘systems immunology’ in preclinical research, during drug development and in patients undergoing therapies affecting the immune system, will enable us to improve clinical results through personalized medicine and help to define clinically relevant patterns of immune reactivity.

Complex protein nanopatterns over large areas via colloidal lithography

The patterning of biomolecules at the nanoscale provides a powerful method to investigate cellular adhesion processes. A novel method for patterning is presented that is based on colloidal monolayer templating combined with multiple and angled deposition steps. Patterns of gold and SiO2 layers are used to generate complex protein nanopatterns over large areas. Simple circular patches or more complex ring structures are produced in addition to hierarchical patterns of smaller patches. The gold regions are modified through alkanethiol chemistry, which enables the preparation of extracellular matrix proteins (vitronectin) or cellular ligands (the extracellular domain of E-cadherin) in the nanopatterns, whereas the selective poly(l-lysine)–poly(ethylene glycol) functionalization of the SiO2 matrix renders it protein repellent. Cell studies, as a proof of principle, demonstrate the potential for using sets of systematically varied samples with simpler or more complex patterns for studies of cellular adhesive behavior and reveal that the local distribution of proteins within a simple patch critically influences cell adhesion.

TRNA Binding, Structure, and Localization of the Human Interferon-Induced Protein IFIT5

Interferon-induced proteins, including the largely uncharacterized interferon-induced tetratricopeptide repeat (IFIT) protein family, provide defenses against pathogens. Differing from expectations for tetratricopeptide repeat (TPR) proteins and from human IFIT1, IFIT2, and IFIT3, we show that human IFIT5 recognizes cellular RNA instead of protein partners. In vivo and in vitro, IFIT5 bound to endogenous 5'-phosphate-capped RNAs, including transfer RNAs. The crystal structure of IFIT5 revealed a convoluted intramolecular packing of eight TPRs as a fold that we name the TPR eddy. Additional, non-TPR structural elements contribute to an RNA binding cleft. Instead of general cytoplasmic distribution, IFIT5 concentrated in actin-rich protrusions from the apical cell surface colocalized with the RNA-binding retinoic acid-inducible gene-I (RIG-I). These findings establish compartmentalized cellular RNA binding activity as a mechanism for IFIT5 function and reveal the TPR eddy as a scaffold for RNA recognition.

Expression de CD158K/KIR3DL2 par les lymphocytes tumoraux chez les patients atteints de lymphome anaplasique cutané à grandes cellules CD30+

Combating diseases such as cancer and virus infection is one of the major goals of the next millennium. Remarkably, our immune system has developed a unique cell type called Natural Killer (NK) cells, able to kill cancer and virus-infected cells. The nature of the lysis/killer receptors expressed on NK cells, mediating this killing, and their ligands, is only little understood. Four lysis receptors for human NK cells were recently identified, including the NKp30, NKp44, NKp46 and CD16, all of which are capable of mediating direct killing of virus-infected and tumor cells. Among these, the NKp46 receptor is considered to be the major lysis receptor for NK cells. The viral hemagglutinin protein was recently identified as the ligand for the NKp46 receptor. However, the cellular (non-viral) ligands recognized by all of the lysis receptors (NKp30, NKp44, NKp46 and CD16) are currently unknown.

Post-transcriptional regulation of CD83 expression by AUF1 proteins

Mature dendritic cells (DC), activated lymphocytes, mononuclear cells and neutrophils express CD83, a surface protein apparently necessary for effective DC-mediated activation of naïve T-cells and T-helper cells, thymic T-cell maturation and the regulation of B-cell activation and homeostasis. Although a defined ligand of CD83 remains elusive, the multiple cellular subsets expressing CD83, as well as its numerous potential implications in immunological processes suggest that CD83 plays an important regulatory role in the mammalian immune system. Lately, nucleocytoplasmic translocation of CD83 mRNA was shown to be mediated by direct interaction between the shuttle protein HuR and a novel post-transcriptional regulatory element (PRE) located in the CD83 transcript’s coding region. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export through the CRM1 protein translocation pathway. More recently, the cellular phosphoprotein and HuR ligand ANP32B (APRIL) was demonstrated to be directly involved in this intracellular transport process by linking the CD83 mRNA:HuR ribonucleoprotein (RNP) complex with the CRM1 export receptor. Casein kinase II regulates this process by phosphorylating ANP32B. Here, we identify another RNA binding protein, AUF1 (hnRNP D) that directly interacts with CD83 PRE. Unlike HuR:PRE binding, this interaction has no impact on intracellular trafficking of CD83 mRNA-containing complexes; but it does regulate translation of CD83 mRNA. Thus, our data shed more light on the complex process of post-transcriptional regulation of CD83 expression. Interfering with this process may provide a novel strategy for inhibiting CD83, and thereby cellular immune activation.

The Stalk Domain and the Glycosylation Status of the Activating Natural Killer Cell Receptor NKp30 Are Important for Ligand Binding

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.

Protein Tyrosine Phosphatase α Phosphotyrosyl-789 Binds BCAR3 To Position Cas for Activation at Integrin-Mediated Focal Adhesions

Integrin-mediated focal adhesions connect the extracellular matrix and cytoskeleton to regulate cell responses, such as migration. Protein tyrosine phosphatase α (PTPα) regulates integrin signaling, focal adhesion formation, and migration, but its roles in these events are incompletely understood. The integrin-proximal action of PTPα activates Src family kinases, and subsequent phosphorylation of PTPα at Tyr789 acts in an unknown manner to promote migration. PTPα-null cells were used in reconstitution assays to distinguish PTPα-Tyr789-dependent signaling events. This showed that PTPα-Tyr789 regulates the localization of PTPα and the scaffolding protein Cas to adhesion sites where Cas interacts with and is phosphorylated by Src to initiate Cas signaling. Linking these events, we identify BCAR3 as a molecular connector of PTPα and Cas, with phospho-Tyr789 PTPα serving as the first defined cellular ligand for the BCAR3 SH2 domain that recruits BCAR3-Cas to adhesions. Our findings reveal a novel role of PTPα in integrin-induced adhesion assembly that enables Src-mediated activation of the pivotal function of Cas in migration.

Tyrosinase-mediated surface grafting of cell adhesion peptide onto micro-fibrous polyurethane for improved endothelialization

The Arg-Gly-Asp (RGD) sequence is one of the most effective cellular adhesion ligands and has been used to improve cellular activity of biomaterials for tissue regenerative medicine. The aim of this study was to develop new facile immobilization methods of RGD on electrospun polyurethane (PU) meshes, using tyrosinase (Ty), to promote attachment and proliferation of human umbilical vein endothelial cells (HUVECs). The RGD peptides were readily immobilized on PU substrates, for 1 h, using 0.4 kU/mL of Ty. Water contact angles and X-ray photoelectron spectroscopy (XPS) were used in order to verify that the RGD peptide was effectively conjugated onto the PU (0.120 nmol/mg-mesh); surface concentration was shown to be proportional to the peptide feed concentration. Enhanced HUVEC attachment onto the polymer substrate was observed after 1 day, and the immobilized RGD was clearly demonstrated to promote the proliferation and spreading of cells on the surface, indicating that the method developed for the conjugation of the peptides on the PU was efficient. These results suggest that this enzyme-triggered immobilization method for the cellular adhesion ligand, RGD, onto the PU mashes may be an efficient tool for improving the biological activity of substrates, especially for vascular tissue engineering.

Enterohaemorrhagic Escherichia Coli Exploits a Tryptophan Switch to Hijack Host F-Actin Assembly

Intrinsically disordered protein (IDP)-mediated interactions are often characterized by low affinity but high specificity. These traits are essential in signaling and regulation that require reversibility. Enterohaemorrhagic Escherichia coli (EHEC) exploit this situation by commandeering host cytoskeletal signaling to stimulate actin assembly beneath bound bacteria, generating "pedestals" that promote intestinal colonization. EHEC translocates two proteins, EspF(U) and Tir, which form a complex with the host protein IRTKS. The interaction of this complex with N-WASP triggers localized actin polymerization. We show that EspF(U) is an IDP that contains a transiently α-helical N-terminus and dynamic C-terminus. Our structure shows that single EspF(U) repeat forms a high-affinity trimolecular complex with N-WASP and IRTKS. We demonstrate that bacterial and cellular ligands interact with IRTKS SH3 in a similar fashion, but the bacterial protein has evolved to outcompete cellular targets by utilizing a tryptophan switch that offers superior binding affinity enabling EHEC-induced pedestal formation.

Progress of LRR Transmembrance Protein Function in Nervous System*

Leucine-rich repeat(LRR) is a common protein domain.LRR domain-containing proteins are present in a large number of cells and tissues in prokaryotes and eukaryotes.The diverse functions of LRR proteins due to their specific locations and the different proteins interacted with them.Many LRR proteins are expressed specially in nerve tissue,and most of the proteins overexpressed in nerve tissue belong to transmembrance protein.As cellular adhesive molecules or ligand receptor proteins,they are involved in a variety of neural physiological activities such as synapse formation,neurite growth,neurotransmitter trafficking and release.The abnormal expression of LRR proteins results in the neurological and psychiatric disorders.

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

Virus-mediated inhibition of natural cytotoxicity receptor recognition.

Natural killer (NK) cells are a part of the innate immune system that functions mainly to kill transformed and infected cells. Their activity is controlled by signals derived from a panel of activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs): NKp30, NKp44, and NKp46 (NCR1 in mice) are prominent among the activating NK cell receptors and they are, notably, the only NK-activating receptors that are able to recognize pathogen-derived ligands. In addition, the NCRs also recognize cellular ligands, the identity of which remains largely unknown. In this review, we summarize the current knowledge regarding viruses that are recognized by the NCRs, focusing on the diverse immune-evasion mechanisms employed by viruses to escape this detection. We also discuss the unique role the NCRs have in regulating NK cell activity with particular emphasis on the in vivo function of NKp46/NCR1.

PCNA: a novel cancer-associated ligand for the NK cell receptor NKp44 (162.10)

Abstract Natural Cytotoxicity Receptors (NCRs: NKp30, NKp44, and NKp46) trigger NK cell-mediated lysis of tumors and virus-infected cells. Previous studies identified BAT3, B7-H6 and Vimentin as cellular ligands for NKp30 and NKp46, respectively. However, our knowledge of NCR-mediated recognition of cancer cells remains incomplete. NKp44 is expressed exclusively in primates and its expression is induced upon activation of NK cells. We have discovered that NKp44 directly recognizes Proliferating Cell Nuclear Antigen (PCNA), which is recruited within target cells to the NK immunological synapse. Over-expression of PCNA was found to inhibit NK cell cytotoxicity in an NKp44-dependent manner. Also, the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) in NKp44 is crucial for PCNA-meditated NK cell inhibition. Since PCNA over-expression can promote cancer survival, our study reveals a novel immune evasion mechanism. Our results also further emphasize the role of nuclear proteins as ligands for Natural Cytotoxicity Receptors.

2110 DISTINGUISHING PROSTATE CANCER FROM BENIGN PROSTATIC HYPERPLASIA BY NANODEVICE DETECTION OF BIOMARKERS ASSOCIATED WITH REACTIVE STROMA

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 20122110 DISTINGUISHING PROSTATE CANCER FROM BENIGN PROSTATIC HYPERPLASIA BY NANODEVICE DETECTION OF BIOMARKERS ASSOCIATED WITH REACTIVE STROMA Elizabeth Singer, Jennifer Linehan, Ashraf Imam, David Smith, Sofia Loera, Timothy Wilson, and Steven Smith Elizabeth SingerElizabeth Singer Duarte, CA More articles by this author , Jennifer LinehanJennifer Linehan Duarte, CA More articles by this author , Ashraf ImamAshraf Imam Pasadena, CA More articles by this author , David SmithDavid Smith Duarte, CA More articles by this author , Sofia LoeraSofia Loera Duarte, CA More articles by this author , Timothy WilsonTimothy Wilson Duarte, CA More articles by this author , and Steven SmithSteven Smith Duarte, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2278AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Reactive stroma (RS) has been shown to be a predictor of biochemical free recurrence in prostate cancer (Ayala et al. 2003;9:2003), however molecular markers of the stromal response have not yet been successfully applied in prognosis. We recently demonstrated that a thioredoxin-targeted nanodevice selectively binds to RS in frozen tumor sections (Singer et al. Nanomedicine 2011;6:659). Thioredoxin was used as the targeting ligand because it is involved in reductive pathways associated with the wound response mounted by the stroma. Our objective was to compare the stromal binding of the Thioredoxin-targeted nanodevice in benign prostatic hyperplasia (BPH) versus prostate cancer (PCa) to determine whether or not they could be distinguished using a stromal marker. METHODS A fluorescein-labeled nanodevice (ND-Trx3) that displays 3 copies of the bacterial Thioredoxin (Trx) as a cellular targeting ligand was generated as described previously (Singer, Nanoletters). Robotic-Assisted radical prostatectomy tissue for frozen sections was obtained from 45 patients. Serial sections were incubated with varying concentrations of nanodevice in PBS and 1% BSA. Binding fluorescence was observed at 100x with a Zeiss Observer microscope and images of the entire tumor section were obtained by raster-tiling of individual pictures. A numerical grading system was given as the level of nanodevice fluorescence in the stroma. 1 - was a lightly visible signal, 2- was a moderately intense visible signal and 3 - was a bright and intense signal within the RS. Slides where then reviewed by a blinded pathologist. RESULTS Specimens were collected from 45 patients with Gleason sums ranging 6-to-10. Adjacent serial sections were stained with ND-Trx3, Trichome and H&E. We compared the stromal binding of the ND-Trx3 in BPH vs. PCa to determine whether or not they could be distinguished using a stromal marker .18 slides contained PCa and 17 only contained BPH. There is a strong correlation p=0.0182 between the intensity of nanodevice binding to the stroma and the presence of PCa compared to BPH (Figure 1). The conditional power of the current sample size (N=33) is 91%. CONCLUSIONS Thioredoxin-linked reductive pathways associated with the wound response may provide useful biomarkers of PCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e852 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Elizabeth Singer Duarte, CA More articles by this author Jennifer Linehan Duarte, CA More articles by this author Ashraf Imam Pasadena, CA More articles by this author David Smith Duarte, CA More articles by this author Sofia Loera Duarte, CA More articles by this author Timothy Wilson Duarte, CA More articles by this author Steven Smith Duarte, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Receptor chimeras demonstrate that the C-terminal domain of the human cytomegalovirus US27 gene product is necessary and sufficient for intracellular receptor localization

BackgroundHuman cytomegalovirus (HCMV) is ubiquitous in the population but generally causes only mild or asymptomatic infection except in immune suppressed individuals. HCMV employs numerous strategies for manipulating infected cells, including mimicry of G-protein coupled receptors (GPCRs). The HCMV US27 gene product is a putative GPCR, yet no ligand or signaling has been identified for this receptor. In the present study, immunofluorescence microscopy was used to examine the cellular distribution of wild type US27, as well as US27 deletion mutants and chimeric receptors.ResultsIn transiently transfected cells, wild type US27 was found primarily in intracellular compartments, in striking contrast to the cell surface distribution seen for the human cellular chemokine receptor CXCR3. When the N-terminal extracellular domains of the two receptors were swapped, no change in protein localization was observed. However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution. A chimera that contained US27 fused to the C-terminal intracellular tail of CXCR3 exhibited surface distribution similar to that of wild-type CXCR3. When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27. In addition, a US27 mutant lacking the C-terminus (US27ΔCT) failed to accumulate inside the cell and exhibited cell surface distribution. Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments.ConclusionsThe results indicate that the C-terminal domain of the HCMV US27 protein, which contains a di-leucine endocytic sorting motif, is both necessary and sufficient for intracellular localization, which may also help explain why no cellular ligands have yet been identified for this viral receptor.

Neutrophil arrest by LFA-1 activation

Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimeric integrin consisting of αL (gene name, Itgal) and β2 (gene name, Itgb2) subunits expressed in all leukocytes. LFA-1 is essential for neutrophil recruitment to inflamed tissue. Activation of LFA-1 by chemokines allows neutrophils and other leukocytes to undergo arrest, resulting in firm adhesion on endothelia expressing intercellular adhesion molecules (ICAMs). In mice, CXCR2 is the primary chemokine receptor involved in triggering neutrophil arrest, and it does so through “inside-out” activation of LFA-1. CXCR2 signaling induces changes in LFA-1 conformation that are coupled to affinity upregulation of the ligand-binding headpiece (extended with open I domain). Unlike naïve lymphocytes, engagement of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils stimulates a slow rolling behavior that is mediated by LFA-1 in a distinct activation state (extended with closed I domain). How inside-out signaling cascades regulate the structure and function of LFA-1 is being studied using flow chambers, intravital microscopy, and flow cytometry for ligand and reporter antibody binding. Here, we review how LFA-1 activation is regulated by cellular signaling and ligand binding. Two FERM domain-containing proteins, talin-1 and Kindlin-3, are critical integrin co-activators and have distinct roles in the induction of LFA-1 conformational rearrangements. This review integrates these new results into existing models of LFA-1 activation.

Immunohistochemical Expression and Prognostic Value of CD97 and Its Ligand CD55 in Primary Gallbladder Carcinoma

Background. CD97 as a member of the EGF-TM7 family with adhesive properties plays an important role in tumor aggressiveness by binding its cellular ligand CD55, which is a complement regulatory protein expressed by cells to protect them from bystander complement attack. Previous studies have shown that CD97 and CD55 both play important roles in tumor dedifferentiation, migration, invasiveness, and metastasis. The aim of this study was to investigate CD97 and CD55 expression in primary gallbladder carcinoma (GBC) and their prognostic significance. Methods. Immunohistochemistry was used to investigate the expression of CD97 and CD55 proteins in 138 patients with GBC. Results. CD97 and CD55 were absent or only weakly expressed in the normal epithelium of the gallbladder but in 69.6% (96/138) and 65.2% (90/138) of GBC, respectively, remarkably at the invasive front of the tumors. In addition, CD97 and CD55 expressions were both significantly associated with high histologic grade (both P = 0.009), advanced pathologic T stage (P = 0.01 and 0.009, resp.) and clinical stage (both P = 0.009), and positive venous/lymphatic invasion (both P = 0.009). Multivariate analyses showed that CD97 (hazard ratio, 3.236; P = 0.02) and CD55 (hazard ratio, 3.209; P = 0.02) expressions and clinical stage (hazard ratio, 3.918; P = 0.01) were independent risk factor for overall survival. Conclusion. Our results provide convincing evidence for the first time that the expressions of CD97 and CD55 are both upregulated in human GBC. The expression levels of CD97 and CD55 in GBC were associated with the severity of the tumor. Furthermore, CD97 and CD55 expressions were independent poor prognostic factors for overall survival in patients with GBC.