Surface Of HEK293 Cells Research Articles

Native adiponectin in serum binds to mammalian cells expressing T-cadherin, but not AdipoRs or calreticulin.

Adiponectin is an adipocyte-derived atypically abundant circulating factor that protects various organs and tissues through its receptors, AdipoRs, calreticulin, and T-cadherin. To identify the major binding partner of circulating native adiponectin, we expressed these receptors on the surface of HEK293 cells. Adiponectin, either that in mouse or human serum, purified from serum, or produced by mammalian cells, bound to cells expressing T-cadherin, but not to those expressing AdipoR1 or calreticulin. The stable introduction of T-cadherin and AdipoR1 into CHO cells resulted in the cell surface localization of these receptors. Native adiponectin in serum bound to cells expressing T-cadherin, not to those expressing AdipoR1. The knockdown of T-cadherin, but not AdipoRs resulted in the significant attenuation of native adiponectin binding to C2C12 myotubes. Therefore, native adiponectin binding depended on the amount of T-cadherin expressed in HEK293 cells, CHO cells, and C2C12 myotubes. Collectively, our mammalian cell-based studies suggest that T-cadherin is the major binding partner of native adiponectin in serum.

Direct stimulation of cellular immune response via TLR2 signaling triggered by contact with hybrid glyco-biointerfaces composed of chitohexaose and cellohexaose.

Recognition of carbohydrates by cell receptors activates various cell signaling processes. In this report, hybrid self-assembled monolayers (SAMs) of chitohexaose (βGlcNAc6) and cellohexaose (βGlc6) are prepared to induce a highly sensitive, glyco-density-dependent inflammatory response by HEK293 cells expressing toll-like receptor 2 (TLR2), which recognizes oligo-βGlcNAc moieties. Thiosemicarbazide-labeled βGlcNAc6 and βGlc6, as a bio-signal inducer and a spacer molecule, respectively, were immobilized on a gold substrate via spontaneous chemisorption to control the βGlcNAc6 density on the SAM surface. The βGlcNAc6 density ranged from 0 to 0.65 chains nm-2. The inflammatory response of HEK293 cells varied as a function of the surface glyco-density, and the hybrid SAM with 0.146 chains nm-2 of βGlcNAc6 induced a stronger response than pure βGlcNAc6-SAM with 0.654 chains nm-2. Thus, direct activation of TLR2-mediated cellular stimulation triggered by as-designed glyco-biointerfaces was possible through a mutual interaction between the fittingly βGlcNAc6 moieties assembled on hybrid glyco-SAMs and TLR2 on the surface of HEK293 cells.

Heterodimerization of Organic Anion Transporting Polypeptide 1B3 can affect function and surface expression

OATP1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes, along with other transporters, including OATP1B1, OCT1 and NTCP. It has a broad spectrum of substrates including endogenous compounds and numerous drugs. Thus, modulation of OATP1B3 function and expression has the potential to affect drug efficacy and disposition. Previous studies in our lab demonstrated that OATP1B3 can form homo‐ and heterodimers, both in cell models and in frozen human liver tissue. However, functional consequences of these interactions have not been investigated so far. In this study, we used transient transfection of HEK293 cells with FLAG‐tagged OATP1B3 in combination with a His‐tagged non‐functional OATP1B3 mutant, or with His‐tagged OATP1B1 or NTCP, surface biotinylation, and cell‐based uptake assays to characterize the functional consequences of the dimerization. Co‐transfection of mutant and wild‐type OATP1B3 resulted in a similar uptake of both estrone‐3‐sulfate and estradiol‐17β‐glucuronide after normalization for surface expression as co‐transfection of empty vector with wild‐type OATP1B3, suggesting that the homodimer of OATP1B3 works as two independent functional subunits. Uptake of the OATP1B3‐specific substrate CCK8 decreased significantly when OATP1B3 was co‐expressed with NTCP, while co‐expression with OATP1B1 had no effect. In contrast, uptake of low concentration estrone‐3‐sulfate by OATP1B1 and taurocholic acid by NTCP was not affected by co‐expression with OATP1B3. Expression of OATP1B3 on the surface of HEK293 cells was reduced when OATP1B3 was co‐expressed with NTCP suggesting that the functional decrease might be a result of disturbed protein production and/or translocation to the plasma membrane. These results indicate that co‐expression of NTCP with OATP1B3 can affect OATP1B3‐mediate drug transport and as a consequence can have an effect on drug therapy.Support or Funding InformationNIH grants GM103549 and GM077336

Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein’s scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

Transduction of human dendritic cells with adenovirus encoding anti-PD-1 reduces PD-1 expression on co-cultured T cells

A potent tumor-specific T cell response is an important part of antitumor immunity. Thus, enhancing T cell responses against tumor cells is a major focus in cancer immunotherapy. Dendritic cells (DC) play a critical role in the induction of T cell responses not only against pathogens but also against tumor cells. Studies have shown that DC-based vaccines are capable of presenting antigens via MHC Class I and MHC Class II molecules resulting in tumor antigen-specific T cell activation in vitro and in vivo. However, T cell responses against tumor antigens can be negatively regulated. For example, PD-1, which is up-regulated in activated T cells, can bind to PD-L1 or PD-L2 expressed in tumor cells as part of the immune suppressive tumor environment and thus inhibiting T cell activation. We wished to determine whether addition of anti-PD-1 to DC vaccines would result in enhanced tumor antigen-specific T cell responses. DCs transduced with recombinant adenovirus (AdV) encoding anti-PD1 (Ad5.hPD1Ab) secreted anti-PD1 in the supernatants which was able to bind to PD-1 expressed on the surface of HEK-293 cells via stable transfection. We examined the expression of surface markers associated with DC function and maturation 48 hours after transduction via flow cytometry. Our results show that Ad5.hPD1Ab transduced DCs had similar expression levels of antigen presentation molecules MHC Class I and II, costimulatory, and maturation related molecules CD40, CD80, CD83 and CD86 compared to DCs that were transduced with adenovirus encoding tumor antigens for hepatocellular carcinoma and melanoma (AdVhAFP and AdVTMM2, respectively). Furthermore, surface expression of inhibitory molecules PD-L1 and PD-L2 were also comparable among the three groups. Cytokine analysis show that 24-48 hours after transduction, Ad5.hPD1Ab transduced DCs secrete more IL-7, IL-15 and IP-10 than AdVhAFP and AdVTMM2 transduced DCs. Importantly, autologous T cells co-cultured with Ad5.hPD1Ab transduced DC results in reduced expression of not only surface PD-1 but also surface CTLA-4 inhibitory molecules in both CD4+ and CD8+ T cells.

Heteromerization of GPR55 and cannabinoid CB2 receptors modulates signalling.

Heteromerization of GPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer-selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55. The direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 cells was assessed by co-immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross-talk on signalling was investigated at downstream levels by label-free real-time methods (Epic dynamic mass redistribution and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays. GPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors. Heteromers, unique signalling units, form in HEK293 cells expressing GPR55 and CB2 receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor.

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.

Specific immunoadsorption of pathogenic autoantibodies in pemphigus requires the entire ectodomains of desmogleins

Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are life-threatening autoimmune blistering skin diseases. They are characterized by circulating autoantibodies which bind to the ectodomains of desmoglein (Dsg) 1 and Dsg3. These antibodies induce acantholysis in skin and mucous membranes. In severe cases of pemphigus, immunoadsorption is applied to remove total IgG from patient plasma using protein A or other ligands. To develop a specific adsorber for anti-Dsg antibodies, epitope mapping studies of Dsg1 and Dsg3 ectodomains were conducted. Dsg variants were expressed on the surface of HEK-293 cells and analysed for reactivity with pemphigus and control sera by indirect immunofluorescence technique. For Dsg1, a construct consisting of domain 1 directly fused to domain 5, seemed to be suitable for specific immunoadsorption of anti-Dsg1 antibodies. The recognized epitopes were mainly conformation-dependent. However, adsorption of pemphigus foliaceus IgG using this protein coupled to a Sepharose matrix did not completely remove pathogenicity from the sera, as proven by a keratinocyte dissociation assay. In contrast, full-length Dsg1 and Dsg3 ectodomains were able to specifically adsorb anti-Dsg antibodies and to efficiently eliminate pathogenicity. Therefore, the complete and correctly folded ectodomains of both desmogleins are required for therapeutic immunoadsorption.

Mammalian Cell Display and Somatic Hypermutation In Vitro for Human Antibody Discovery

Human therapeutic antibody discovery has utilized a variety of systems, from in vivo immunization of human immunoglobulin-expressing mice, to in vitro display of antibody libraries. Of the in vitro antibody display technologies, mammalian cell display provides a number of advantages with the ability to co-select immunoglobulin molecules for high expression level in mammalian cells, native folding, and biophysical properties appropriate for drug development. Mammalian cell display has been achieved using either transient or stable expression systems, using a number of alternate transmembrane domains to present antibody on the cell surface. The unique capability of mammalian cells to present IgG in its fully post-translationally modified format also allows selection of antibodies for functional properties. One limitation of mammalian cell based systems, however, has been the smaller library size that can be presented compared to phage display approaches. Until recently, this has necessitated the use of libraries biased toward a particular antigen, such as libraries derived from immunized donors, to achieve success. An alternative approach has now been developed which recapitulates key aspects of the in vivo immune system through reproducing somatic hypermutation (SHM) in vitro. Libraries representing a naïve human B lymphocyte antibody repertoire are created by PCR amplification of the rearranged (D)J segments of heavy and light chain variable regions from human donors and incorporating the resulting sequence diversity into panels of human germline VH and VL genes. The resulting antibodies are presented as full length IgG on the surface of HEK293 cells. After isolation of antibodies binding to individual target antigens, subsequent affinity maturation using in vitro SHM is induced by expression of activation-induced cytidine deaminase (AID). Selection of antibodies from naïve fully human libraries using mammalian cell display coupled with in vitro SHM is an efficient methodology for the generation of high affinity human antibodies with excellent properties for drug development.

Mechanisms of homologous and heterologous phosphorylation of FFA receptor 4 (GPR120): GRK6 and PKC mediate phosphorylation of Thr347, Ser350, and Ser357 in the C-terminal tail

Free fatty acid receptor 4 (FFA4), previously known as GPR120, is a G protein-coupled receptor that promotes numerous anti-inflammatory and antidiabetic effects upon its agonism by long chained unsaturated fatty acids. We have previously demonstrated that agonism of FFA4 with docosahexaenoic acid (DHA) and alpha-linoleic acid (ALA) facilitates rapid and transient phosphorylation of FFA4 expressed ectopically on the surface of HEK293 cells. However, the precise mechanisms that promote FFA4 phosphorylation remain elusive. In the current study, we examined the mechanisms behind both heterologous and homologous phosphorylation of FFA4 and set out to identify the foci of FFA4 phosphorylation. Our results demonstrate that basal and heterologous phosphorylation of FFA4 are mediated by protein kinase C (PKC), while G protein-coupled receptor kinase 6 (GRK6) plays the predominant role in DHA-mediated phosphorylation of FFA4. Furthermore, we identify Thr347, Ser350, and Ser357 in the C-terminal tail as major sites of FFA4 phosphorylation. Concurrent mutation of these three sites leads to a FFA4 receptor that seemingly affects Gαq/11 signaling in a positive manner as demonstrated by heightened intracellular Ca2+ responses following agonism with DHA. Importantly, this phosphodefective FFA4 mutant lacked the ability to promote β-arrestin-2 recruitment to the cell membrane. Since many of the functionally beneficial physiological effects of FFA4 are noted to be β-arrestin mediated, these findings could provide insight into the structural requirements for FFA4 function.

Abstract 2138: Targeting cancer-associated fibroblasts using antibodies recognizing fibroblast activation protein-alpha.

Abstract An expanding body of work suggests that the tumor stroma plays an integral role in the initiation and development of epithelial cancers. Within the stroma, cancer-associated fibroblasts (CAFs) are involved in several functions that form integral aspects of tumor progression, including immune suppression, metabolism, and invasion and metastasis. Despite the demonstrated importance of CAFs within the tumor microenvironment, targeting CAFs in a therapeutic context remains a challenge. The work presented here details the isolation and characterization of antibody fragments targeting multiple epitopes of fibroblast activation protein-alpha (FAP), a membrane-bound protease selectively overexpressed on CAFs in numerous epithelial cancers. The combination of a minimalist antibody fragment library and yeast surface display allowed for the isolation of more than 50 unique antibodies recognizing the human or murine forms of FAP, with close to 30 exhibiting cross-reactivity. To narrow the pool of cross-reactive antibodies to a biologically relevant subset, the fragments were converted to an antibody-like format that could be secreted from yeast. Without purification, the soluble proteins were first used to investigate the abilities of individual fragments to recognize FAP transiently expressed on the surface of HEK293 cells. Proteins for which binding to FAP could be confirmed were then used in combination with antibody fragments displayed on the surface of yeast in order to determine which sets of fragments bind distinct epitopes of FAP. Flow cytometry-based assays revealed that the isolated fragments recognize two or more distinct epitopes of the antigen. A handful of fragments is currently being subjected to affinity maturation and conversion to IgGs, and work to further investigate the in vitro and in vivo characteristics of the proteins is ongoing. These proteins may allow for the ablation of cancer-associated fibroblasts or interference with the proteolytic activity of FAP in the tumor microenvironment, providing biological insights into the roles of CAFs and therapeutic approaches to cancer treatment. Furthermore, the general antibody isolation and characterization approaches used in this work are accessible to many single-investigator laboratories and should be applicable to the targeting of numerous cancer antigens. Citation Format: James A. Van Deventer, Karl D. Wittrup. Targeting cancer-associated fibroblasts using antibodies recognizing fibroblast activation protein-alpha. [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 2138. doi:10.1158/1538-7445.AM2013-2138

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.

Selective Covalent Labeling of Tag-Fused GPCR Proteins on Live Cell Surface with a Synthetic Probe for Their Functional Analysis

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.

Molecular characterization of a human cation‐Cl− cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport

Cation-Cl- cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl(-)-coupled Na+ and/or K+ movement at the cell surface. The CCCs are also included in a larger family termed amino acid-polyamine-organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA-amino acid transport at the surface of HEK-293 cells. We have also found that the influx of PAs in CCC9a-expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na+, K+, or Cl-. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation.

Tissue Factor Pathway Inhibitor Anticoagulant and Anti-PAR Signaling Activity Requires GPI-Anchor Attachment to the Cell Surface in Chinese Hamster Ovary (CHO) Cells.

Introduction: TFPI is an endothelial associated protein that prevents intravascular thrombosis. It produces active site directed inhibition of the TF-fVIIa catalytic complex. TF-fVIIa activates blood coagulation via cleavage of fIX and fX. TF-fVIIa also initiates inflammation via activation of protease activated receptors (PARs). TFPI is attached to the endothelial surface through a GPI-anchor that localizes it to lipid raft/caveolae microdomains. We examined how GPI-anchor mediated localization of TFPI to these microdomains alters its ability to inhibit TF-fVIIa mediated coagulation and signaling events in vitro and in vivo.Methods: CHO cells were transfected with TF to create CHO-TF cells. CHO-TF cells were transfected with chimeric forms of TFPI containing either a GPI-anchor or a transmembrane (TM) attachment sequence at the C-terminus to create CHO-TF/TFPI-GPI and CHO-TF/TFPI-TM cells. Clones expressing equal amounts of TFPI-GPI and TFPI-TM were selected using flow cytometry. TF-fVIIa activity on the surface of these cells was compared to that of CHO-TF cells in three assays. TF-fVIIa coagulation and PAR signaling activity were examined in vitro using fXa activation assays and ERK1/2 phosphorylation assays, respectively. A TF-mediated tumor metastasis assay was used to assess TF activity in vivo.Results: GPI-anchored TFPI displays significantly more anti-TF-fVIIa activity than TM-anchored TFPI in the in vitro assays. In the anticoagulant activity assays, when compared to CHO-TF cells, the amount of fXa generated on the cell surface for 30 min was reduced by 64% in the CHO-TF/TFPI-GPI cells but only 8% in the CHO-TF/TFPI-TM cells (CHO-TF cells generated fXa at 6.3 pmol/min; CHO-TF/TFPI-GPI at 2.3 pmol/min; CHO-TF/TFPI-TM at 5.8 pmol/min). The signaling assays were performed using flow cytometry. When compared to CHO-TF cells, ERK1/2 phosphorylation was reduced 73% in CHO-TF/TFPI-GPI cells while no effect was observed in CHO-TF/TFPI-TM cells (MFI for CHO-TF cells at 3.17±2.04; CHO-TF/TFPI-GPI at 0.86±1.9; CHO-TF/TFPI-TM at 3.5±0.95). To assess TFPI inhibitory activity in vivo, cell lines were injected into the tail vein of SCID mice and the lungs examined for tumors at 10 days. TF-fVIIa activity is required for tumor formation in this assay. Consistent with the in vitro results, the CHO-TF/TFPI-GPI cells had greatly decreased tumor burden while CHO-TF/TFPI-TM had much less effect. Interestingly, transfection of CHO-TF cells with wild type TFPI that is secreted by CHO cells had no effect on tumor burden indicating that cell surface association of TFPI is required for anti-TF-fVIIa activity in this assay. These CHO cell results contrast with our previously published data where we found no difference in the anticoagulant activity of TFPI-GPI and TFPI-TM on the surface of HEK293 cells (Thromb. Hemost. 2003, 89:65). Since CHO cells express abundant caveolin-1 while HEK293 cells express very little, we considered that caveolin-1 may be responsible for the differential activity observed. In support of this hypothesis we found that TFPI and caveolin-1 co-immunoprecipitate from human placental lysates.Conclusions: A CHO cell model was used to examine the inhibition of TF-fVIIa by TFPI-GPI or TFPI-TM. GPI-anchor attachment of TFPI to the cell surface appears to be critical for its anti-TF activity. Localization to caveolae microdomains and/or a direct interaction with a protein complex containing caveolin-1 may be required for full TFPI activity in vivo.

ANP-NPRA Signaling Modulates Immunity By Altering TLR-2 Expression In Human Dendritic Cells (DCs)

RATIONALE: The interaction between atrial natriuretic peptide (ANP) and its receptor (atrial natriuretic peptide receptor (NPRA) in DCs was reported to modulate immunity, but the mechanism remains poorly understood. Since toll-like receptor-2 (TLR-2) plays an important role in shaping innate immune response, the role of TLR-2 in ANP signaling was assessed in DCs. METHODS: The presence of TLRs and the signal protein, MyD88, was detected in pANP transfected DC lysates by immunoblotting, and secreted cytokines were assayed using the Bio-Plex array system. Cytokines were also measured in pANP-transfected DCs where TLR-2 expression was blocked by siTLR-2. TLR-2 interaction with NPRA was examined in TLR-2-transfected HEK 293 cells and NPRA and TLR-2 colocalization and protein-protein interaction was investigated by using immunohistochemistry and immunoprecipitation, respectively. RESULTS: ANP-NPRA signaling upregulates TLR-2 expression and downregulates MyD88 in DCs compared to controls. Treatment of pANP-transfected DCs with NPRA inhibitor isatin leads to an upregulation of IL-10. Also, pANP-transfected DCs treated with siTLR-2 show upregulation of IL-10, suggesting that TLR-2 is involved with NPRA signaling. Colocalization studies indicate that NPRA and TLR-2 are expressed together on the surface of HEK293 cells. CONCLUSIONS: These results indicate that ANP regulation of innate immunity in human DCs may involve TLR-2 signaling.

Genetic Targeting of Individual Cells with a Voltage‐Sensitive Dye through Enzymatic Activation of Membrane Binding

Optical recording of the electrical activity of individual neurons in culture or in a tissue requires cell-selective staining with a fluorescent voltage-sensitive dye. In a proof-of-principle experiment, we implement a novel approach to genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme that transforms the precursor into a hydrophobic dye that binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general-purpose membrane anchor, and the fusion protein was expressed on the surface of HEK293 cells, as was corroborated by immuno- and histochemical staining. We next synthesised an amphiphilic hemicyanine dye containing two enzymatically cleavable phosphate groups at its hydrocarbon tails. When the phosphate groups were removed, the binding to membranes was enhanced by a factor of a thousand, as shown by titration with lipid vesicles. We observed selective staining of enzymatically active cells by fluorescence microscopy in a mixed population of phosphatase-transfected and untransfected HEK293 cells. The critical parameters of enzyme-induced cell-selective staining were elucidated by a simple kinetic model to guide further developments of the method.

The Role of Integrin αDβ2 in Macrophage Migration.

Integrin αDβ2 (CD11d/CD18), the most recently discovered member of the β2 sub-family of adhesion receptors, is strongly upregulated on macrophage foam cells which underscores its potential role in atherosclerosis. However, the contribution of αDβ2 to monocyte/macrophage adhesive reactions and the significance of its overexpression on these cells remain unknown. Recently we characterized αDβ2 as a multiligand receptor capable of binding many extracellular matrix proteins with the recognition specificity overlapping that of the major myeloid-specific integrin αMβ2 (Mac-1). We hypothesized that the αDβ2 ability to bind numerous ligands in the extracellular matrix and its capacity to be upregulated to high density on the surface of macrophages may modulate cell adhesiveness and, thus, affect migration. To evaluate the role of αDβ2 in migration, we generated model and natural cells expressing different densities of αDβ2 and tested their migration to different extracellular matrix proteins. In vitro studies demonstrated that αDβ2 expressed at low densities, either on the surface of HEK293 cells or the mouse macrophage cell line IC-21, supported migration which was partially inhibited by anti-αD function-blocking antibodies. Furthermore, β1 and β3 integrins expressed on HEK293 cells and IC-21 macrophages, respectively, contributed to migration because anti-β1 and anti-β3 antibodies inhibited migration. Increased expression of αDβ2 on the surface of HEK293 cells and its upregulation by PMA on IC-21 macrophages resulted in the inhibition of cell migration. Ligation of αDβ2 with anti-αD antibodies restored β1- and β3-driven cell migration by means of removing restraints imposed by the excess of the αDβ2-ligand adhesive bonds. To test the possibility that progressive upregulation of αDβ2 can block macrophage migration in vivo, we assessed the effect of anti-αD function blocking antibodies using the thioglycollate-induced peritonitis model. More than 4-fold upregulation of αDβ2 was detected on macrophages in 72 h after thioglycollate stimulation and, similar to in vitro studies, the numbers of migration macrophages increased in the presence of anti-αD antibodies. These results demonstrate that the density of αDβ2 can modulate cell migration and suggest that low levels of αDβ2 can contribute to monocyte migration while αDβ2 upregulation on differentiated macrophages might facilitate their retention at the site of inflammation.

The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.

Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1 class.

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain.

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.