Selenium overexposure induces insulin resistance: In silico study.

BACKGROUND AND AIMS: Several studies raise concerns about the possible association of high selenium exposure with insulin resistance and type 2 diabetes. This in silico study proposes a possible mechanism of insulin resistance in the case of overexposure to selenium. METHOD: A study was carried out using molecular modeling, where cysteines of the insulin-receptor are replaced by selenocysteines. Calculation of the interaction energy of the receptor was performed in both cases with Auto Dock Tools and Vina 4.2 software to predict whether the substitution of amino acid could lead to destabilization of the protein-ligand complex and therefore possibly insulin resistance. Finally, the docked complex was analyzed by using BIOVIA Discovery Studio Visualizer to show the type of interactions between the ligands and insulin-receptor, and to determine the distance of the ligands from the binding site on insulin-receptor. RESULTS: The results show that the substitution of cysteine by selenocysteine in the insulin receptor does not lead to stabilization of the complex receptor/insulin, but to its disruption.In addition, the types and the number of bonds between insulin and its receptor in the two cases are different, where 7 strong bonds between insulin and its receptor were found in the case of the cysteine complex compared to 6 weak bonds in the second case. CONCLUSION: Findings of this study suggest that misincorporation of selenocysteines in insulin receptor could lead to destabilization of the insulin-receptor complex and therefore may possibly cause an insulin resistance.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Muscone/RI7217 co-modified upward messenger DTX liposomes enhanced permeability of blood-brain barrier and targeting glioma.

Rationale: The dual-targeted drug delivery system was designed for enhancing permeation of the blood-brain barrier (BBB) and providing an anti-glioma effect. As transferrin receptor (TfR) is over-expressed by the brain capillary endothelial (hCMEC/D3) and glioma cells, a mouse monoclonal antibody, RI7217, with high affinity and selectivity for TfR, was used to study the brain targeted drug delivery system. Muscone, an ingredient of traditional Chinese medicine (TCM) musk, was used as the "guide" drug to probe the permeability of the BBB for drug delivery into the cerebrospinal fluid. This study investigated the combined effects of TCM aromatic resuscitation and modern receptor-targeted technology by the use of muscone/RI7217 co-modified docetaxel (DTX) liposomes for enhanced drug delivery to the brain for anti-glioma effect. Methods: Cellular drug uptake from the formulations was determined using fluorescence microscopy and flow cytometry. The drug penetrating ability into tumor spheroids were visualized using confocal laser scanning microscopy (CLSM). In vivo glioma-targeting ability of formulations was evaluated using whole-body fluorescent imaging system. The survival curve study was performed to evaluate the anti-glioma effect of the formulations. Results: The results showed that muscone and RI7217 co-modified DTX liposomes enhanced uptake into both hCMEC/D3 and U87-MG cells, increased penetration to the deep region of U87-MG tumor spheroids, improved brain targeting in vivo and prolonged survival time of nude mice bearing tumor. Conclusion: Muscone and RI7217 co-modified DTX liposomes were found to show improved brain targeting and enhanced the efficacy of anti-glioma drug treatment in vivo.

High affinity sugar ligands of C-type lectin receptor langerin.

BACKGROUND: Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application. METHODS: We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin. RESULTS: We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system. CONCLUSIONS: L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin. GENERAL SIGNIFICANCE: These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs.

Molecular Remodeling of the Insulin Receptor Pathway by Thiazolidinediones in Type 2 Diabetes Mellitus: A Brief Review.

Type 2 diabetes mellitus (T2DM) is characterized by abnormalities in carbohydrate, lipoprotein and lipid metabolism, leading to hyperglycemia and several other complications. Insulin is the major hormone regulating these facets by eliciting various biological responses through its receptor. Insulin exerts diverse effects on cells by targeting distinct functions such as gene expression, fatty acid synthesis, glucose transport and receptor translocation. Insulin mediates these effects through signaling pathways utilizing adapter molecules like small Gproteins, lipid and tyrosine kinases. The anomalous cell response in diabetic condition is due to altered expression/function of these molecules. Thiazolidinediones (TZD's), a class of oral hypoglycemic drugs, have shown to modify these responses, leading to insulin sensitizing effect(s). The TZD's are not only PPARγ agonists, but substantial insulin sensitizing activity is observed through its direct and indirect targets of the insulin receptor pathway, which contributes to its overall performance. TZD's alter(s) cell response via downstream players, primarily IRS, Akt/PKB, PKC, GLUT4, MEK, ERK and transcription factor PGC1α. Thus, this review will focus on the alteration(s) of these molecules in various cell types in diabetic condition and their regulation by TZD's. The physiological changes that occur at the molecular level in T2DM and their modulation by TZD's will provide insights into the key players involved and the potential drug targets for future drug development. The review further highlights the key markers to be evaluated in screening of any potential anti-diabetic agent, and to standardize therapy for T2DM based upon its modulation of the various signaling pathways.

Treating Diabetes Mellitus: Pharmacophore Based Designing of Potential Drugs from Gymnema sylvestre against Insulin Receptor Protein.

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders which can affect the quality of life severely. Injectable insulin is currently being used to treat DM which is mainly associated with patient inconvenience. Small molecules that can act as insulin receptor (IR) agonist would be better alternatives to insulin injection. Herein, ten bioactive small compounds derived from Gymnema sylvestre (G. sylvestre) were chosen to determine their IR binding affinity and ADMET properties using a combined approach of molecular docking study and computational pharmacokinetic elucidation. Designing structural analogues were also performed for the compounds associated with toxicity and less IR affinity. Among the ten parent compounds, six were found to have significant pharmacokinetic properties with considerable binding affinity towards IR while four compounds were associated with toxicity and less IR affinity. Among the forty structural analogues, four compounds demonstrated considerably increased binding affinity towards IR and less toxicity compared with parent compounds. Finally, molecular interaction analysis revealed that six parent compounds and four analogues interact with the active site amino acids of IR. So this study would be a way to identify new therapeutics and alternatives to insulin for diabetic patients.

CD71 targeting boosts immunogenicity of sublingually delivered influenza haemagglutinin antigen and protects against viral challenge in mice.

The delivery of vaccines to the sublingual mucosa is an attractive prospect due to the ease and acceptability of such an approach. However, novel adjuvant and delivery approaches are required to optimally vaccinate at this site. We have previously shown that conjugation of protein antigen to the iron transport molecule, transferrin, can significantly enhance mucosal immune responses. We tested whether conjugating influenza haemagglutinin to transferrin could improve the immune response to sublingually delivered antigen. Transferrin conjugated haemagglutinin induced a significant antibody and T cell response in both naïve animals and previously immunized animals. The immune response generated was able to protect mice against influenza virus challenge. Sublingually administered antigen dispersed more widely through the gastro-intestinal tract than intranasally delivered antigen and transferrin conjugation had a more marked effect on sublingually delivered antigen than intranasal immunisation. From these studies we conclude that transferrin conjugation of antigen is effective at boosting immune responses to sublingually delivered antigen and may be an attractive approach for influenza vaccines, particularly when mass campaigns are required.

Nanovehicles as a novel target strategy for hyperthermic intraperitoneal chemotherapy: a multidisciplinary study of peritoneal carcinomatosis.

In general, detection of peritoneal carcinomatosis (PC) occurs at the late stage when there is no treatment option. In the present study, we designed novel drug delivery systems that are functionalized with anti-CD133 antibodies. The C1, C2 and C3 complexes with cisplatin were introduced into nanotubes, either physically or chemically. The complexes were reacted with anti-CD133 antibody to form the labeled product of A0-o-CX-chem-CD133. Cytotoxicity screening of all the complexes was performed on CHO cells. Data showed that both C2 and C3 Pt-complexes are more cytotoxic than C1. Flow-cytometry analysis showed that nanotubes conjugated to CD133 antibody have the ability to target cells expressing the CD133 antigen which is responsible for the emergence of resistance to chemotherapy and disease recurrence. The shortest survival rate was observed in the control mice group (K3) where no hyperthermic intraperitoneal chemotherapy procedures were used. On the other hand, the longest median survival rate was observed in the group treated with A0-o-C1-chem-CD133. In summary, we designed a novel drug delivery system based on carbon nanotubes loaded with Pt-prodrugs and functionalized with anti-CD133 antibodies. Our data demonstrates the effectiveness of the new drug delivery system and provides a novel therapeutic modality in the treatment of melanoma.

Coupling to a glioblastoma-directed antibody potentiates antitumor activity of curcumin.

Current therapies for glioblastoma are largely palliative, involving surgical resection followed by chemotherapy and radiation therapy, which yield serious side effects and very rarely produce complete recovery. Curcumin, a food component, blocked brain tumor formation but failed to eliminate established brain tumors in vivo, probably because of its poor bioavailability. In the glioblastoma GL261 cells, it suppressed the tumor-promoting proteins NF-κB, P-Akt1, vascular endothelial growth factor, cyclin D1 and BClXL and triggered cell death. Expression of exogenous p50 and p65 subunits of NF-κB conferred partial protection on transfected GL261 cells against curcumin insult, indicating that NF-κB played a key role in protecting glioblastoma cells. To enhance delivery, we coupled curcumin to the glioblastoma-specific CD68 antibody in a releasable form. This resulted in a 120-fold increase in its efficacy to eliminate GL261 cells. A very similar dose response was also obtained with human glioblastoma lines T98G and U87MG. GL261-implanted mice receiving intratumor infusions of the curcumin-CD68 adduct followed by tail-vein injections of solubilized curcumin displayed a fourfold to fivefold reduction in brain tumor load, survived longer, and about 10% of them lived beyond 100 days. Hematoxylin-eosin staining of brain sections revealed a small scar tissue mass in the rescued mice, indicating adduct-mediated elimination of glioblastoma tumor. The tumor cells were strongly CD68+ and some cells in the tumor periphery were strongly positive for microglial Iba1, but weakly positive for CD68. This strategy of antibody targeting of curcumin to tumor comes with the promise of yielding a highly effective therapy for glioblastoma brain tumors.

Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases.

Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.

Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity.

The interaction between 4-1BB ligand (CD137L), a member of the tumor necrosis factor superfamily, and its receptor 4-1BB provides a co-stimulatory signal for T lymphocyte proliferation and survival. However, the structure of 4-1BBL has not been thoroughly investigated, and none of the human recombinant 4-1BBL molecules available have been described as capable of co-stimulating T cells. The present work provides a model of the three-dimensional structure of the tumor necrosis factor homology domain of 4-1BBL and describes the production of a recombinant human soluble 4-1BBL whose originality lies in that it contains the whole extracellular tail preceding the tumor necrosis factor homology domain and an AviTag peptide (AviTag-4-1BBL) allowing enzymatic biotinylation and multimerization via streptavidin. We provide evidence that this chimeric protein exists as a homotrimer, whereas commercial FLAG-tagged 4-1BBL does not. This resulted in a much higher affinity for 4-1BB (1.2 nM) as compared with FLAG-4-1BBL (55.2 nM). We demonstrate that the single extracellular cysteine residue in the tail (Cys-51) could form a disulfide bond, both in our recombinant protein and in physiologically expressed 4-1BBL. The mutation of this cysteine residue exerted no effect on trimerization but increased the dissociation rate of AviTag-4-1BBL from 4-1BB. In its soluble form, AviTag-4-1BBL did not stimulate purified T cells but dramatically inhibited proliferation of peripheral blood mononuclear cells stimulated with anti-CD3 mAb. In contrast, a very significant co-stimulatory effect was observed on purified T cells once AviTag-4-1BBL was immobilized onto streptavidin beads. In addition, we show that the cross-linking of two trimeric AviTag-4-1BBL molecules was the minimum step required to elicit significant costimulatory activity.

Epitope mapping of monoclonal antibody to integrin alphaL beta2 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.

Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization.

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acalpha2-6Galbeta1-4GlcNAc-residues are receptors for the anticancer drug rViscumin.

The anticancer drug rViscumin, currently under clinical development, has been shown in previous studies to be a sialic acid specific ribosome inactivating protein (RIP). Comparative binding assays with the CD75s-specific monoclonal antibodies HB6 and J3-89 revealed rViscumin to be a CD75s-specific RIP due to identical binding characteristics toward CD75s gangliosides. The receptor gangliosides are IV6nLc4Cer, VI6nLc6Cer, and the newly characterized ganglioside VIII6nLc8Cer, all three carrying the Neu5Acalpha2-6Galbeta1-4GlcNAc motif. To elucidate the clinical potential of the rViscumin targets, CD75s gangliosides were determined in several randomly collected gastrointestinal tumors. The majority of the tumors showed an enhanced expression of CD75s gangliosides compared with the unaffected tissues. The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N-glycans. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, revealed specific recognition of type II N-glycans with CD75s determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s motifs and others. This strict binding specificity of rViscumin and the increased expression of CD75s gangliosides in various tumors suggest this anticancer drug as a promising candidate for an individualised adjuvant therapy of human tumors.

Identification of allelic variants of the bovine immune regulatory molecule CEACAM1 implies a pathogen-driven evolution.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), the primordial member of the carcinoembryonic antigen (CEA) family, functions as a MHC-independent natural killer (NK) cell inhibitory receptor, regulates T and B cell proliferation, and induces dendritic cell (DC) maturation. Despite these fundamental functions, CEACAM1 and most of the CEA family members differ significantly in primates and rodents. A number of diverse murine and human pathogens use CEACAM1 as a cellular receptor, indicating that the observed species-specific differences are the result of divergent molecular pathogen/host coevolution. To gain deeper insight into its evolution and function, we cloned CEACAM1 cDNA from cattle as a representative of a third mammalian order. Bovine CEACAM1 differs considerably from rodent and primate CEACAM1 due to deletion of the B domain exon which was most likely caused by insertion of LINE/SINE sequences and reveals alternative splicing within the transmembrane exon. However, the characteristic long and short isoforms exist which contain or lack the typical immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tails, respectively. Bovine peripheral blood lymphocytes (PBL) express only ITIM-containing CEACAM1 isoforms, and upregulate their expression upon stimulation, suggesting an inhibitory function in these cells. As found in rodents, two clearly distinct CEACAM1 alleles exist in cattle. In the a allele, a unique deletion of three amino acids is found in the N domain, which is important for pathogen binding in mice and humans. This is consistent with the notion that CEACAM1 serves or has served as a pathogen receptor in cattle.

Neuronal roles of the integrin-associated protein (IAP/CD47) in developing cortical neurons.

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.

Fc receptor gamma chain residues at the interface of the cytoplasmic and transmembrane domains affect association with FcalphaRI, surface expression, and function.

The assembly of multiple subunit immunoreceptors is dependent on transmembrane interactions. The Fc receptor gamma (FcR-gamma) chain is a ubiquitous immune receptor tyrosine-based activation motif-containing dimeric subunit, gamma(2), which in humans associates with both the activating members of the leukocyte receptor cluster, including the IgA receptor FcalphaRI, and the classical Fc receptors, including the IgE receptor FcepsilonRI. This study identifies a new site in the transmembrane region of FcR-gamma that affects receptor assembly and surface expression with FcalphaRI but not with FcepsilonRI. The wild type complex, FcalphaRI-gamma(2)WT, remains robustly associated in both Brij-96 and Thesit detergent conditions. However, mutation of either Tyr(25) or Cys(26) of FcR-gamma, near the interface of the transmembrane and cytoplasmic regions, resulted in impaired FcR-gamma association with FcalphaRI. This association was disrupted in the presence of the detergent Brij-96 but was preserved in milder conditions using the detergent Thesit. Ligand-mediated cross-linking of the FcalphaRI-gamma(2)Y25F mutant receptor resulted in diminished signal transduction, including an abnormal calcium response, compared with the FcalphaRI-gamma(2)WT receptor. Furthermore, the FcalphaRI-gamma(2)Y25F mutant receptor was expressed at the cell surface at approximately 10% of that of the wild type, whereas the surface expression of FcepsilonRI-gamma(2)Y25F was not significantly different from the wild type. In contrast, although the FcalphaRI-gamma(2)C26S mutant was also less stably associated, it was not reduced in surface expression or function. Thus, these TM residues of FcR-gamma are important for association with FcalphaRI and probably other activating LRC members but not with the classical FcR, FcepsilonRI.

CD109 represents a novel branch of the alpha2-macroglobulin/complement gene family.

We report here the genomic organization and phylogenic relationships of CD109, a member of the the alpha2-macroglobulin/complement (AMCOM) gene family. CD109 is a GPI-linked glycoprotein expressed on endothelial cells, platelets, activated T-cells, and a wide variety of tumors. We cloned full-length CD109 cDNA from the mammalian U373 cell line by RT-PCR and performed analysis of its corresponding genomic sequence. The CD109 cDNA spans 128 kb of chromosome 6q with its 33 exons constituting approximately 3.3% of the total CD109 genomic sequence. Sequence analysis revealed that CD109 contains specific motifs in its N-terminus, that are highly conserved in all AMCOM members. CD109 also shares motifs with certain other AMCOM members including: (1) a thioester 'GCGEQ" motif, (2) a furin site of four positively charged amino acids, and (3) a double tyrosine near the C-terminus. Based on a phylogenic analysis of human CD109 with other human homologs as well as orthologs from other mammalian species, C. elegans (ZK337.1) and E. coli homologs, we propose CD109 represents a novel and independent branch of the alpha2-macroglobulin/complement gene family (AMCOM) and may be its oldest member.

The tyrosine phosphatase 1B regulates linker for activation of T-cell phosphorylation and platelet aggregation upon FcgammaRIIa cross-linking.

Human platelets express the receptor for immunoglobulin G, FcgammaRIIa, that triggers cell aggregation upon interaction with immune complexes. Here, we report that the rapid tyrosine phosphorylation of the Linker for Activation of T-cell (LAT) in human platelets stimulated by FcgammaRIIa cross-linking was followed by its complete dephosphorylation in an alphaIIb/beta3 integrin-dependent manner. Concomitant to LAT dephosphorylation, the protein tyrosine phosphatase 1B (PTP1B) was activated through a mechanism involving its proteolysis by calpains downstream of integrins. Both PTP1B and LAT were associated with the actin cytoskeleton complex formed during platelet aggregation. Moreover, phospho-LAT appeared as a good substrate of activated PTP1B in vitro and these two proteins interacted upon platelet activation by FcgammaRIIa cross-linking. The permeant substrate-trapping PTP1B (TAT-PTP1B D181A) partly inhibited LAT dephosphorylation in human platelets, strongly suggesting that this tyrosine phosphatase was involved in this regulatory pathway. Using a pharmacological inhibitor, we provide evidence that PTP1B activation and LAT dephosphorylation processes were required for irreversible platelet aggregation. Altogether, our results demonstrate that PTP1B plays an important role in the integrin-mediated dephosphorylation of LAT in human platelets and is involved in the control of irreversible aggregation upon FcgammaRIIa stimulation.