Home About us Contact
|
Home About us Contact | ||
|
|
||
Cytoplasmic Tail (cytoplasmic + tail)
Selected AbstractsIdentification and characterization of Xenopus OMP25DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2004Masafumi Inui This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube. [source] Analysis of conserved residues in the ,pat-3 cytoplasmic tail reveals important functions of integrin in multiple tissuesDEVELOPMENTAL DYNAMICS, Issue 3 2010Xiaojian Xu Abstract Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans ,pat-3 cytoplasmic tail. The ,1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the ,1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a ,1C-like variant, which results in a frameshift, was constructed. The ,pat-3(,1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin. Developmental Dynamics 239:763,772, 2010. © 2010 Wiley-Liss, Inc. [source] Cytosolic protein-protein interactions that regulate the amyloid precursor proteinDRUG DEVELOPMENT RESEARCH, Issue 2 2002Shasta L. Sabo Abstract Alzheimer disease (AD), a progressive neurodegenerative disease, is the most common cause of dementia in the elderly and is among the leading causes of death in adults. AD is characterized by two major pathological hallmarks, amyloid plaques and neurofibrillary tangles. For a number of reasons, amyloid plaque accumulation is widely thought to be the probable cause of AD. The amyloid plaque core is largely composed of an approximately 4-kDa peptide referred to as A,. A, is derived from its precursor, the Alzheimer amyloid protein precursor (APP), by endoproteolytic processing. APP is a type I integral membrane protein, with a long extracellular domain, one transmembrane domain, and a short (,50 amino acid) cytoplasmic tail. Despite intense efforts to decipher the function of APP, its normal physiological role has remained elusive. The carboxy-terminus of APP contains the sequence YENPTY, which is absolutely conserved across APP homologues and across species. The YENPTY sequence is important for regulation of APP processing and trafficking. Given the importance of the cytoplasmic domain in APP physiology, a number of laboratories have hypothesized that proteins that bind to the YENPTY sequence in the cytoplasmic domain of APP might regulate APP processing, trafficking, and/or function. In this article, we will discuss data revealing which proteins bind to the cytoplasmic domain of APP, how these binding-proteins regulate APP metabolism and function, and why such protein-protein interactions provide an exciting new target for therapeutic intervention in AD. Drug Dev. Res. 56:228,241, 2002. © 2002 Wiley-Liss, Inc. [source] Cathepsin X cleaves the ,2 cytoplasmic tail of LFA-1 inducing the intermediate affinity form of LFA-1 and ,-actinin-1 bindingEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2009Zala Jevnikar Abstract The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the ,2 chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S769, E768 and A767 from the C-terminal of the ,2 cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein ,-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with ,-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration. [source] Recombinase-deficient T cell development by selective accumulation of CD3 into lipid raftsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2008Denise Ferrera Abstract The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR ,,chain locus by signaling in a ligand-independent fashion. The TCR ,,chain associates with the invariant pre-T, (pT,) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pT, chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated ,,selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pT, chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that ,,selection by oligomerized pre-TCR is implemented by the pT, cytoplasmic tail. [source] A pseudosymmetric cell adhesion regulatory domain in the ,7 tail of the integrin ,4,7 that interacts with focal adhesion kinase and srcEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2006Geoffrey Abstract The ,7 integrins ,4,7 and ,E,7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The ,4,7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of ,4,7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735,740 of the cytoplasmic tail of the ,7 subunit inhibit the adhesion of T cells to ,7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of ,4,7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr735Phe and Tyr740Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of ,4,7. The quasi-palindromic sequence YDRREY within the ,7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of ,7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease. [source] The cytoplasmic tail of invariant chain modulates antigen processing and presentationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2003Abstract The MHC class II-associated invariant chain (Ii) has several important functions in antigen presentation. In this study, we have examined the effect of Iip33 expression on endocytic transport and antigen presentation. We find that degradation of both endocytosed antigen and Ii itself is delayed in cells expressing high levels of Ii, whereas a mutant Ii with an altered charge distributionin the cytoplasmic tail was unable to exert this effect. Furthermore, the Ii mutant did not enhance the presentation of an Ii-dependent MHC class II-restricted epitope to the same extent as the wild type. In a parallel study, we investigated the effect of charge in the cytoplasmic tail of Ii. We find that due to exposed negative charges, it promotes endosome fusion events, and we suggest thatthis causes endosomal retention (Nordeng et al., Mol. Biol. Cell 2002). Together, the data reveal an additional property of the Iip33 cytoplasmic tail that contributes to the modulation of antigen processing and presentation. [source] N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel,HUMAN MUTATION, Issue 5 2008G.G. Gené Abstract Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure,function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology. Hum Mutat 29(5), 738,749, 2008. © 2008 Wiley-Liss, Inc. [source] Initiation of TCR signaling: regulation within CD3 dimersIMMUNOLOGICAL REVIEWS, Issue 1 2003Balbino Alarcón Summary: The number of possible T cell activation outcomes resulting from T cell receptor (TCR) engagement suggests that the TCR is able to differentially activate a myriad of signaling pathways depending on the nature of the stimulus. The complex structural organization of the TCR itself could underlie this diversity of responses. Assembly and stoichiometric studies have helped us to shed some light on the initiation of TCR signaling. The TCR is composed of TCR and CD3 dimers. Changes in the interaction between CD3 subunits within the CD3 dimers and in the interaction of these dimers with the TCR heterodimer could be the triggering mechanism that initiates the first activation events. One of the hallmarks of these early changes in TCR conformation is the induced recruitment of the adapter protein Nck to a proline-rich sequence of the cytoplasmic tail of CD3,, but there may be others. According to our most recent observations, the TCR is organized in pre-existing clusters within plasma membrane microdomains, exhibiting a complexity above and beyond that of dimer composition complexity. How the presence of TCR in clusters influences TCR avidity and propagation of TCR signals is something that has yet to be investigated. [source] C-terminal 37 residues of LRP promote the amyloidogenic processing of APP independent of FE65JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2008Madepalli K. Lakshmana Abstract The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid , protein (A,), a small peptide derived from ,- and ,-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that the Low-density lipoprotein receptor-related protein (LRP) plays a pivotal role in the trafficking of APP and generation of A,. In particular, we recently showed that the soluble cytoplasmic tail of LRP (LRP-ST) without a membrane tether was sufficient to promote A, generation. In this study, we demonstrate that the last 37 residues of LRP cytoplasmic tail (LRP-C37) lacking the NPxY motifs and FE65 binding mediate the core pro-amyloidogenic activity of LRP-ST. Moreover, we show that the conserved dileucine motif within the LRP-C37 region is a key determinant of its A, promoting activity. Finally, results from a yeast two-hybrid screen using LRP-C37 region as bait reveal four new LRP-binding proteins implicated in intracellular signalling and membrane protein trafficking. Our findings indicate that the LRP-C37 sequence represents a new protein-binding domain that may be useful as a therapeutic target and tool to lower A, generation in AD. [source] Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the ,V,3 integrinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Laura A. Maile Abstract The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor ,V,3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of ,3 and that this interaction is required for the positive effects of ,V,3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the ,V,3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the ,3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the ,V,3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates. J. Cell. Biochem. 105: 437,446, 2008. © 2008 Wiley-Liss, Inc. [source] BT-IgSF, a novel immunoglobulin superfamily protein, functions as a cell adhesion moleculeJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Hideki Harada BT-IgSF is a newly identified cell surface glycoprotein belonging to the immunoglobulin superfamily (IgSF). We have previously shown that the expression of the BT-IgSF gene was highly restricted to brain and testis, and its transcript was detected in both neurons and glial cells. In this study, to explore its function, we generated cells overexpressing BT-IgSF proteins and analyzed their phenotypes. We found that the constitutive expression of BT-IgSF in the myeloid leukemia cell line TF-1 -fms did not alter the growth rates, but caused the formation of large cell aggregates. The cell aggregates were also observed with mutant BT-IgSF lacking its cytoplasmic tail, the amino acid sequences of which were highly conserved among the BT-IgSF subgroup proteins. The neutralizing antibody to ,1 integrin did not diminish the cell aggregate formation. These results indicate that BT-IgSF functions as a cell adhesion molecule, that its cytoplasmic tail is not essential for the function, and that ,1 integrin is not involved in the function. We confirmed the cell adhesion function using NIH/3T3 fibroblastic cells expressing BT-IgSF in an inducible system. Flow cytometric analyses with the cells demonstrated that the cell aggregation mediated by BT-IgSF was through homophilic molecular interaction, and in a Ca2+/Mg2+ -independent manner. Coupled with its restricted pattern of the expression, the cell adhesion-inducing function of BT-IgSF suggests a role of the cell surface molecule in the development/function of the central nervous system and spermatogenesis. © 2005 Wiley-Liss, Inc. [source] Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugateJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2001Bruce R. Conway Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor,green fluorescent protein (PTHR,GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR,GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR,GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR,GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR,GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR,GFP back to the plasma membrane was complete within 1,2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization. © 2001 Wiley-Liss, Inc. [source] LIM-only protein 4 interacts directly with the repulsive guidance molecule A receptor NeogeninJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Gregor Schaffar Abstract Repulsive guidance molecule A (RGM A) was recently described as a potent inhibitor of neuroregeneration in a rat spinal cord injury model. The receptor mediating RGM A's repulsive activity was shown to be Neogenin, a member of the Deleted in Colorectal Cancer (DCC) family of netrin receptors. Binding of RGM A to Neogenin induces activation of the small GTPase RhoA and of its effector Rho-kinase by an unknown mechanism. Here we show, that the cytoplasmic tail of Neogenin interacts directly with the transcriptional coactivator LIM domain only 4 (LMO4) in human SH-SY5Y cells, human Ntera neurons, and in embryonic rat cortical neurons. RGM A binding to Neogenin but not binding of Netrin-1, induces release of LMO4 from Neogenin. Down-regulation of LMO4 neutralizes the repulsive activity of RGM A in neuronal cell lines and embryonic rat cortical neurons and prevents RhoA activation. These results show for the first time that an interaction of Neogenin with LMO4 is involved in the RGM A , Neogenin signal transduction pathway for RhoA activation. [source] The cytoplasmic tail of the ,3 integrin subunit promotes neurite outgrowth in PC12 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Nadja Mechai Abstract Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein,protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the ,3 integrin subunit of ,3,1 integrin is required for ,3,1 integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) , subunit and the cytoplasmic sequence of the ,3 integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic ,3 tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic ,3 sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-,3 can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase ,. © 2005 Wiley-Liss, Inc. [source] The ,3 integrin cytoplasmic tail: protein scaffold and control freakJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009S. J. SHATTIL Summary., Platelet integrin ,IIb,3 plays an essential role in thrombus formation through interactions with adhesive ligands. Successful parenteral blockade of these interactions has validated ,IIb,3 as a therapeutic target in cardiovascular medicine. However, oral ,IIb,3 antagonists have not been successful and there is an unmet need for more effective anti-platelet drugs. Growing evidence points to the cytoplasmic tails of ,IIb and ,3, and the ,3 tail in particular, as scaffolds for intracellular proteins that mediate inside-out signaling and regulate ,IIb,3 affinity for ligands. Intracellular protein interactions with the integrin cytoplasmic tails also regulate outside-in signals to the actin cytoskeleton. Here we focus on recent studies that illustrate the relevance of the ,3 cytoplasmic tail as a regulatory scaffold in vivo. We speculate that this scaffold or its interacting proteins may serve as therapeutic targets for the development of future anti-thrombotic drugs. [source] A 13-bp deletion in ,IIb gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombastheniaJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2005N. ROSENBERG Summary. Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of ,IIb,3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13-bp deletion in the ,IIb gene that causes in-frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13-bp deletion, and determined the mechanism by which the 13-bp deletion abolishes ,IIb,3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13-bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300,600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective ,IIb,3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in ,IIb gene, TT1616-7 deletion in ,3 gene, and IVS14: ,3C , G in ,3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of ,3 and was expressed as dysfunctional ,IIb,3. None of 15 unrelated Jordanian patients carried any of the described mutations. [source] Involvement of the ,3 E749ATSTFTN756 region in stabilizing integrin ,IIb,3 -ligand interactionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2003P. E. M. H. Litjens Summary., Platelet integrin ,IIb,3 must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the ,-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756 domain (E,N peptide) and the T755NITYRGT762 domain (T,T peptide) of ,3 in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E,N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E,N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E,N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E,N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on ,IIb,3. T,T peptide did not affect these processes. In a model for outside-in integrin activation, E,N peptide disrupted the binding of CHO cells expressing ,IIb,3 to surface-bound ligand. Again, T,T peptide had no effect. We conclude that the E749ATSTFTN756 region of the ,3 -tail stabilizes the binding of soluble and surface-bound ligand to integrin ,IIb,3 via a mechanism that involves the phosphorylation of FAK. [source] Glycoprotein Ib,IX-mediated activation of integrin ,IIb,3: effects of receptor clustering and von Willebrand factor adhesionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2003M. Arya Summary., The interaction between the platelet glycoprotein (GP) Ib,IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to ,IIb,3, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib,IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF,GP Ib,IX and fibrinogen,,IIb,3 bonds using optical tweezers. In our system, fusion of tandem repeats of FK506-binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib,IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell-permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma-derived VWF or the VWF A1 domain and GP Ib,IX(FKBP)2, and those between fibrinogen-coated beads and ,IIb,3 expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib,IX(FKBP)2 and A1 or plasma-derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and ,IIb,3 was not changed by the clustering agent; however, the strength of single fibrinogen,,IIb,3 bonds increased significantly after ligation of GP Ib,IX(FKBP)2 by A1. These results demonstrate that GP Ib,IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib,IX can activate ,IIb,3, thereby increasing the strength of its interaction with fibrinogen. [source] Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastomaTHE JOURNAL OF GENE MEDICINE, Issue 6 2004Sergio Gonzalez Abstract Background Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. Methods The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-, chain (scFvFc:,). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. Results CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8+ CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and Tc1 cytokine production. Conclusions These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R+CD8+ CTL clones to children with recurrent/refractory neuroblastoma. Copyright © 2004 John Wiley & Sons, Ltd. [source] Acceleration of the onset of collagen-induced arthritis by a deficiency of platelet endothelial cell adhesion molecule 1ARTHRITIS & RHEUMATISM, Issue 11 2003Yoshifumi Tada Objective Platelet endothelial cell adhesion molecule 1 (PECAM-1; CD31) is a member of the immunoglobulin superfamily that is expressed in platelets, leukocytes, and endothelial cells. PECAM-1 has been shown to play a role in transendothelial migration of leukocytes and contains immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail and inhibits cellular responses. We examined the role of PECAM-1 in the development of collagen-induced arthritis (CIA). Methods CIA was induced in PECAM-1,deficient DBA/1 mice. The incidence of arthritis and the arthritis index were examined. Anti,type II collagen (anti-CII) antibody levels and interferon-, (IFN,) production by lymph node cells and spleen cells were determined. Lymphocytes from arthritic PECAM-1,deficient and wild-type mice were labeled with dye, transferred to arthritic PECAM-1+/, mice, and cell migration to inflamed joints was examined. Results PECAM-1,deficient mice showed accelerated onset of arthritis and increased severity only during the early phase. Anti-CII antibody levels were also increased during the early phase. IFN, production by lymph node cells and spleen cells from PECAM-1,deficient mice in response to CII was higher than that in wild-type mice. Lymphocytes from arthritic PECAM-1,deficient mice showed accelerated migration to inflamed joints, but not lymph nodes or spleen. The development of anti-CII antibody,induced arthritis was similar in PECAM-1,deficient and wild-type mice. Conclusion These results indicate that PECAM-1 negatively regulates humoral and cell-mediated immune responses and lymphocyte migration into joints and, consequently, the development of CIA. In addition, the role of PECAM-1 in the transendothelial migration of leukocytes appears to be redundant in this model. [source] Expression, purification, crystallization and preliminary crystallographic analysis of the diarrhoea-causing and virulence-determining region of rotaviral nonstructural protein NSP4ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004Rotaviral nonstructural protein NSP The region spanning the tetrameric coiled-coil domain and the interspecies-variable virulence-determining region of the cytoplasmic tail of rotaviral nonstructural protein NSP4 has been crystallized. The crystals belong to space group I222, with unit-cell parameters a = 30.70, b = 38.07, c = 181.62,Å, and contain two molecules in the asymmetric unit. Diffraction data have been collected utilizing a MAR imaging plate to a resolution of 2.2,Å. The tetramer is generated by the crystallographic dyad along the c axis. [source] Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of the adhesion protein ICAM-2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001Keisuke Hamada Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The FERM domains located at the N-terminal regions of ERM proteins are responsible for membrane association through direct interactions with the cytoplasmic domains of integral membrane proteins. Here, crystals of the complex between the radixin FERM domain and the full-length cytoplasmic tail (28-residue peptide) of intercellular adhesion molecule 2, ICAM-2, have been obtained. The crystals were found to belong to space group P3121 or P3221, with unit-cell parameters a = b = 100.44,(9), c = 99.49,(6),Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.60,Å. [source] Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherenceCELLULAR MICROBIOLOGY, Issue 3 2001Helena Källström Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell-surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus-binding site within the CD46 molecule, a set of CD46,BC1 deletion constructs were transfected into COS-7 cells. Piliated N. gonorrhoeae attached well to CD46,BC1-expressing COS-7 cells. We show that the complement control protein repeat 3 (CCP-3) and the serine,threonine,proline (STP)-rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells. [source] Cytoplasmic tail motifs mediate endoplasmic reticulum localization and export of transmembrane reporters in the protozoan parasite Toxoplasma gondiiCELLULAR MICROBIOLOGY, Issue 6 2000Heinrich C. Hoppe In mammalian cells and yeasts, amino acid motifs in the cytoplasmic tails of transmembrane proteins play a prominent role in protein targeting in the early secretory pathway by mediating localization to or rapid export from the endoplasmic reticulum (ER). However, early sorting events are poorly characterized in protozoan parasites. Here, we show that a C-terminal QKTT sequence mediates the ER localization of chimeric reporter constructs consisting of bacterial alkaline phosphatase (BAP) fused to the transmembrane domain (TMD) and truncated cytoplasmic tail of the human low-density lipoprotein receptor (LDL) receptor or of murine lysosome-associated membrane protein (lamp-1) in Toxoplasma gondii. The cytoplasmic tail of human TGN46 also determines ER localization of BAP chimeras in the parasite, but this can be overcome by the addition at the C-terminus of the tail of an acidic patch, which functions as an ER export signal in conjunction with an upstream tyrosine motif. These results suggest that COPI-dependent ER retrieval and COPII-dependent export mechanisms mediated by KKXX and DXE motifs of mammalian cells are generally conserved in T. gondii. In contrast, the failure of the QKTT motif and TGN46 cytoplasmic tail to induce steady-state ER localization of vesicular stomatitis virus glycoprotein (VSVG) chimeras in HeLa and NRK cells indicates that significant differences in early secretory trafficking also exist. [source] Analysis of conserved residues in the ,pat-3 cytoplasmic tail reveals important functions of integrin in multiple tissuesDEVELOPMENTAL DYNAMICS, Issue 3 2010Xiaojian Xu Abstract Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans ,pat-3 cytoplasmic tail. The ,1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the ,1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a ,1C-like variant, which results in a frameshift, was constructed. The ,pat-3(,1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin. Developmental Dynamics 239:763,772, 2010. © 2010 Wiley-Liss, Inc. [source] The cytoplasmic tail of the ,3 integrin subunit promotes neurite outgrowth in PC12 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Nadja Mechai Abstract Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein,protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the ,3 integrin subunit of ,3,1 integrin is required for ,3,1 integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) , subunit and the cytoplasmic sequence of the ,3 integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic ,3 tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic ,3 sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-,3 can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase ,. © 2005 Wiley-Liss, Inc. [source] The ,3 integrin cytoplasmic tail: protein scaffold and control freakJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009S. J. SHATTIL Summary., Platelet integrin ,IIb,3 plays an essential role in thrombus formation through interactions with adhesive ligands. Successful parenteral blockade of these interactions has validated ,IIb,3 as a therapeutic target in cardiovascular medicine. However, oral ,IIb,3 antagonists have not been successful and there is an unmet need for more effective anti-platelet drugs. Growing evidence points to the cytoplasmic tails of ,IIb and ,3, and the ,3 tail in particular, as scaffolds for intracellular proteins that mediate inside-out signaling and regulate ,IIb,3 affinity for ligands. Intracellular protein interactions with the integrin cytoplasmic tails also regulate outside-in signals to the actin cytoskeleton. Here we focus on recent studies that illustrate the relevance of the ,3 cytoplasmic tail as a regulatory scaffold in vivo. We speculate that this scaffold or its interacting proteins may serve as therapeutic targets for the development of future anti-thrombotic drugs. [source] Platelet integrin ,IIb,3: activation mechanismsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007Y.-Q. MA Summary., Integrin ,IIb,3 plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of ,IIb,3 depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of ,IIb,3 and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of ,IIb and ,3 cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of ,IIb,3 activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation. [source] Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2007Yumiko Takai Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4322, with unit-cell parameters a = b = 68.72, c = 201.39,Å, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P212121, with unit-cell parameters a = 80.74, b = 85.73, c = 117.75,Å, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9,Å for the FERM,CD43 complex and 2.8,Å for the FERM,PSGL-1 complex. [source] | ||||||||||||||||