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PubMed2002

Clustering the mast cell function-associated antigen (MAFA) leads to tyrosine phosphorylation of p62Dok and SHIP and affects RBL-2H3 cell cycle.

The mast cell function-associated antigen (MAFA) is a type II membranal glycoprotein expressed by rat mast cells and basophils. MAFA clustering by its specific monoclonal antibody, (mAb) G63, efficiently inhibits the FcvarepsilonRI induced secretory response of mucosal-type mast cells of the RBL-2H3 line, as well as bone marrow-derived mast cells. Here we present results which suggest that MAFA has also a capacity of modulating the cell cycle of the RBL-2H3 line. We found that MAFA clustering, by mAb G63 or by its F(ab')2 fragments, reduces the cell proliferation rate. Cell cycle analysis by flow cytometry revealed that the number of cells in sub-G phase is considerably higher for cells on which MAFA was clustered. Results of biochemical experiments established that MAFA clustering leads to a marked increase in the transient tyrosine phosphorylation of the adaptor protein p62(Dok) and the inositol phosphatase SHIP. Concomitantly, their respective binding to RasGAP and Shc was increased. Furthermore, the GTP binding protein Sos1 was found to dissociate from Shc upon MAFA clustering, suggesting that SHIP and Sos1 compete for Shc binding. We therefore suggest that MAFA has also a role in regulating RBL-2H3 cell proliferation rate by inhibiting RasGTP formation in the Ras signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Interaction of HLA-DR with actin microfilaments.

Capping of HLA-DR on the surface of a human lymphoblastoid cell line (RAJI) and a transfectant human fibroblast cell line (M1DR1) was studied by confocal microscopy. Capping was induced at 22 degrees C after treating cells with an HLA-DR specific monoclonal antibody, L243, followed by a secondary antibody conjugated with FITC. Cytoskeletal actin filaments (F-actin) accumulated under the caps were detected by rhodamine-phalloidin fluorescence. Two processes appear to take place: in the round lymphoblastoid cells, actin, initially distributed uniformly at the cell periphery, redistributes and becomes concentrated underneath HLA-DR patches or caps. In the non-round, substrate-attached fibroblasts, actin was organized in tightly packed filaments along the plasma membrane. It was observed that crosslinked HLA-DR receptors were associated with these filaments and were dragged toward the perinuclear area of the cells, where they coalesce to form a cap. The cytoskeleton-disrupting drugs that inhibit actin polymerisation were used to investigate the mechanism of capping of HLA-DR molecules. Sodium nitroprusside, a nitric oxide releasing agent, cytochalasin D both inhibited the percentage of capping in a dose-dependent manner. These data suggest that on antigen presenting cells, such as B cells and fibroblasts, actin microfilaments acts as a regulator of the movement and capping of HLA-DR receptors.

Actins↗
PubMed1999

Role of abnormal integrin-cytoskeletal interactions in impaired beta1 integrin function in chronic myelogenous leukemia hematopoietic progenitors.

Abnormal circulation and unregulated proliferation of chronic myelogenous leukemia (CML) progenitors is related, at least in part, to BCR/ABL induced abnormalities in beta1 integrin-mediated adhesion and signaling. The BCR/ABL oncogene has several potential interactions with cytoskeletal elements that are important for normal integrin signaling. In the present study, we evaluated whether abnormalities in beta1 integrin-cytoskeletal interactions were present in primary CML progenitors and contributed to defective integrin function. beta1 integrin-cytoskeletal interactions were studied in CML and normal CD34+ primary hematopoietic progenitors as well as BCR/ABL-transfected or mock-transfected M07e cells. In normal CD34+ progenitors, antibody-mediated cross-linking of beta1 integrins resulted in their redistribution into caps via a process requiring receptor-cytoskeletal interactions. CML CD34+ cells demonstrated significantly impaired beta1 integrin capping. This defect was related to the presence of the BCR/ABL gene, because capping also was impaired in BCR/ABL-transfected M07e cells. Defective receptor capping was not seen for non-integrin receptors. In addition, CML CD34- and M07eBCR/ABL cells also demonstrated increased actin polymerization and altered actin cytoskeletal organization. Further studies suggested that impaired beta1 integrin capping and defective integrin-mediated adhesion and proliferation inhibition in CML cells were related to abnormally enhanced integrin-cytoskeletal association and restricted receptor mobility. Finally, interferon alpha, which restores integrin-mediated adhesion and signaling in CML progenitors, also enhanced integrin capping in CD34+ cells. These studies suggest that p210BCR/ABL induces abnormal association of integrin receptors with the cytoskeleton and restricted receptor mobility and provide new insights into mechanisms underlying abnormal integrin function in CML progenitors.

Actins↗
PubMed1999

Outwardly rectifying K+ channels display clustering in guinea pig retinal Müller cells.

The cell-attached configuration of the patch-clamp technique was used to characterize the outward currents in acutely isolated Müller cells from the guinea pig retina. Sixty-five of 353 patches displayed macroscopic, outwardly rectifying currents due to depolarizing voltage steps. Single channel transitions were found in only two patches. The remaining patches did not reveal any voltage-dependent currents. Tail current analysis revealed a reversal potential close to the resting membrane potential. The currents disappeared if internal K+ was replaced by Cs+ in inside-out patches. From these results we conclude that guinea pig Müller cells possess voltage-dependent K+ channels that are distributed in clusters.

Animals↗
PubMed1999

Intracellular mechanisms of L-selectin induced capping.

Leucocyte adhesion to endothelial cells is a tightly regulated process involving selectins, integrins and immunoglobulin-like proteins. Cell adhesion and communication are controlled by membrane dynamics like receptor capping. Capping of surface receptors is an ubiquitous mechanism but still not well understood. Employing immunofluorescence techniques, we demonstrate that L-selectin triggering results in receptor capping of the L-selectin molecules in lymphocytes. Using pharmacological inhibitors and genetic deficient cell lines we show that this process involves intracellular signalling molecules. L-Selectin capping seems to be independent on activation of p56lck-kinase, but requires the neutral sphingomyelinase, small G proteins and the cytoskeleton. Therefore, capping of L-selectin upon stimulation might play an important role in the very early phase of lymphocyte trafficking.

B-Lymphocytes↗
PubMed2000

Tuning chemotactic responses with synthetic multivalent ligands.

BACKGROUND: Multivalent ligands have been used previously to investigate the role of ligand valency and receptor clustering in eliciting biological responses. Studies of multivalent ligand function, however, typically have employed divalent ligands or ligands of undefined valency. How cells respond to multivalent ligands of distinct valencies, which can cluster a signaling receptor to different extents, has never been examined. The chemoreceptors, which mediate chemotactic responses in bacteria, are localized, and clustering has been proposed to play a role in their function. Using multivalent ligands directed at the chemoreceptors, we hypothesized that we could exploit ligand valency to control receptor occupation and clustering and, ultimately, the cellular response. RESULTS: To investigate the effects of ligand valency on the bacterial chemotactic response, we generated a series of linear multivalent arrays with distinct valencies by ring-opening metathesis polymerization. We report that these synthetic ligands elicit bacterial chemotaxis in both Escherichia coli and Bacillus subtilis. The chemotactic response depended on the valency of the ligand; the response of the bacteria can be altered by varying chemoattractant ligand valency. Significantly, these differences in chemotactic responses were related to the ability of the multivalent ligands to cluster chemoreceptors at the plasma membrane. CONCLUSIONS: Our results demonstrate that ligand valency can be used to tune the chemotactic responses of bacteria. This mode of regulation may arise from changes in receptor occupation or changes in receptor clustering or both. Our data implicate changes in receptor clustering as one important mechanism for altering cellular responses. Given the diverse events modulated by changes in the spatial proximity of cell surface receptors, our results suggest a general strategy for tuning biological responses.

Bacillus subtilis↗
PubMed1996

Association between HLA-DM and HLA-DR in vivo.

The exchange of HLA class II-associated invariant chain peptides (CLIP) for cognate peptide is catalyzed by HLA-DM under acidic conditions in vitro by an unknown mechanism. Here, we show an association between HLA-DM and HLA-DR in vivo by coprecipitation of the two heterodimers. The association is favored at low pH and in the nonionic detergent digitonin. Most DM-DR complexes are isolated from dense subcellular fractions. Recovery of HLA-DM by the conformation-dependent DR3 monoclonal antibody 16.23 suggests an association with HLA-DR heterodimers beyond the stage at which CLIP is released. The additional N-linked glycan on mutant DR3 molecules isolated from the 10.24.6 cell line, which interferes with DM-enhanced CLIP release from DR3 in vitro, also affects the DM-DR interaction.

HLA-D Antigens↗
PubMed1999

Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20.

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilization. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 micrograms/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2-3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomolgus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

Animals↗
PubMed1991

Aggregation of IGF-I receptors or insulin receptors and activation of their kinase activity are simultaneously caused by the presence of polycations or K-ras basic peptides.

Several groups including us reported that basic proteins and polycations activate the insulin receptor tyrosine-specific protein kinase (TPK) in vitro. However, some inconsistency has become obvious in the observations. The most intriguing was the brief description by Morrison et al. [(1989) J. Biol. Chem. 264, 9994-10001] that polylysine had no effect on the IGF-I receptor TPK despite its 84% identity to the insulin receptor TPK. In the present study, we used highly purified IGF-I and insulin receptor TPKs in an effort to solve the discrepancies noted in the recent publications and to reveal the mechanism by which polycations stimulate the receptor TPKs. We report that the IGF-I receptor TPK is stimulated by polycations and basic proteins in a manner similar to their effects on the insulin receptor TPK. When effects of polylysine and polyarginine on both receptor TPKs were closely compared, subtle qualitative differences were found: Polylysine stimulated autophosphorylation and exogenous substrate phosphorylation activities of both insulin receptor TPK and IGF-I receptor TPK similarly. In contrast, another polycation, polyarginine, affected both TPKs in a manner quite different from polylysine: Polyarginine stimulated insulin receptor autophosphorylation to a greater extent than polylysine did while it had a very small effect on the IGF-I receptor autophosphorylation as well as the exogenous substrate phosphorylation activities of the two receptor TPKs. We have further extended the studies to include the domains of natural proteins which contain a polylysine-like sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Rotational mobility of high-affinity epidermal growth factor receptors on the surface of living A431 cells.

The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated with a phosphorescent label, erythrosin, or on the subpopulation of high-affinity EGF receptors by using a low concentration of labeled EGF. At 4 degrees C, the rotational correlation times for both the high-affinity and total (mostly low affinity) receptor populations were in the range of 60-100 microns. Elevation of the temperature to 37 degrees C resulted in a lengthening of the rotational correlation time of the total receptor population to 200-300 microns, confirming a previous study of receptor microaggregation. The high-affinity EGF receptors were completely immobilized at 37 degrees C (rotational correlation time greater than 500 microns). The data are consistent with a model involving association of the cytoskeleton with the high-affinity receptors at 37 degrees C, but not at 4 degrees C.

Animals↗
PubMed1990

Synthesis and alpha 2-adrenoceptor effects of substituted catecholimidazoline and catecholimidazole analogues in human platelets.

It is known that the steric requirements for the interactions of catecholamines and catecholimidazolines with alpha 1- and alpha 2-adrenoceptors are different. New analogues of desoxycatecholimidazoline (1), desoxycatecholimidazole (3), benzylic hydroxyl substituted imidazole (4), and the aromatic fluorine substitution analogues of 1 at the 2 (5), 5 (6), and 6 (7) positions, and a set of asymmetric 4-substituted catecholimidazolines, S-8 and R-8, were prepared and tested for interaction with alpha 2-adrenoceptors in human platelets. With the exception of 3, all compounds were selective for alpha-adrenoceptor-mediated responses in human platelets. Introduction of a double bond in imidazoline 1 to give an imidazole 3 or the introduction of a benzylic hydroxyl group to 3, as in 4, reduced the inhibition of platelet aggregation with a rank order potency of 1 greater than 3 greater than 4. Fluorine atom substitution at the 2-, 5-, or 6-positions only slightly modified the inhibitory activity of 1. Each analogue (1, 3-7) produced alpha 2-mediated inhibition of platelet adenylate cyclase and can be classified as a partial agonist. The inhibition potency of S-8 and R-8 against epinephrine-induced aggregatory responses were greatly different, and only R-8 and 4 were alpha 2-agonists on human platelet function. Our studies provide further evidence for the differential interaction of catecholamines and catecholimidazolines in alpha 1- and alpha 2-adrenoceptor systems.

Blood Platelets↗
PubMed2000

Isoxazolines as potent antagonists of the integrin alpha(v)beta(3).

Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.

Cell Adhesion↗
PubMed1990

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.

Antibodies, Monoclonal↗
PubMed2000

Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity.

Integrin adhesion receptors transduce signals that control complex cell functions which require the regulation of gene expression, such as proliferation, differentiation and survival. Their intracellular domain has no catalytic function, indicating that interaction with other transducing molecules is crucial for integrin-mediated signalling. Here we have identified a protein that interacts with the cytoplasmic domain of the beta2 subunit of the alphaL/beta2 integrin LFA-1. This protein is JAB1 (Jun activation domain-binding protein 1), a coactivator of the c-Jun transcription factor. We found that JAB1 is present both in the nucleus and in the cytoplasm of cells and that a fraction of JAB1 colocalizes with LFA-1 at the cell membrane. LFA-1 engagement is followed by an increase of the nuclear pool of JAB1, paralleled by enhanced binding of c-Jun-containing AP-1 complexes to their DNA consensus site and increased transactivation of an AP-1-dependent promoter. We suggest that signalling through the LFA-1 integrin may affect c-Jun-driven transcription by regulating JAB1 nuclear localization. This represents a new pathway for integrin-dependent modulation of gene expression.

Animals↗
PubMed1991

Structural studies of membranes and surface layers up to 1,000 A thick using X-ray standing waves.

The X-ray standing wave (XSW) method, developed in the 1960s, was used originally to determine heavy atom positions in and on silicon and germanium single crystals. An X-ray standing wave generated by the interference of coherent incident and reflected beams excites X-ray fluorescence from the heavy atom, the intensity of which as a function of incident angle provides an indication of the atom's distance from the X-ray reflecting surface. The availability of X-ray mirrors and the ability to prepare layered synthetic microstructures has made possible the study of biologically relevant structures using the XSW technique on length scales of typically tens to hundreds of ångströms, allowing heavy atoms in such structures to be located with ångström or subångström resolution. Many model biological systems (such as Langmuir-Blodgett films, which mimic membranes) require access to still larger scales, but it is not obvious that an XSW will remain coherent over such length scales. Here we report studies of a lipid multilayer system using the XSW method, in which we have been able to locate the metal atoms in a zinc arachidate bilayer with ångström resolution at a distance of almost 1,000 A above the surface of a gold mirror. Our results indicate that the XSW technique should be useful for structural studies of supramolecular aggregates, receptor-ligand interactions and multi-membrane stacks, in which length scales of this order are encountered.

Arachidonic Acids↗

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