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PubMed1995

'Tethered ligand' derived pentapeptide agonists of thrombin receptor: a study of side chain requirements for human platelet activation and GTPase stimulation.

Proteolytic action of alpha-thrombin on human thrombin receptor results in cleavage of a portion of the N-terminus, thereby generating a 'tethered ligand' at the newly exposed N-terminus, which then activates the receptor in an intramolecular fashion. Agonist peptides incorporating the amino acid sequence of the newly exposed N-terminal portion of the cleaved receptor cause receptor activation without requiring prior cleavage of the receptor by thrombin. The pentapeptide amide Ser-Phe-Leu-Leu-Arg-NH2, which retains the N-terminal sequence of the 'tethered ligand' of the receptor, has been shown to be the minimum sequence to cause receptor activation. To understand the importance of the side chains of various residues within the pentapeptide amide, we carried out an extensive structure-activity study of the ability of peptides to stimulate gel-filtered platelet aggregation. In this study 106 pentapeptide amides were synthesized, utilizing naturally occurring L-amino acids, unnatural amino acids, D-amino acids and N-methyl amino acids for replacements. At position-1, charged residues (acidic or basic) were not tolerated, and the size and shape of the residue were important. Position-2 tolerated only aromatic residues. Position-3 accommodated various residues. A significant finding of this study was that two very different residues, [3-(2-naphthyl)]-L-alanine and L-arginine, when substituted for leucine residue at position-3, resulted in more active agonists. At position-4 aromatic and aliphatic residues were well tolerated, whereas basic and acidic residues were less tolerated. Position-5 mimicked position-3 in its ability to tolerate a wide range of residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts.

Epidermal growth factor (EGF) receptors extracted with Triton X-100 from human skin fibroblasts and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin fibroblasts and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = 16 h) than that (t1/2 = 9 h) of fibroblasts. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from 16 to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.

Carcinoma, Squamous Cell↗
PubMed1999

Collagen-platelet interaction: platelet non-integrin receptors.

Platelet-collagen interaction is a complex event that involves ligand-receptor interaction. There are many adhesive non-integrin receptors for platelets to interact with various types of collagens. These non-integrin receptors also serve as signal transducers both from the outside of platelets to the inside and possibly vice versa. The present review covers basic aspects of non-integrin receptor function and various signal transduction pathways.

Animals↗
PubMed1999

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation.

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.

Animals↗
PubMed1994

Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.

Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein beta-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with beta 1 integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells.

Animals↗
PubMed2001

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

Agrin↗
PubMed1999

Platelet glycoprotein IIb/IIIa blockade with tirofiban: effect on aggregation caused by P256, an antibody to human IIb/IIIa receptors.

AIMS: P256 is a divalent antibody which aggregates human platelets by interaction with glycoprotein (GP) IIb/IIIa receptors. We investigated the effect of tirofiban, an antagonist of the GP IIb/IIIa receptor, on P256-mediated platelet aggregation. METHODS: Responses to agonists were measured turbidometrically at 37 degrees C in stirred citrated platelet-rich plasma from venous blood samples from healthy human volunteers. Inhibitory effects were determined by comparison with aggregation to the same concentration of agonist in a vehicle treated sample. RESULTS: Tirofiban inhibited a near maximally effective dose of P256 (10-7 mol l-1 ) with an IC50 of 9. 3x10-8 mol l-1. Tirofiban (10-7 mol l-1 ) inhibited responses to arachidonic acid, U46619 and P256 similarly, whereas aspirin (1. 1x10-4 mol l-1 ) inhibited arachidonic acid more effectively than P256 (P<0.007 by anova ). CONCLUSIONS: Tirofiban potently and selectively inhibits P256-stimulated aggregation of human platelets.

Abciximab↗
PubMed1994

Focal contact assembly through cytoskeletal polymerization: steady state analysis.

For many cell types, initial receptor-mediated attachment to a ligand-coated surface is followed by the formation of focal contacts--strong, specialized, discrete adhesive connections between cell and substrate in which receptors are clustered and simultaneously linked to extracellular ligand and cytoskeletal proteins. Since adhesion affects many aspects of cellular physiology including growth, differentiation, and motility, understanding the biochemical factors which regulate focal contact assembly should enhance our understanding of these phenomena. In this paper, we present a mathematical model to examine how receptor-ligand, receptor-cytoskeleton, and cytoskeleton-cytoskeleton interactions affect the formation of receptor clusters which serve as precursors to mature focal contacts. Receptor clustering is presumed to occur through self-recognition of cytoskeletal elements which induce the polymerization of ligand-receptor-cytoskeleton complexes. Polymerization only occurs when the ligand density is above a critical value and a decrease in the receptor-ligand affinity shifts the critical ligand density to higher values. While cytoskeletal protein expression and receptor-cytoskeleton affinity influence the concentration of monomeric complexes, the formation of polymeric ligand-receptor-cytoskeleton aggregates is most sensitive to changes in the self-association affinity between cytoskeletal proteins. We find that a 100-fold enhancement in the affinity between cytoskeletal elements can produce a substantial increase in the total fraction of adhesion receptors associated with focal contact precursors (from 5% to over 90%). Our results suggest that under physiological conditions, cellular control of focal contact assembly most likely occurs through modulation of specific cytoskeletal proteins to solidify cytoskeleton-cytoskeleton connections within precursor focal contact structures.

Amino Acid Sequence↗
PubMed2004

Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase.

Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin alpha(v) receptor than aggregates. Blocking alpha(v) function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin alpha(v). Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.

Animals↗
PubMedDate not supplied

Death receptor ligands in tumors.

Activation of apoptosis via death receptors is a tightly regulated event, and the death pathway itself is open to interference on the part of soluble or membrane-bound decoy receptors. The aggregation state of the death-inducing ligand is a crucial factor, particularly when these molecules are used as recombinant drugs against tumors. Whether tumors are sensitive to such ligands is determined by both the net abundance of death receptors versus decoy receptors and the balance between intracellular apoptotic and antiapoptotic mechanisms. This means that in vivo elimination of tumor cells by effector arms such as T lymphocytes, natural killer cells, macrophages, and dendritic cells is dependent on both the function of activated lymphoid cells and the genetic properties of tumor cells. Death receptor ligands, however, may be a double-edged sword. When expressed on cytotoxic T lymphocytes, natural killer cells, monocytes, and dendritic cells, they induce the apoptosis of many tumor cells, whereas their expression on tumor cells induces the apoptosis of killer cells. The in vivo result is influenced by the number of infiltrating cells, their state of activation, the cytokine repertoire in the tumor microenvironment, and the ability of the tumor to produce soluble factors inhibiting their cytolytic functions.

Animals↗
PubMed1995

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.

Animals↗
PubMed1976

The specificity of the Fc receptor on murine lymphocytes for immunoglobulins of the IgG and IgM classes.

It is shown that the Fc receptor for IgG on lymph node cells recognizes IgG molecules of mouse IgG1, IgG2a and IgG2b subclasses. The presence of an intact CH3 domain is required to bind IgG to the Fc receptors. While monomeric IgG molecules are shown to bind to Fc receptors, heat aggregated IgG was found to bind more efficiently. The various forms of IgM molecules investigated show only weak or no binding in comparison. The significance of these results is discussed with relevance to the possible biological function of the Fc receptors on lymphocytes.

Animals↗
PubMed1998

Engagement of ICAM-3 activates polymorphonuclear leukocytes: aggregation without degranulation or beta 2 integrin recruitment.

ICAM-3 is a preferred counterreceptor for the leukocyte alpha(L)beta2 integrin. It activates T cells through outside-in signaling, but polymorphonuclear leukocytes (PMN) are reported to be refractory to ICAM-3 stimulation. We found that engagement of ICAM-3 by a mAb (CAL3.10), which binds in the region where alpha(L)beta2 integrin binds, activates PMN homotypic aggregation and adhesion to surfaces. These functional changes were due to ICAM-3 outside-in signaling because aggregation and adhesion were beta2 integrin-dependent, tyrosine kinase and protein kinase C activities were activated, and there was a reorganization of the cytoskeleton. This reorganization and kinase activity was required for ICAM-3-, but not FMLP-, induced aggregation. This is not an Fc-mediated event as an appropriate anti-ICAM-3 F(ab')2 fragment still induced aggregation. Another anti-ICAM-3 Ab (HP2/19), which activates T cells, did not activate PMN. Strikingly, anti-ICAM-3 did not induce degranulation or cause an increase in surface beta2 integrin expression, so adhesion and aggregation were due solely to the activation of the constitutively expressed beta2 integrins. Aggregation in response to ICAM-3, but not FMLP, was compromised at lower cell densities, showing that beta2 integrin recruitment enhances aggregation under suboptimal conditions. We conclude that engagement of ICAM-3 stimulates PMN as well as T cells, but that the appropriate epitope varies between these two cells. ICAM-3 outside-in signaling reorganizes the cytoskeleton without causing degranulation, induces serine and tyrosine kinase activation, and activates existing surface beta2 integrins to a proadhesive state.

Antibodies, Monoclonal↗
PubMed1992

Nerve growth factor (NGF) receptor on rat glial cell lines. Evidence for NGF internalization via p75NGFR.

Two types of nerve growth factor (NGF) receptors have been described: high affinity (class I) and low affinity (class II). Biological responses to NGF are thought to be mediated by class I receptors, whereas the role of class II receptors is less clear. While some neuronal cells express both receptor types, only class II receptors have been detected on glial cells. Two glial cell lines, peripheral Schwannoma D6P2T and central 33B glioma cells, were employed to investigate the properties of class II receptors in the absence of class I receptors. These cell lines were found to express NGF receptors identified as class II by a low nanomolar dissociation constant, rapid dissociation kinetics at 4 degrees C, and trypsin sensitivity. The receptor was found to bind brain-derived neurotrophic factor with similar affinity as NGF. The responsible binding molecule appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a heterogeneously glycosylated protein of 60-80 kDa with a tendency to aggregate. All receptor bands affinity-labeled with radioiodinated NGF were immunoprecipitated with anti-p75NGFR antibody, but not with anti-p140prototrk antiserum. In these cells, which express p75NGFR as only NGF receptor, a time- and temperature-dependent appearance of a nondisplaceable, trypsin-resistant, acid wash-stable ligand fraction, followed by an increase of trichloroacetic acid-soluble radiolabel in the medium was observed. This sequestration resembled receptor-mediated internalization with subsequent degradation of NGF. Whether this ligand processing indicates a functional role of p75NGFR in glial cells remains to be shown.

Animals↗