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PubMed2006

Determinants of chemoreceptor cluster formation in Escherichia coli.

Chemotactic stimuli in bacteria are sensed by large sensory complexes, or receptor clusters, that consist of tens of thousands of proteins. Receptor clusters appear to play a key role in signal processing, but their structure remains poorly understood. Here we used fluorescent protein fusions to study in vivo formation of the cluster core, which consists of receptors, a kinase CheA and an assisting protein CheW. We show that receptors aggregate through their cytoplasmic domains even in the absence of other chemotaxis proteins. Clustering is further enhanced by the binding of CheW. Surprisingly, we observed that some fragments of CheA bind receptor clusters well in the absence of CheW, although the latter does assist the binding of full-length CheA. The resulting mode of receptor cluster formation is consistent with an experimentally observed flexible stoichiometry of chemosensory complexes and with assumptions of recently proposed computer models of signal processing in chemotaxis.

Bacterial Proteins↗
PubMed1987

Transduction of signals in the activation of T lymphocytes: relation to leukemia.

The biochemical events initiated by mitogen in T lymphocytes are the subject of this paper. Following interaction of the mitogen with its receptors, a transmembrane 'trigger-type' signal is propagated which has both positive and negative correlates. The negative signal occurs with high mitogen concentrations and is associated with membrane freezing, microtubular aggregation, receptor capping, adenylate cyclase activation, and cellular cyclic AMP increases. The positive signal occurs with optimal mitogen concentrations and is associated with changes in membrane permeability and transport with influx of calcium and potassium ion and efflux of sodium, in transport processes for glucose, amino acids, and nucleosides, and in a collected series of early membrane lipid changes which can be considered essential for the positive signal. These lipid changes include the uptake of arachidonic acid and other fatty acids, choline, phosphate and other molecules, their incorporation into membrane phospholipids, particularly phosphatidylinositol (PI), and a turnover of PI with the production of inositol triphosphate, which can be related to calcium mobilization and diacylglycerol which activates a cytoplasmic protein kinase C. A key event associated with mitogen action is arachidonic acid release. Arachidonic acid may give rise to prostaglandins and thromboxanes as part of negative components of the signal through effects on the adenylate cyclase/cyclic AMP system. Arachidonic acid gives rise to eicosanoids like 5-, 11-, possibly 12- and 15-hydroxyperoxy and hydroxy eicosatetraenoic acids and leukotrienes B4 and C4. The activation of the 5-lipoxygenase, a critical calcium-dependent step, leads via the production of 5-HPETE and 5-HETE to the activation of membrane and soluble guanylate cyclase and the production of cyclic GMP. Cyclic GMP appears to be essential for mitogen activation and is associated with cyclic GMP-dependent protein kinase activation and the phosphorylation of a number of substrates. Calcium ion influx is clearly central to mitogen action. Calcium through its influx and mobilization from cellular stores is thought to contribute directly and indirectly through the action of calmodulin and protein kinase C to the activation of a number of enzymatic processes involved in the positive signal including phospholipase C, diglyceride kinase and lipase, 5-lipoxygenase, and guanylate cyclase. Cyclic GMP and calcium ion both participate in nuclear processes leading to RNA and protein synthesis. Interleukin 2 is associated with midcycle increases in cyclic GMP and entry into DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗
PubMed2001

Role of chaperones in nuclear translocation and transactivation of steroid receptors.

Molecular chaperones assist proteins to reach their mature and functional conformation. It has become apparent in recent years that chaperones function as part of a multiprotein heterocomplex that is potentially involved not only in protein folding, but also in intracellular trafficking and in targeting proteins for degradation. In the case of steroid receptors, the activity of the chaperone heterocomplex, as well as the proteins comprising the heterocomplex, has an effect on the observed ligand-dependent transcriptional activity of the receptor. The direct interaction between chaperones and steroid receptors makes them potential therapeutic targets in a number of pathologic conditions. In the case of cancers with steroid receptor involvement, such as breast and prostate cancer, the inhibition of chaperone activity may inhibit tumor cell growth. Conversely, enhancement of chaperone activity may be beneficial in disorders of protein misfolding, as in the case of androgen receptor aggregates found in Spinal and Bulbar Muscular Atrophy.

Biological Transport↗
PubMed1996

Changes in the composition of the platelet cytoskeleton in response to ADP: effects of MK-852 and ARL 66096.

Platelet activation by adenosine diphosphate (ADP) results in an alteration in the composition of the cytoskeleton. Here we have determined the effects of MK-852 and ARL 66096 on the cytoskeletal changes that occur. MK-852 is a GPIIb/IIIa antagonist that inhibits aggregation by interfering with fibrinogen binding ARL 66096 is a P2T antagonist that selectively inhibits ADP-induced aggregation. Neither agent inhibits the shape change response. Experiments were performed in hirudinized platelet-rich plasma. Platelet activation led to a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin, alpha-actinin, a 66K protein and actin, and a significant decrease in a 31K protein. In the presence of MK-852 there was no increase in ABP or the 66K protein and no decrease in the 31K protein. The increase in myosin and alpha-actinin became reversible but there was still incorporation of actin into the cytoskeleton. In the presence of ARL 66096 there was no increase in ABP or the 66K protein and no decrease in the 31K protein. ARL 66096 also prevented incorporation of alpha-actinin and actin. As with MK-852, myosin incorporation became reversible. The results suggest that (1) myosin is incorporated into the cytoskeleton transiently during shape change, (2) ADP interaction with the ADP aggregation receptor (but not that for shape change) is associated with alpha-actinin and actin incorporation into the cytoskeleton, and (3) further changes that occur are consequent to fibrinogen binding and platelet aggregation.

Actinin↗
PubMed1987

Identification of a very large nuclear estrogen receptor complex.

A high mol wt estrogen receptor complex (mol wt: greater than 669,000) has been isolated from chromatin prepared from mouse uteri with the use of multiple protease inhibitors, exposure to DNAase I, and size exclusion HPLC (SEHPLC). This complex is stable in the presence of molybdate and 0.4 M KCl. Because receptors in the cytosol do not elute similarly to this large complex when investigated under a wide variety of conditions, it is unlikely that random receptor aggregation is responsible for the formation of this nuclear complex. Consequently, this complex may represent a collection of chromatin components that participate in the mechanisms of hormonal regulation. Since the large nuclear receptor complex can be isolated, this hypothesis can be readily subjected to extensive investigation.

Animals↗
PubMed1999

Decay-accelerating factor and cytoskeleton redistribution pattern in HeLa cells infected with recombinant Escherichia coli strains expressing Dr family of adhesins.

Escherichia coli strains expressing Dr fimbriae are able to enter epithelial cells by interacting with a complement-regulatory protein, decay-accelerating factor. This model of bacterial internalization, with a well-characterized bacterial ligand and host receptor, provides a unique opportunity to investigate the early stages of invasion. We used immunofluorescence staining techniques to examine the distribution of receptor and cytoskeletal proteins in HeLa cells infected with E. coli recombinant strains that expressed Dr family of adhesins: Dr, Dr-II, F1845, AFA-I, and AFA-III. A major rearrangement of decay-accelerating factor was found at the adherence sites of recombinant strains expressing Dr, Dr-II, and F1845 adhesins. The changes in the distribution of receptor were significantly smaller on HeLa cells infected with E. coli bearing AFA-I or AFA-III afimbrial adhesins. Receptor aggregation was associated with the redistribution of cytoskeleton-associated proteins such as actin, alpha-actinin, ezrin, and occasionally tropomyosin. Purified Dr fimbriae coated on polystyrene beads were capable of triggering clustering of receptor and accumulating actin at the adhesion sites of beads to HeLa cells. Using scanning and transmission electron microscopic techniques, we have shown that beads coated with Dr fimbriae, as opposed to beads coated with bovine serum albumin, were enwrapped by cellular microvilli and ultimately internalized into HeLa cells. This indicates that interaction of Dr fimbriae with decay-accelerating factor is associated with redistribution of receptor and is sufficient to promote bacterial internalization.

Actins↗
PubMed1998

Surface aggregation patterns of LDL receptors near coated pits. I. The radially convective diffusion and generalized insertion mechanism.

In this paper we formulate a mathematical model for the receptor mediated endocytotic cycle under the influence of diffusion, radial convection, and generalized receptor reinsertion. The steady state radial concentration function of unbound receptors admits an explicit representation. This can be expressed as a functional of the insertion rate, the diffusion coefficient, and the flow strength. Using the referred functional we study the influence of the aforementioned mechanisms on the surface aggregation pattern of low density lipoprotein (LDL) receptors near coated pits. We perform that analysis on both a theoretical level and by means of simulated receptor aggregation patterns obtained by computer graphics techniques. We conclude that radially convective diffusion in combination with suitable characterizations of the insertion mode are consistent with reported cell surface aggregation patterns.

Coated Pits, Cell-Membrane↗
PubMed1976

Cooperative properties of hormone receptors in cell membranes.

The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady-state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.

Animals↗
PubMed1984

Estrogen receptor transformation to a high-affinity state without subunit-subunit interactions.

We have tested the ability of monomeric rat estrogen receptor (generated by dissociating the native receptor aggregate with KCl and binding to hydroxylapatite) to undergo heat-induced transformation to a state with altered chromatographic properties and estradiol dissociation kinetics. Participation of nonadsorbed cytosolic components in the transformation process was eliminated by washing the adsorbed receptor prior to incubation at 28-30 degrees C for 30 min. We found that monomeric estradiol-receptor complexes can be converted to a form which coelutes with transformed receptor during hydroxylapatite chromatography and which has a slow estradiol dissociation rate characteristic of transformed receptor. Monomeric receptor does not, however, display cooperative ligand-binding behavior, strongly suggesting adsorbed monomers cannot dimerize or interact with other adsorbed cytosol proteins. Maintenance of the monomer form was further verified by the sedimentation of eluted receptor solely as a 4S species. Although conversion of monomers to the slow-dissociating state was incomplete (congruent to 80%) on hydroxylapatite, eluted receptor showed only a slow dissociation rate component. The data are consistent with a model in which receptor monomers are in equilibrium between low-affinity and high-affinity conformations. Dimerization of the high-affinity receptor may shift the equilibrium to greatly favor the high-affinity (transformed) state.

Animals↗
PubMed1989

Receptor cross-linking restores an insulin metabolic effect altered by mutation on tyrosine 1162 and tyrosine 1163.

The pivotal role that the tyrosine residues in positions 1162 and 1163 play in the control of the insulin action has been clearly established by substitution of these tyrosine residues for phenylalanine [Ellis, L. (1986) Cell 45, 721-732]. We have recently found that this type of mutation, which abolishes the effects of insulin on glucose metabolism, was without any effect on the mitogenic effect of the hormone [Debant, A. (1988) Proc. Natl. Acad. Sci. U.S.A. (in press)]. Here, we provide evidence that a polyclonal antibody, raised against the human insulin receptor, can restore the receptor-mediated stimulation of glycogen synthesis that was abolished by the mutation. Stimulation of the biological effect by the anti-receptor antibody did not necessitate, whatsoever, the activation of the tyrosine kinase activity and/or receptor autophosphorylation. Furthermore, the antibody-induced reversal of the mutation was not observed when we used Fab fragments alone, but addition of anti-(Fab')2 IgG in a second step resulted in a similar effect as that observed with intact IgG. We propose that Tyr 1162 and Tyr 1163 exert their control on the metabolic effects of insulin through the modulation of receptor aggregation.

Animals↗
PubMed1988

Activation of rat basophilic leukemia cells. Temporal identification of the signal calcium influx mediated by the receptor-operated channel pathway.

Antigen-induced 45calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45Ca2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE-receptor occupancy at low receptor occupancies, and is sensitive to the anti-allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin-2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45Ca2+ uptake curve. These qualities suggest that the early peak at 2-3 min, rather than any later 45Ca2+ uptake, comprises the initial signalling Ca2+ pool. Maximal apparent inhibition by cromolyn for 45Ca2+ uptake was about 65% and required a 10-15-min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5-6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn-binding protein [Corcia, A. et al. (1986) EM BO J. 5, 849-854]. This, as well as the effects of cromolyn, support the assumption that cromolyn-binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.

Animals↗
PubMed2002

The adapter molecule Gab2 regulates Fc epsilon RI-mediated signal transduction in mast cells.

The recently cloned scaffolding molecule Gab2 can assemble multiple molecules involved in signaling pathways. Bone marrow-derived mast cells isolated from Gab2(-/-) mice have defective signaling probably due to the lack of the activation of phosphatidylinositol-3 kinase (PI3-kinase). In this study, we investigated the role of Gab2 using the rat basophilic leukemia 2H3 cell line mast cells. Fc epsilon RI aggregation induced the tyrosine phosphorylation of Gab2 and translocation of a significant fraction of it from the cytosol to the plasma membrane. As in other cells, Gab2 was found to associate with several signaling molecules including Src homology 2-containing protein tyrosine phosphatase 2, Grb2, Lyn, and phospholipase C gamma (PLC gamma). The association of Gab2 with Lyn and PLC gamma were enhanced after receptor aggregation. Overexpression of Gab2 in rat basophilic leukemia 2H3 cell line cells inhibited the Fc epsilon RI-induced tyrosine phosphorylation of the subunits of the receptor, and the phosphorylation and/or activation of Syk and mitogen-activated protein kinase. Downstream events such as calcium mobilization, degranulation, and induction of TNF-alpha and IL-6 gene transcripts were decreased in Gab2 overexpressing cells, although Akt phosphorylation as a measure of PI3-kinase activation was unaffected. These results suggest that in addition to the positive effects mediated by PI3-kinase that are apparent in Gab2(-/-) mast cells, Gab2 by interacting with Lyn and PLC gamma may have negative regulatory effects on Fc epsilon RI-induced mast cell signaling and functions.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Quantification of G-Protein Coupled Receptor Internatilization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScantrade mark High-Content Screening System.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScantrade mark (Cellomicstrade mark, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathyroid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a beta(2)-adrenergic receptor (beta(2) AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate ³spots² within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

Journal Article↗
PubMed1995

Glial endothelin/nitric oxide system participates in hippocampus CA1 neuronal death of SHRSP following transient forebrain ischaemia.

1. When delayed neuronal death occurred in the hippocampus CA1 pyramidal cell layer of stroke-prone spontaneously hypertensive rats (SHRSP) at 4 and 7 days after a 10 min bilateral carotid occlusion and reperfusion, intense endothelin-1 (ET-1)- and ET-3-like immunoreactivities became evident in astrocytes in the damaged hippocampus CA1 subfields. 2. We also observed that microglia equipped with an ETB receptor aggregated within the CA1 pyramidal cell layer with neuronal death. 3. There was a dramatic increase in nitric oxide synthase (NOS) activity in astrocytes and microglia in the damaged hippocampus CA1 subfields. 4. Thus, the possibility that microglia with the ETB receptor are activated to produce NO, a neurotoxic factor, by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischaemia would have to be considered.

Animals↗
PubMed1983

Temperature-dependent inactivation of nucleic acid binding and aggregation of the 1,25-dihydroxyvitamin D3 receptor.

The interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with immobilized calf thymus DNA has been compared with its sedimentation properties on hypotonic sucrose gradients. Forty to sixty percent of total hormone:receptor complexes formed at 4 degrees C were retained by DNA-cellulose and could be eluted by 0.18 to 0.2 M KCl. In contrast, heating preparations to 25 degrees C rapidly and irreversibly converted receptor to a form which bound hormone and DEAE-cellulose normally, but was unable to associate with DNA. Similarly, the ability of receptor to aggregate to a 6 S species was labile at 25 degrees C. Stabilization of receptor in the DNA binding aggregating form was accomplished using Ca2+, Mg2+, Mn2+, or Na2MoO4 while several protease and phosphatase inhibitors were ineffective. An examination of DNA binding properties of aggregating and nonaggregating receptor forms revealed that only receptor competent to enter into aggregates could bind DNA suggesting that a functional nucleic acid binding site, and, hence, a nucleic acid interaction is necessary for aggregate formation. Consistent with this view, an RNA:receptor interaction appears to be involved in formation of the 6 S complex since removal of RNA by ribonuclease treatment or purification of receptor reduced aggregation, an effect that could be reversed by addition of purified RNA.

Animals↗
PubMed2001

Pharmacodynamics and pharmacokinetics of higher-dose, double-bolus eptifibatide in percutaneous coronary intervention.

BACKGROUND: Pharmacodynamics of eptifibatide, a cyclic heptapeptide antagonist of platelet glycoprotein IIb/IIIa, are substantially altered by anticoagulants that chelate calcium, resulting in overestimation ex vivo of the in vivo effects of this agent. We conducted a dose-ranging study to characterize the pharmacodynamics and pharmacokinetics of eptifibatide under physiological conditions. METHODS AND RESULTS: Patients (n=39) undergoing elective percutaneous coronary intervention were randomly assigned to an eptifibatide bolus followed by an infusion (180-microgram/kg bolus followed by 2 microgram/kg per minute or 250-microgram/kg bolus followed by 3 microgram/kg per minute) for 18 to 24 hours. In a 2:1 ratio, these patients received either a second bolus of eptifibatide (90 microgram/kg or 125 microgram/kg for the initial 180-microgram/kg or 250-microgram/kg groups, respectively) or placebo 30 minutes after the initial bolus. Bleeding times, ex vivo platelet aggregation, receptor occupancy, and plasma eptifibatide levels at baseline and at 1, 2, 3, 4, 6, and 8 hours were evaluated. Platelet inhibition was dose dependent and >80% in all groups by steady state. The single-bolus regimens had a transient loss of inhibition at 1 hour, consistent with rapid distribution and drug elimination. Pharmacokinetic modeling suggested that optimal dosing of eptifibatide would be obtained with a 180-microgram/kg bolus and a 2-microgram/kg per minute infusion followed by a second 180-microgram/kg bolus 10 minutes later. CONCLUSIONS: A novel higher-dose, double-bolus regimen of eptifibatide in coronary intervention attains and maintains >90% inhibition of platelet aggregation in >90% of patients, providing the pharmacodynamic construct for the design of the Enhanced Suppression of the Platelet IIb/IIIa Receptor with Integrilin Therapy (ESPRIT) trial of adjunctive eptifibatide in coronary stent implantation.

Angioplasty, Balloon, Coronary↗
PubMed1999

Membrane organization in immunoglobulin E receptor signaling.

The structure and dynamics of the plasma membrane are proposed to be critical for the initial steps of signal transduction by the high-affinity immunoglobulin E receptor. Recent experimental advances indicate that interactions between the high-affinity immunoglobulin E receptor and the tyrosine kinase Lyn with cholesterol- and sphingolipid-rich regions within the plasma membrane are important for receptor function. This accumulating evidence points to spatio-temporal control of immunoglobulin E receptor signaling by the organization of the plasma membrane; an attractive hypothesis is that ligand-dependent receptor aggregation causes the segregation of Lyn-containing ordered regions of the plasma membrane from disordered regions.

Animals↗
PubMed1980

Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine.

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.

Animals↗