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PubMed2000

Cytoplasmic localization and the choice of ligand determine aggregate formation by androgen receptor with amplified polyglutamine stretch.

Polyglutamine tract expansion in androgen receptor is a recognized cause of spinal and bulbar muscular atrophy (SBMA), an X-linked motor neuronopathy. Similar mutations have been identified in proteins associated with other neurodegenerative diseases. Recent studies have shown that amplified polyglutamine repeat stretches form cellular aggregates that may be markers for these neurodegenerative diseases. Here we describe conditions that lead to aggregate formation by androgen receptor with polyglutamine stretch amplification. In transfection experiments, the mutant, compared with the wild-type receptor, was delayed in its cytoplasmic-nuclear translocation and formed large cytoplasmic aggregates in the presence of androgen. The cytoplasmic environment appears crucial for this aggregation, since retention of both the wild-type and mutant receptors in this cellular compartment by the deletion of their nuclear localization signals resulted in massive aggregation. Conversely, rapid nuclear transport of both receptors brought about by deletion of their ligand binding domains did not result in aggregate formation. However, androgen antagonists that altered the conformation of the ligand binding domain and promoted varying rates of cytoplasmic-nuclear translocation all inhibited aggregate formation. This demonstrates that in addition to the cytoplasmic localization, a distinct contribution of the ligand binding domain of the receptor is necessary for the aggregation. The finding that antiandrogens inhibit aggregate formation may provide the basis for in vivo determination of the role of these structures in SBMA.

Animals↗
PubMed1977

Studies of the mechanism of the human platelet release reaction induced by immunologic stimuli. III. Relationship between the binding of soluble IgG aggregates to the Fc receptor and cell response in the presence and absence of plasma.

Aggregated IgG coupled covalently with bis-diazobenzidine (BDB-IgG) and labeled with 3H-diazobenzene (3H-BDB-IgG) has been used to study the binding of soluble IgG aggregates to human platelets in relationship to the release of the contents of intracellular granules (e.g., serotonin). In washed cell suspensions a minimum of 0.14 to 0.2 mug 3H-BDB-IgG per 5 X 10(8) platelets (40 to 70 aggregates per cell) was required for the triggering of the release reaction and cell aggregation. Binding was independent of divalent cations. The Arrhenius plot gave a straight line between 0 to 37 degrees C and a Q10 of 1.6. Neither inhibitors of the release reaction nor energy metabolism, nor formaldehyde fixation of the platelets affected binding. Bound 3H-BDB-IgG was not significantly eluted by IgG, bovine albumin (BSA), buffer, or plasma. Binding to washed platelets was more strongly inhibited by human IgG than by F(ab')2, bovine IgG, human albumin (HSA), or BSA. Plasma was an even more effective inhibitor of both binding and release. Plasma deficient in IgG or depleted of complement retained its inhibitory capacity. In the presence of plasma, at physiologic ratios of plasma and platelets, no release of serotonin was observed. Binding, although inhibited in rate, nevertheless occurred. It was enhanced by divalent cation chelation and had a Q10 of 2.5. The release reaction of washed platelets to which 3H-BDB-IgG had been bound in the presence of HSA or BSA was also inhibited by the subsequent addition of plasma or plasma proteins (human IgG being more effective than bovine IgG, F(ab')2, HSA, or BSA). 3H-BDB-IgG bound in the presence of either plasma or human IgG did not induce release when the platelets were subsequently suspended in media lacking these proteins. Thus, it appears that the platelet Fc receptor binds 3H-BDB-IgG by a process which is effectively inhibited by plasma, or by free IgG with an intact Fc, and to some extent by high concentrations of other proteins. The effects of bound IgG aggregates are dependent on the other proteins present both during binding and subsequently added. The mechanism of such receptor modulation and its implications in vivo are discussed.

Antigen-Antibody Complex↗
PubMed1995

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors.

Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP-induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP-induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca2+ influx. Binding studies, using the non-hydrolysable ligand [33P]2MeSADP, have shown that human platelets contain about 600 high-affinity binding sites for 2MeSADP (Kd approximately 5 nM). These sites present pharmacological characteristics of a P2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP-induced platelet aggregation but normal shape change display very low levels of [33P]2MeSADP binding sites. The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca2+ influx and the other a Gi protein-coupled receptor responsible for Ca2+ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.

Adenosine Diphosphate↗
PubMed2001

Adaptor ADAP (adhesion- and degranulation-promoting adaptor protein) regulates beta1 integrin clustering on mast cells.

Aggregation of the high-affinity IgE receptor (FcepsilonRI) activates a tyrosine phosphorylation cascade needed for enhanced adhesion and degranulation events on mast cells. We previously identified the adaptor ADAP (otherwise known as FYB/SLAP) as a modulator of integrin-mediated adhesion of T-cells and mast cells. However, the molecular basis for the effect on beta1 integrin adhesion on mast cells was unclear. In this study, we demonstrate that ADAP can promote the kinetics of beta1 integrin clustering on mast cells. By contrast, the clustering of the FcepsilonRI receptor was unaffected by transfected ADAP, indicating that the promoting effect on clustering was selective for beta1 integrins. These findings extend the modulatory effect of ADAP from beta2 to beta1 members of the integrin family, and provide a molecular basis for the modulatory effect of ADAP on mast cell adhesion.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Human anti-streptokinase antibodies induce platelet aggregation in an Fc receptor (CD32) dependent manner.

Exposure to streptokinase (SK) elicits anti-SK antibodies (Abs), which inhibit fibrinolysis and induce platelet aggregation. The mechanism of the latter is not fully understood, although it seems to involve platelet binding by a plasminogen streptokinase and anti-SK ternary complex. Anti-SK Abs were purified by affinity chromatography from serum of patients having received SK for acute myocardial infarction (AMI), and were shown to be of the IgG type. Their effects were studied with (i) human platelets in citrated plasma in the presence of SK or acetylated plasminogen-SK activator complex (APSAC), and (ii) in washed platelets, resuspended in Tyrode buffer after lowering the ionic strength, in the presence of APSAC (which provides both SK and plasminogen). An antibody concentration-response curve was obtained, showing a plateau in the presence of 0.1 mg/ml IgG. By increasing the concentration of APSAC, we obtained a unimodal response curve, the optimal concentration of APSAC being 0.05 U/ml. Aggregation was suppressed by chelating calcium with EDTA, blocking fibrinogen binding by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), and raising intraplatelet cAMP with Iloprost (a prostacyclin analogue). Aggregation required the interaction of the anti-SK Ab Fc domain with the platelet Fc-gamma receptor type II, also known as CD32, since: (i) it was blocked by the monoclonal antibody IV-3 directed against CD32, (ii) it did not occur with F(ab)'2 fragments, which block the response to the intact IgG. The clinical relevance of these platelet-activating anti-SK antibodies remains to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation, deaggregation and shape change responses mediated by the purinergic P2Y1 receptor in vitro.

Platelet shape change (SC), aggregation and deaggregation responses are integral components of hemostasis that are elicited and modulated in vivo by the simultaneous activation of several membrane receptors. Selective activation of the purinergic P2Y1 receptor in vivo elicits a sustained SC and a small, transient aggregation response that is reversed rapidly by a robust deaggregation response (Platelets 2003; 14: 89). Using a kinetics-based turbidimetric approach to study the modulation of these concurrent components of human platelet responses, we demonstrate that these P2Y1 receptor-related responses and a number of their kinetic and steady-state characteristics are differentially elicited and modulated. P2Y1 receptor agonist concentrations that elicited aggregation (pEC50 for ADP, 2-MeSADP; 5.88, 6.69) were 10-fold greater than those that elicited SC (7.33, 7.67). The magnitude of the aggregation response was agonist concentration-dependent, saturable and was associated with an agonist concentration-dependent deceleration of the deaggregation response. Gi-coupled receptor (alpha 2A-adrenoceptor, EP3 and P2Y12 receptors) agonists also enhanced aggregation through deceleration of the deaggregation response, and an inhibitor of PI3K activity (wortmannin) inhibited aggregation through acceleration of the deaggregation response. Neither treatment affected the extent or the kinetics of the SC response. The aggregation but not the SC response was rapidly desensitized by P2Y1 receptor activation by ADP. The affinity of the presence of a single P2Y1 receptor subtype. The differential characteristics and modulation of the SC and aggregation responses by a single receptor support the idea that different signaling pathways activated at different occupancy states of the same receptor underlie the two responses. P2Y1 receptor-mediated platelet aggregation and SC responses provide a convenient model for studying the phenomenon of agonist-directed signaling by differential occupancy of the same membrane receptor.

Adenosine Diphosphate↗
PubMed1997

The unique domain as the site on Lyn kinase for its constitutive association with the high affinity receptor for IgE.

Aggregation of the high affinity receptor for IgE (FcepsilonRI) leads to the phosphorylation of tyrosines on the beta and gamma chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcepsilonRI in vivo using a transfection-based approach. FcepsilonRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcepsilonRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the beta chain but not with the receptor's other cytoplasmic domains.

Animals↗
PubMed1981

Receptors for fibrinogen and aggregated beta 2-microglobulin detected in strains of group B streptococci.

Binding of radiolabeled human fibrinogen and aggregated beta-microglobulin was measured in 60 strains of beta-hemolytic group B streptococci. Positive fibrinogen binding was detected in seven of the strains. Six of the group B strains showed an uptake of aggregated beta 2-microglobulin. Four individual strains carried both receptors, indicating a positive correlation between their occurrence. Inhibition studies showed that fibrinogen competed sterically with beta 2-microglobulin binding. Receptors for both proteins were trypsin sensitive. The presence of receptors did not correlate with the serological type of the 49 group B strains tested. However, all seven type II strains were negative. No uptake of fibrinogen was noted in any of 40 group D strains tested. Binding structures for fibrinogen and aggregated beta 2-microglobulin detected in group B streptococci were similar to receptors for the same proteins in group A, C, and G streptococci in terms of mutual correlation and steric interference of binding. The occasional occurrence of these receptors also in group B strains might reflect a common origin of some types of surface proteins in gram-positive cocci.

Beta-Globulins↗
PubMed2001

Identification, characterization, and inhibition of the platelet ADP receptors.

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis, and its receptors are potential targets for antithrombotic drugs. Two G-protein-coupled P2 receptors contribute to platelet aggregation: the P2Y1 receptor initiates aggregation through mobilization of calcium stores, whereas the P2Y12 receptor coupled to adenylyl cyclase inhibition is essential for a full aggregation response to ADP and the stabilization of aggregates. The latter is defective in certain patients with a selective congenital deficiency of aggregation to ADP. It is also the target of the antithrombotic drug clopidogrel and of adenosine triphosphate analogues and other compounds currently under evaluation. In addition, the P2X1 ionotropic receptor is present in platelets, but its role is not yet completely known. Studies in P2Y1-knockout mice and experimental thrombosis models using selective P2Y1 antagonists have shown that the P2Y1 receptor, like the P2Y12 receptor, is a potential target for new antithrombotic drugs.

Animals↗
PubMed1999

Thermally induced aggregation of human transferrin receptor studied by light-scattering techniques.

The thermal stability of transferrin receptor isolated from human placenta in detergent-free solution has been investigated by static light-scattering and photon correlation spectroscopy. In detergent-free solution at 293.2 K, human transferrin receptor (hTfR) forms stable associates with a hydrodynamic radius of 16 nm. With increasing temperature the particles get more compact, above 340 K a phase transition takes, place and spontaneous aggregation of the receptor occurs. Under these conditions large clusters are formed that lead to fractal aggregates, coexisting with dendritic crystalline structures. The experimental findings are compatible with a model, which involves a reaction limited cluster-cluster aggregation mechanism in conjunction with a nucleation process. The molar enthalpy change associated with the phase transition was determined to be (1860 +/- 150) kJ/mol(-1) at a transition temperature of (341.3 +/- 0.2) K.

Biophysical Phenomena↗
PubMed1991

Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein.

Agrin is a synapse-organizing protein that is concentrated in embryonic motor neurons and the synaptic basal lamina of the neuromuscular junction. Agrin or closely related proteins are also associated with most other basal laminae. Here I report that the major agrin-like proteins from the nervous system and other tissues of the chicken are immunochemically and biochemically similar. Four major agrin-like proteins of approximately 60, 72, 80, and 90 kDa were identified on immunoblots of agrin preparations from both neural and non-neural tissues. Agrin-like proteins from embryonic chicken brain and adult kidney were similar in amino acid composition. Rabbit antisera against each of the kidney proteins labeled basement membranes of several tissues, as well as spinal cord motor neurons. These antibodies specifically precipitated and inhibited acetylcholine receptor (AChR)-aggregating activity from the chicken nervous system and Torpedo electric organ. Thus, the agrin-like proteins of non-neural tissues in the chicken are closely related to agrin from the nervous system. However, the AChR-aggregating activity of chicken agrin preparations differed depending on the tissue of origin. Agrin enriched by immunoaffinity chromatography from the central nervous system induced large numbers of AChR aggregates on cultured myotubes. In contrast, agrin preparations from other chicken tissues induced dramatically fewer and smaller AChR aggregates. The difference in biological activity between these agrin preparations may reflect differential inactivation or the existence of tissue- or cell-specific isoforms of agrin.

Agrin↗
PubMed2000

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: refinements and applications.

Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve-muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better-defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon-induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon-induced AChR aggregation on muscle cell species, by the use of chick-rat chimeric co-cultures. (5) We provide evidence for the role of alternatively-spliced agrin isoforms in synapse formation by using single cell RT-PCR with neurons collected from co-cultures after observation of axon-induced AChR aggregation. Microsc. Res. Tech. 49:26-37, 2000. Published 2000 Wiley-Liss, Inc.

Agrin↗
PubMed1999

Regulation of the size and distribution of agrin-induced postsynaptic-like apparatus in adult skeletal muscle by electrical muscle activity.

We compared actylcholine receptor (AChR) aggregates induced by neural agrin released from transfected muscle fibers with AChR aggregates induced by transplanted axons in extrajunctional regions of denervated rat soleus muscles. Both neural agrin and transplanted axons induced multiple, irregularly distributed AChR aggregates on muscle fibers. Direct electrical muscle stimulation of transfected muscles for up to 10 weeks removed all agrin-induced AChR aggregates (the losers) except one (the winner) on many fibers. Axon-induced AChR aggregates underwent comparable selection of winners and losers. The results suggest that agrin and acetylcholine-driven muscle activity provided by transplanted axons are sufficient to elicit in a denervated adult muscle fiber processes that regulate the size and distribution of ectopic neuromuscular junctions.

Agrin↗
PubMed2004

Signaling assemblies formed in mast cells activated via Fcepsilon receptor I dimers.

Although aggregation of the Fcepsilon receptor I (FcepsilonRI) is necessary for Ag-mediated mast cell triggering, the relationship between the extent of the FcepsilonRI aggregation and subsequent biochemical and topographical events is incompletely understood. In this study, we analyzed the activation events induced by FcepsilonRI dimers, elicited by binding of anti-FcepsilonRI mAb to rat basophilic leukemia cells. We found that, in contrast to extensively aggregated FcepsilonRI, receptor dimers (1) induced a less extensive association of FcepsilonRI with detergent-resistant membranes, (2) delayed the tyrosine phosphorylation and membrane recruitment of several signaling molecules, (3) triggered a slower but more sustained increase in concentration of free cytoplasmic calcium, (4) induced degranulation which was not inhibited at higher concentrations of the cross-linking mAb, and (5) failed to produce clusters of FcepsilonRI, Syk kinase and Grb2 adapter in osmiophilic membranes, as detected by immunogold electron microscopy on membrane sheets. Despite striking differences in the topography of FcepsilonRI dimers and multimers, biochemical differences were less pronounced. The combined data suggest that FcepsilonRI-activated mast cells propagate signals from small signaling domains formed around dimerized/oligomerized FcepsilonRI; formation of large FcepsilonRI aggregates in osmiophilic membranes seems to promote both strong receptor triggering and rapid termination of the signaling responses.

Actins↗
PubMed1992

Early appearance of and neuronal contribution to agrin-like molecules at embryonic frog nerve-muscle synapses formed in culture.

Antibodies against chicken and Torpedo agrin were used for immunofluorescent staining in order to assess the spatial distribution and temporal appearance of agrin-like molecules at newly formed synaptic contacts in cultures of embryonic Xenopus nerve and muscle cells. The antibodies stained Xenopus neuromuscular junctions and removed ACh receptor (AChR)-aggregating activity from extracts of Xenopus brain. Immunofluorescence was observed at almost all nerve-induced AChR aggregates, even at microaggregates in cocultures as young as 7.5 hr and at nerve-muscle contacts less than 2 hr old. Microdeposits of immunofluorescence extended as far distally as, or farther than, the microaggregates of AChRs along young nerve-muscle contacts. They also occurred along portions of growing neurites that were not in contact with muscle. By contrast, immunofluorescence was rarely observed at the nonsynaptic aggregates of AChRs that form on noninnervated muscle cells. These results raise the possibility that neuronally derived microaggregates of agrin-like molecules may be primary sites of nerve-induced clustering of AChRs, and they indicate that these molecules are present at embryonic nerve-muscle synapses from the very onset of AChR aggregation. The cellular origin of the agrin-like molecules at synapses was examined in cross-species cocultures in which the neurons and muscle cells were obtained from embryos of Xenopus laevis and Rana pipiens. Immunofluorescent staining with anti-agrin antibodies reactive at both Rana and Xenopus neuromuscular junctions revealed immunofluorescence at AChR aggregates along nerve-muscle contacts involving both cross-species combinations. Immunofluorescent staining with an anti-agrin antibody reactive at Rana but not at Xenopus neuromuscular junctions was positive only at cross-species nerve-muscle contacts involving Rana neurons. These results provide the first demonstration that embryonic neurons supply agrin-like molecules to the synapses they form with embryonic muscle cells.

Agrin↗
PubMed1993

Muscle agrin: neural regulation and localization at nerve-induced acetylcholine receptor clusters.

The synapse-organizing protein agrin is expressed by muscle cells. In this study we begin to characterize the role of muscle agrin in synapse formation by investigating its distribution and expression during the formation of nerve-induced ACh receptor (AChR) aggregates in vitro. We have used species-specific anti-agrin antibodies to show that muscle agrin colocalizes with nerve-induced AChR clusters in chimeric nerve-muscle cocultures. Furthermore, quantitation by radioimmune assay shows that the expression of muscle agrin by cultured chick myotubes is increased more than twofold by coculture with cells from the motoneuron-rich ventral spinal cord. These data suggest a role for muscle agrin in neuromuscular junction development.

Agrin↗
PubMed1999

Regulation of the size and distribution of ectopic neuromuscular junctions in adult skeletal muscle by nerve-derived trophic factor and electrical muscle activity.

Transplanted axons induced multiple, irregularly distributed acetylcholine receptor (AChR) aggregates on muscle fibers at early stages of ectopic neuromuscular junction formation in denervated adult rat soleus muscles. Subsequently, most AChR aggregates disappeared (the losers). A few aggregates survived (the winners) and, as part of the surviving junctions, reached a certain size and spatial separation along the fibers. This elimination of losers and development of winners occurred only in electrically active muscles whether the activity was elicited by intact axons or by electrical muscle stimulation after the axons had been cut early. We conclude that electrical muscle activity regulates the size and distribution of ectopic neuromuscular junctions by acting in conjunction with a nerve-derived priming influence that does not require the continued presence of nerve terminals.

Animals↗
PubMed1981

Effects of isoproterenol on the development of beta-adrenergic receptors in brain cell aggregates.

The development of binding sites for the beta-adrenergic receptor antagonist [125I]iodohydroxybenzylpindolol in reaggregate cultures of fetal rat brain has been investigated. beta-Adrenergic receptor density increased rapidly between 6 and 22 days in culture at which time maximal density was attained. When cultures were grown in the presence of the beta-adrenergic receptor agonist isoproterenol, the development of receptors was markedly inhibited. Cultures were also grown for 15 days in the presence of isoproterenol and then for 7-14 days in the absence of isoproterenol. Following the removal of the agonist, binding sites developed, but to only approximately 50% of the density seen in control cultures.

Animals↗