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At least 19 recordsLinked to original sources
PubMed1992

Interpretation of Scatchard plots for aggregating receptor systems.

Aggregation of cell surface receptors, with each other or with other membrane proteins, occurs in a variety of experimental systems. The list of systems where receptor aggregation appears to be important in understanding ligand binding and cellular responses is growing rapidly. In this paper we explore the interpretation of equilibrium binding data for aggregating receptor systems. The Scatchard plot is a widely used tool for analyzing equilibrium binding data. The shape of the Scatchard plot is often interpreted in terms of multiple noninteracting receptor populations. Such an analysis does not provide a framework for investigating the role of receptor aggregation and will be misleading if there is a relation between receptor aggregation and ligand binding. We present a general model for the equilibrium binding of a ligand with any number of aggregating receptor populations and derive theoretical expressions for observable Scatchard plot features. These can be used to test particular models and estimate model parameters. We develop particular models and apply the general results in the cases of six aggregating receptor systems where ligand binding and receptor aggregation are related: cross-linking of monovalent cell surface proteins by monoclonal antibodies, cross-linking of cell surface antibodies by bivalent ligand, antibody-induced co-cross-linking of cell surface antibodies and Fc gamma receptors, ligand-enhanced aggregation of identical epidermal growth factor receptors, aggregation of heterologous receptors for interleukin 2 to form a high-affinity receptor, and association of receptors, including those for interleukins 5 and 6, with nonbinding accessory proteins that influence receptor affinity or effector function.

Animals↗
PubMed1983

Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor.

The effect of several basement membrane components on the aggregation of acetylcholine (ACh) receptors on cultured myotubes was studied. Cultures were incubated for 16 to 24 hr with laminin, a heparan sulfate proteoglycan, collagen types IV and V, or fibronectin, alone, or together with medium conditioned by NG108-15 neuroblastoma X glioma hybrid cells (NCM). The number of ACh receptor aggregates per myotube was assayed by fluorescence microscopy of cultures stained with tetramethylrhodamine-labeled alpha-bungarotoxin. Laminin induced ACh receptor aggregation on primary rat myotubes and on myotubes formed by G8-1 clonal rat muscle cells. Laminin enhanced the receptor-aggregating activity of NCM in a concentration-dependent manner (0.6 to 6.0 micrograms/ml) and the number of aggregates formed in the presence of laminin and NCM together was greater than the sum of the aggregates induced by NCM and laminin separately. The aggregation factor in NCM is probably not laminin, since less than 10 ng/ml of laminin-like immunoreactivity was detected in NCM, and antiserum against laminin blocked the effects of laminin but had little effect on NCM aggregation activity. Collagen type V enhanced the receptor aggregation activity of NCM, but less strongly than laminin, and had little or no effect by itself. The other basement membrane components did not induce receptor aggregation or enhance the effect of NCM. Experiments in which ACh receptors were labeled before exposure of cultures to NCM and laminin indicated that laminin enhanced the rearrangement of receptors at the cell surface. Immunofluorescence microscopy indicated that laminin binds to the myotubes within 30 min and forms patches on the cell surface over a period of hours. Laminin bound to the myotube surface enhanced receptor aggregation as well as laminin continuously present in the culture medium. The results suggest the possibility that laminin could enhance the receptor aggregation activity of a neuronal factor(s) released at the developing neuromuscular junction.

Animals↗
PubMed2002

Kinetic proofreading in receptor-mediated transduction of cellular signals: receptor aggregation, partially activated receptors, and cytosolic messengers.

Signaling by the T cell receptor (TCR), and the related immunoreceptor Fc epsilon RI, is sensitive to ligand-receptor binding kinetics. Differences in the rate at which a ligand dissociates from a receptor cause disproportionate differences in signaling events and cellular responses to ligand-receptor engagement. Analysis of a simple mathematical model, developed by McKeithan (1995, Proc. Natl. Acad. Sci. USA, 92, 5042-5046), has indicated that such sensitivity to binding kinetics is expected if a bound receptor must complete a cascade of modifications before generating a productive signal. However, recent experiments show that some cellular responses mediated by immunoreceptors escape from the control of kinetic proofreading, in the sense that these responses do not exhibit the expected sensitivity to the lifetime of a ligand-receptor bond. Here, we use an extended form of the McKeithan model to investigate possible explanations for such exceptions to the kinetic proofreading rule. We examine cellular responses triggered by cytosolic messengers, which are activated by modified receptors, and responses triggered by receptors in intermediate states of modification, i.e., receptors that have not progressed through the full series of potential modifications. Receptor aggregation is also considered. We find that the expected relationship between ligand-receptor binding kinetics and cellular responses can change significantly when signal transduction depends on a messenger or a partially modified receptor. In particular, cellular responses triggered by a messenger, such as a transcription factor that translocates from the membrane to the nucleus after receptor-mediated activation, can be sensitive or insensitive to a change in the lifetime of a ligand-receptor bond, depending on the parameters that govern the activation and decay of a messenger.

Animals↗
PubMed2002

Distribution of postsynaptic GABA(A) receptor aggregates in the deep cerebellar nuclei of normal and mutant mice.

In the central nervous system, the aggregation of receptors is crucial for synapse formation and function. To study the role of presynaptic terminals in the maintenance of postsynaptic specializations, we analyzed the synaptic contacts between Purkinje cells and neurons of the deep cerebellar nuclei in two in vivo models: the Lurcher and Purkinje cell-deficient (PCD) mutant mice. These mutants lose their Purkinje cells at different postnatal stages. By using confocal scanner microscopy and immunohistochemistry, we studied the distribution of the alpha subunit of the gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)Ralpha1) and gephyrin, one of its anchoring proteins, in relation to the distribution of presynaptic markers, glutamic acid decarboxylase (GAD), or synaptophysin. In Lurcher the distribution of GABA(A) receptor aggregates on the membrane of postsynaptic neurons was not affected by the important loss of GAD-positive terminals, whereas in PCD, the number of large GABA(A) receptor aggregates increased. In both mutants the number of aggregates of gephyrin decreased. Most of these remaining aggregates were clustered to form groups, some of which were in front of GAD-positive terminals. This study shows, for the first time, the localization of GABA(A)R alpha 1 in Lurcher and PCD mutant mice. It clearly establishes that GABA(A)R alpha 1 and gephyrin are differentially affected by deafferentation. Because the receptor aggregates are maintained while the gephyrin aggregates are lost, as a result some receptor aggregates are not associated with any gephyrin. These two postsynaptic components appeared to be regulated by different mechanisms.

Aging↗
PubMed2001

Ultrastructure of acetylcholine receptor aggregates parallels mechanisms of aggregation.

BACKGROUND: Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. RESULTS: In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. CONCLUSIONS: The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

Animals↗
PubMed1989

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor.

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.

Amino Acids↗
PubMed1982

Embryonic brain extract induces collagen biosynthesis in cultured muscle cells: involvement in acetylcholine receptor aggregation.

The involvement of extracellular matrix components in induction of the aggregation of acetylcholine (AcCho) receptors by factor(s) present in embryonic brain extract was investigated. Embryonic brain extract induced a three-fold increase in the number of AcCho receptor aggregates on the surface of cultured myotubes and a 5- to 10-fold increase in the synthesis of procollagen, which was secreted into the medium and converted to collagen. Adult brain extract, embryonic serum, and embryonic liver extract were less active in stimulating both collagen synthesis and AcCho receptor aggregation. A physiological connection between the two processes is suggested, since the number of AcCho receptor aggregates could be reduced to control levels by treating brain extract-stimulated myotubes with purified bacterial collagenase. In addition, stimulation of collagen secretion by ascorbic acid (50 micrograms/ml) promoted a 1.6-fold increase in AcCho receptor aggregation. When ascorbic acid was added together with the brain extract, further increases in both collagen synthesis and AcCho receptor aggregation were observed.

Animals↗
PubMed1992

Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are also important in shaping the binding curves. Because we have controlled experimentally for many sources of receptor heterogeneity, we have limited the potential explanations for residual Scatchard plot curvature.

Animals↗
PubMed1986

Luteinizing hormone triggers two opposite regulatory pathways through an initial common event, receptor aggregation.

LH receptor internalization was studied with an antireceptor monoclonal antibody (aLHR) which induces Leydig cells to produce testosterone. To follow receptor-mediated aLHR internalization, cells were incubated with aLHR at 10 C for 3 h to generate an aLHR complex; this was followed by a second incubation with fluorescent labeled antimouse immunoglobulin at 34 C, a temperature which allows internalization. Within 15 min at 34 C, cytoplasmic fluorescent staining was detectable; this staining was strongly visible after 60 min. At no time was nuclear staining observable. Employing such an approach, it has also been possible to follow the fate of unoccupied receptors when cells are stimulated with a submaximal dose of LH. The results show that LH interactions with 20% of its receptors produces microaggregation, patching, capping, and internalization of free receptor sites. The results further demonstrate that cells with receptors in the state of capping are less sensitive to a second LH stimulation, suggesting that in this state receptors are no longer coupled to the adenylate cyclase system.

Adenylyl Cyclases↗
PubMed1976

Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface.

An aggregation receptor from the siliceous sponge Geodia cydonium has been isolated and purified in an almost pure form. It sediments at about 2-6s, has a buoyant density of 1-51 g/ml in CsCl and elutes from Sephadex G-50 at a Ve/V0 value of 1-311. Chemical analysis revealed that the receptor consists of 81% neutral carbohydrate and 7-5% protein. The activity of the receptor is rapidly destroyed by Na-periodate. The receptor is released from the cell surface after removal of Ca2+ from the medium or after incubation of the cells with trypsin. The depleted cells can be charged again with isolated receptor molecules. The binding of the receptor molecules on the cell surface is prevented in the presence of trypsin. For optimal binding, physiological salt concentrations with respect to NaCl (540 mM NaCl) and Ca2+ ions are necessary. The receptor whose isolation is described in this report, is involved in secondary aggregation processes, which are initiated by a soluble aggregation factor. The primary aggregation of the cells is not influenced by the receptor. Time-course studies with receptor-depleted cells revealed that new aggregation receptor molecules are formed during the aggregation process. By competition experiments it could be shown that high concentrations of soluble aggregation receptor molecules inhibit secondary aggregation. The soluble receptor molecules can complete with surface-bound receptor molecules only if these are not linked with the aggregation factor.

Animals↗
PubMed1999

Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells.

Syk has been shown to be activated by osmotic stress, however, the mechanisms involved are largely unknown. In this study, we demonstrated that cell shrinkage, rather than osmolarity, was responsible for osmotic stress-induced Syk activation. Osmotic stress-induced Syk activation depended partly upon aggregation of surface receptors. Moreover, intracellular reactive oxygen species were involved in mediating osmotic stress-induced Syk activation, with osmotic stress-induced Syk activation being inhibited by the pretreatment of cells with N-acetyl-cysteine and reduced glutathione. When cells were treated with the combination of sodium chloride and hydrogen peroxide, there was a synergistic activation of Syk. In conclusion, osmotic stress-induced Syk activation required suramin-inhibitable surface receptor aggregation and accumulation of intracellular reactive oxygen species.

Acetylcysteine↗
PubMed1986

Receptor aggregation is necessary for activation of the soluble insulin receptor kinase.

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.

Animals↗
PubMed1994

Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight.

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.

Acetylcholine↗
PubMed1988

Retinal neurons lack an acetylcholine receptor aggregating factor.

Spinal cord neurons form stable synapses on muscle cells in culture, whereas retinal neurons, an inappropriate presynaptic partner for muscle cells, form synapses that are transient. We have hypothesized that a trophic influence of neurons on muscle is involved in the stabilization of synapses. Because other neural tissues that form stable synapses on muscle cells contain factors that aggregate acetylcholine receptors (AChR) into clusters on the surface of muscle cells, it may be that these aggregation factors are necessary for stabilization of neuron-muscle synapses. Therefore, we determined the AChR-aggregating activity of retinal neurons. The results showed that cocultures of retinal neurons and muscle cells and retinal-conditioned medium do not show increases in the number of AChR on muscle cells. Conversely, spinal cord-muscle cocultures and spinal cord-conditioned medium produce increases in the number of AChR clusters. These data, along with previous studies demonstrating that retinal neurons are unable to affect the electrical membrane properties of cultured muscle cells, whereas spinal cord neurons do elicit such changes, add support to the above hypothesis of a trophic influence of neurons in synapse stabilization.

Animals↗
PubMed1983

Receptor aggregation induced by antilutropin receptor antibody and biological response in rat testis Leydig cells.

Antibodies against the lutropin receptor have been obtained by the monoclonal antibody technique. Mice were immunized with luteal membrane from ovaries from pseudopregnant rats, containing high lutropin receptor concentration. Hybridoma cells were obtained by fusing mouse myeloma cells with spleen cells from the immunized animal. Five clones were produced that secreted monoclonal antibodies that specifically inhibited lutropin binding to its receptor in a competitive fashion. Antibodies from three clones were capable of blocking biological response to lutropin (e.g., testosterone production by isolated rat Leydig cells). Antibodies secreted by two other clones, however, were capable of acting as Leydig cell stimulators. Immunofluorescence studies demonstrated the presence of receptor capping which may be associated with receptor-mediated testosterone production. Antagonist antibodies could be transformed into agonist by the addition of a second crosslinking anti-mouse IgG. The discovery of agonist antibodies against the receptor molecule proves that the biological information of the lutropin-receptor complex resides in the receptor and not in the hormone.

Animals↗
PubMed1996

Graded fibronectin receptor aggregation in migrating cells.

We have examined the distribution of the integrin fibronectin receptor in migrating NIH 3T3 fibroblasts to test the hypothesis that cell locomotion involves regional differences of adhesive receptor aggregation. A distinct asymmetry of fibronectin receptor aggregation was observed on the surface of migrating NIH 3T3 fibroblasts. Direct current electric fields were used to stimulate directional cell migration and thus allow the quantitative correlation of receptor asymmetries to the direction of cell locomotion. Digital particle analysis of fluorescent confocal micrographs demonstrates that the leading half of cells has a higher proportion of receptors contained in small clusters than does the trailing half. Conversely, larger receptor aggregates are more prevalent in the rear of the cell than in the front. We have also observed a gradient of extracellular fibronectin fibril assembly, similar to that described for the fibronectin receptor. Extracellular fibronectin appears in progressively larger fibrils across the ventral cell surface from front to rear, with dense meshworks often deposited as a trail behind the cell. Large fibronectin receptor clusters toward the rear of the cell generally do not correlate with focal contacts, and are thus most likely aggregated by cell surface-bound fibronectin fibrils and not by adhesion to the substratum. These results suggest that spatial variations in the degree of adhesive receptor aggregation are created in fibroblasts during the processes of migration and matrix synthesis.

3T3 Cells↗
PubMed1978

A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells.

There is an increase in the number of acetylcholine (AcCho) receptor aggregates on striated embryonic mouse myotubules when they are cocultured with clonal neuroblastoma-glioma hybrid cells. Medium conditioned by hybrid cells contains a factor which increases the number of AcCho receptor aggregates on myotubes cultured from mouse, rat or chick muscle. AcCho receptor-aggregating activity was present in medium conditioned by the neuroblastoma parent clone but was not detected in medium conditioned by cells of the parent glioma clone, fibroblasts, or HeLa cells. The factor increased the aggregation of AcCho receptors within 24 hr without a significant increase in the total number of AcCho receptors, and its action did not depend on myotube protein synthesis. The factor appears to rearrange the distribution of myotube AcCho receptors either by aggregating mobile AcCho receptors or by stabilizing labile receptor aggregates.

Cells, Cultured↗