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PubMed1989

Internalization of IgE receptors on rat basophilic leukemic cells by phorbol ester. Comparison with endocytosis induced by receptor aggregation.

Phorbol myristate acetate (PMA) can induce a rapid and significant decrease in the expression of IgE receptors on RBL-2H3 cells. Fluorescence microscopy confirmed that the down-regulation is due to internalization of receptors. The endocytotic response to PMA shares several characteristics with endocytosis induced by immunochemical aggregation of surface-bound monomeric IgE: the rates of internalization both have a t1/2 of about 5 min, a maximum of 35% of the surface-bound IgE can be endocytosed by the action of PMA (50% by receptor aggregation), endocytosis is sustained for at least up to 60 min, neither stimulus requires extracellular Ca2+ and endocytosis induced by either stimulus is an active process, i.e., is dependent on temperature and cellular energy. Biochemical studies revealed some differences between the endocytotic responses to the two stimuli. After prolonged treatment of cells with dexamethasone, only endocytosis induced by PMA is inhibited. Cells depleted of protein kinase C by prolonged exposure to PMA can sustain a significant endocytotic response to aggregation of IgE receptors, but become completely desensitized to PMA. These data suggest that different biochemical pathways mediate the signals from the two stimuli and that protein kinase C is directly involved in endocytosis induced by PMA but does not have a major role in endocytosis induced by receptor aggregation.

Animals↗
PubMed1982

Role of receptor aggregation in triggering IgE-mediated reactions.

The first event in the IgE-mediated triggering of basophils and mast cells is the binding of serum IgE to membrane IgE receptors; quantitative relationships have been found among the serum IgE concentration, the number of IgE molecules per cell, the total receptor number, and the degree of endogenous receptor occupancy. Once bound, these antibody molecules must be cross-linked by multivalent antigen to activate the cell. Receptor juxtaposition is necessary throughout both activation and desensitization of these cells. Two types of desensitization occur. Antigen-specific desensitization alters only the function of certain IgE molecules; the cell can respond normally to other antigens. Specific desensitization is not reversed by removal of the antigen. Also, the affected antibody-receptor complexes remain on the cell surface, can rebind antigen, but cannot trigger the cell. Nonspecific desensitization is less well understood but is directly related to the number of cross-links on the basophils. Inasmuch as the cells become insensitive to all IgE-mediated stimuli, the total depletion of some intermediate has been postulated. In the presence of calcium the stimulated cells normally degranulate and release mediators. We have shown that for the simple antigens, both the release process and desensitization are a function of the number of cross-links present on the cell surface, and we have generated a mathematical model that quantitatively fits the experimental data. Thus, after many studies that span decades, the role of receptor aggregation in triggering mast cells and basophils is becoming qualitatively and quantitatively defined.

Animals↗
PubMed1990

Localization of actin, beta-spectrin, 43 x 10(3) Mr and 58 x 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes.

I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4-6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 x 10(3) Mr and 58 x 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.

Actins↗
PubMed1998

alpha-Dystroglycan functions in acetylcholine receptor aggregation but is not a coreceptor for agrin-MuSK signaling.

alpha-dystroglycan (alpha-DG) is an agrin-binding protein that has been implicated in acetylcholine receptor (AChR) clustering, but it is unclear whether it acts as a coreceptor involved in initial agrin signaling or as a component involved in later events. To investigate its role, we have generated antisense derivatives of the C2 mouse muscle cell line, which have reduced alpha-DG expression. When compared with wild-type cells, the alpha-DG-deficient myotubes have a dramatic reduction in the number of spontaneous and agrin-induced AChR clusters. Several findings suggest that this decrease in AChR clustering is likely not because of a defect in agrin signaling through the MuSK receptor tyrosine kinase. Compared with wild-type cells, the alpha-DG-deficient cell lines showed only a transient reduction in the level of agrin-induced MuSK tyrosine phosphorylation and no reduction in AChR beta-subunit tyrosine phosphorylation. Additionally, agrin-induced phosphorylation of MuSK in wild-type myotubes was not decreased using agrin fragments that lack the domain primarily responsible for binding to alpha-DG. Finally, neural agrin-induced phosphorylation of MuSK was unaffected by treatments such as excess muscle agrin or anti-alpha-DG antibodies, both of which block agrin-alpha-DG binding. Together, these results suggest that alpha-DG is not required for agrin-MuSK signaling but rather that it may play a role elsewhere in the clustering pathway, such as in the downstream consolidation or maintenance of AChR clusters.

Agrin↗
PubMed1995

Fc alpha receptors mediate release of tumour necrosis factor-alpha and interleukin-6 by human monocytes following receptor aggregation.

The functional capacity of the human monocyte receptor for the Fc portion of IgA (Fc alpha R) in mediating signal transduction was evaluated by cytokine release. F(ab')2 fragments of anti-Fc alpha R monoclonal antibodies (mAb) were used as specific probes to induce release of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Multivalent cross-linking by a secondary anti-mouse antibody [F(ab')2 fragments] induced a significant release of TNF-alpha and IL-6 by human blood mononuclear cells, indicating requirements for Fc alpha R aggregation on the cell surface to transmit signals. Both cytokines were released exclusively by adherent cells, identifying monocytes as the responding cells within the mononuclear cell population. This cytokine release could not be due to contaminating endotoxins, because it was not abolished by polymyxin B, a lipopolysaccharide (LPS) inhibitor. Moreover, purified recombinant soluble Fc alpha R inhibited the anti-Fc alpha R mAb-mediated cytokine release from blood monocytes, demonstrating that TNF-alpha and IL-6 were released in a receptor-specific manner. Our data suggest that Fc alpha R, through its capacity to mediate secretion of IL-6, may play an important role in B-cell proliferation and immunoglobulin production. On the other hand, release of TNF-alpha following stimulation of Fc alpha R molecules directly implicates these receptors in amplification and regulation of the inflammatory process occurring during IgA-mediated host defence.

Animals↗
PubMed1984

Luteinizing hormone (LH) receptor aggregation: modification of ferritin-LH binding and aggregation by prostaglandin F2 alpha and ferritin-LH.

The interaction of LH and its receptor was investigated by ultrastructural analysis of ferritin-LH (FELH) binding to isolated rat luteal cells in the absence and presence of prostaglandin F2 alpha (PGF2 alpha), an inhibitor of LH-stimulated cAMP production. FELH, with a molar ratio of FE to LH of 1:1, bound specifically to LH receptors, either singly or in small groups (microaggregates), at intervals on luteal cell surfaces. FELH elicited a dose-dependent increase in progesterone production, and its binding increased with increased FELH concentration. The number of LH receptors per cell, estimated from particle counts, was about 6.2 +/- 0.6 X 10(4), similar to estimates from Scatchard analysis of [125I]iodo-hCG binding. Microaggregate size increased in parallel with FELH binding. Only partial aggregation was seen at concentrations of FELH that elicited near-maximal progesterone secretion. Aggregation continued to increase at FELH concentrations beyond that required to elicit maximal progesterone secretion. In the presence of PGF2 alpha, FELH-stimulated progesterone production was attenuated, and FELH binding decreased from 2.9 +/- 0.4 X 10(4) to 2.1 +/- 0.3 X 10(4) receptors/cell. PGF2 alpha did not alter microaggregate size on cells labeled with FELH at 4 C when membrane fluidity was already reduced, but did substantially reduce microaggregate size at 37 C. We conclude that FELH binds initially at random sites on membranes of isolated luteal cells and that as binding increases, receptors aggregate into small groups. Furthermore, microaggregates are related in part to receptor occupancy and possibly also to levels of cAMP or activation of the adenylate cyclase mechanism.

Animals↗
PubMed1998

The putative sponge aggregation receptor. Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats.

Porifera (sponges) are the oldest extant metazoan phylum. Dissociated sponge cells serve as a classic system to study processes of cell reaggregation. The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR). In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR. The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, as detected by northern blotting. Two cDNA clones corresponding to the shorter forms were already reported earlier; here we present an analysis of the largest. All three putative polypeptides feature scavenger receptor cysteine-rich (SRCR) domains. The largest form, SRCR-SCR-Car, is a cell-surface receptor of molecular mass 220 kDa, which is assumed to be the cell-adhesion receptor AR; the second form, SRCR-Re, is also a putative receptor of 166 kDa, while the third form, SRCR-Mo, is a soluble molecule of 129 kDa. The SRCR-SCR-Car molecule consists of fourteen SRCR domains, six short consensus repeats (SCRs), a C-terminal transmembrane domain and a cytoplasmic tail; its fourteenth SRCR domain features an Arg-Gly-Asp tripeptide. To obtain monoclonal antibodies, a 170-amino-acid-long polypeptide that is found in all three forms of the SRCR-containing proteins was expressed in E. coli. In a western blot of sponge cells lysate the monoclonal antibody raised against the recombinant polypeptide recognized two major immuno-reacting polypeptides (220 and 117 kDa) and two minor bands (36 and 32 kDa). The antibody was found to react with antigen(s) predominantly localized on the plasma membranes of cells, especially those of spherulous cells. In a functional assay Fab' fragments of the antibodies suppressed AF-mediated cell-cell reaggregation. Additionally, a recombinant SRCR-soluble fragment effectively inhibited AF-mediated cell-cell reaggregation. We conclude that the 220 kDa SRCR-containing protein of the sponge G. cydonium is very likely the AR.

Amino Acid Sequence↗
PubMed2000

Alpha-galactosidase stimulates acetylcholine receptor aggregation in skeletal muscle cells via PNA-binding carbohydrates.

Aggregation of nicotinic acetylcholine receptors (AChRs) in skeletal muscle is an essential step in the formation of the mammalian neuromuscular junction. While proteins that bind to myotube receptors such as agrin and laminin can stimulate AChR aggregation in cultured myotubes, removal of cell surface sialic acids stimulates aggregation in a ligand-independent manner. Here, we show that removal of cell surface alpha-galactosides also stimulates AChR aggregation in the absence of added laminin or agrin. AChR aggregation stimulated by alpha-galactosidase was blocked by peanut agglutinin (PNA), which binds to lactosamine-containing disaccharides, but not by the GalNAc-binding lectin Vicia villosa agglutinin (VVA-B4). AChR aggregation stimulated by alpha-galactosidase potentiated AChR clustering induced by either neural agrin or laminin-1 and could be inhibited by muscle agrin. These data suggest that capping of cell surface lactosamines or N-acetyllactosamines with alpha-galactose affects AChR aggregation much as capping with sialic acids does.

Acetylgalactosamine↗
PubMed1993

Local induction of acetylcholine receptor aggregates on cultured rat myotubes by a partially purified protein from fetal pig brain.

It has been suggested that at the time of innervation, developing neurites release one or more soluble factors that locally induce acetylcholine receptor (AChR) aggregate formation at the synaptic site. To test this hypothesis, we developed a model system that mimics the local, neural induction of AChR aggregation at developing synapses. Partially purified protein derived from fetal pig brain was applied locally to the surface of cultured myotubes via a micropipet. We found that local application of this factor for as little as 30 min induced the formation of AChR aggregates that were restricted to a region within 30 microns around the release site. In addition, the locally applied factor induced a local AChR aggregation response, but did not cause a detectable change in the myotube resting membrane potential at the release site. Our data support a soluble factor hypothesis and suggest that neither cell-cell contact nor local electric fields are necessary for the initial induction of AChR aggregation.

Animals↗
PubMed1993

Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation.

Protein-tyrosine phosphorylation plays a critical role in the high-affinity IgE receptor (Fc epsilon RI) signaling. Here we investigated the involvement of the tyrosine kinase p72syk in Fc epsilon RI signaling in the rat mast cell line RBL-2H3. Specific antibodies were raised against peptides synthesized on the basis of the deduced peptide sequence of an essentially full-length rat syk cDNA. The expression of p72syk in RBL-2H3 cells was demonstrated with these antibodies. The aggregation of Fc epsilon RI led to the tyrosine phosphorylation of p72syk that was detected after 15 s of stimulation, reached a plateau by 5 min, and was not induced by calcium influx or protein kinase C activation. Association of p72syk with the tyrosine phosphorylated Fc epsilon RI gamma chain was detected only after receptor aggregation. We previously demonstrated that aggregation of the Fc epsilon RI on mast cells results in the tyrosine phosphorylation of a 72-kDa protein (pp72) involved in IgE signaling. The depletion of p72syk from RBL-2H3 cell lysates resulted in only a slight decrease in the amount of pp72. These results demonstrate that pp72 is composed of several phosphoproteins and identify p72syk as one component of pp72. These data, together with recent observations in T cells, indicate that the interaction between p72syk-related tyrosine kinases and zeta-related proteins could play an important role in signal transduction.

Amino Acid Sequence↗
PubMed1984

Three cholinergic neuroblastoma hybrid cell lines that form few synapses on myotubes are deficient in acetylcholine receptor aggregation molecules and large dense core vesicles.

Three neuroblastoma X glioma hybrid cell lines that synthesize and release acetylcholine but that form few or no synapses with cultured skeletal muscle cells lack two characteristics of neuroblastoma or hybrid cell lines that do form many synapses with myotubes: large dense core vesicles and the ability to increase the number of nicotinic acetylcholine receptor clusters on co-cultured myotube membranes. Functional synapse formation on myotubes was increased by co-culturing myotubes and cells from one of the defective lines with neuroblastoma cells that induce myotube acetylcholine receptor aggregation but which synthesize little or no acetylcholine.

Animals↗
PubMed1989

A point mutation in the neu oncogene mimics ligand induction of receptor aggregation.

The rat neu gene, which encodes a protein closely related to the epidermal growth factor receptor, is a proto-oncogene that can be converted into an oncogene by a point mutation. Both genes encode proteins with a relative molecular mass of 185,000 but the question of why the neu gene product, p185neu, is oncogenic, whereas the product of c-neu, p185c-neu, is not, remains unanswered. The proteins have several features common to the family of tyrosine kinase growth-factor receptors, including cysteine-rich external domains, a hydrophobic transmembrane region and a cytoplasmic tyrosine kinase domain. The oncogenic p185neu differs from p185c-neu by an amino-acid substitution in the transmembrane region of the glycoprotein: this replacement of valine by glutamic acid at position 664 induces increased intrinsic tyrosine kinase activity which is associated with transformation. Many glycoproteins with charged amino acids in the transmembrane region exist as multimeric complexes at the plasma membrane. We have therefore investigated the association state of both products of the neu gene and show that the oncoprotein p185neu is organized at the plasma membrane primarily in an aggregated form, but that p185c-neu is not. Induction of an aggregated state may mimic aspects of ligand-induced receptor aggregation resulting in enzymatic activation that leads to cellular transformation.

Animals↗
PubMed2005

Ig-independent Ig beta expression on the surface of B lymphocytes after B cell receptor aggregation.

In order for humoral immune responses to develop, B cells must be able to recognize, bind, and internalize Ags. These functions are performed by the BCR, which is also responsible for initiating and transducing activation signals necessary for B cell proliferation and differentiation. We have examined surface expression patterns of individual components of the BCR following anti-Ig- and Ag-induced aggregation. Specifically, the localization and expression levels of the Ag-binding component, surface Ig (sIg), and the Igbeta component of the Igalpha/Igbeta signaling unit were investigated to determine their individual participation in the internalization and signal transduction. Using primary murine B cells, we found that while >95% of the sIg is internalized following anti-Ig-induced aggregation, 20-30% of Igbeta remains on the surface. These results suggest that sIg and Igbeta may function independently following the initial stages of signal transduction.

Animals↗
PubMed2005

Death of a dogma or enforcing the artificial: monomeric IgE binding may initiate mast cell response by inducing its receptor aggregation.

Several recent reports have suggested that binding monomeric IgE (mIgE) to its type 1 receptor, Fc epsilon RI, on mast cells induces important responses. These observations contradict the notion that it is the aggregation of this receptor that is essential for initiating mast cell response. In the present study, we suggest that the most probable causes for the reported observations are the experimental protocol used combined with the high expression levels of the Fc epsilon RI by mast cells. Specifically, we suggest using the published data and physicochemical calculations that the exceptionally high number of cell surface Fc epsilon RI-bound monoclonal IgE yields, in the two-dimensions of the cells' membranes, a situation where even a low affinity of these mIgE for epitopes on their own structure or on another cell surface component may lead to their aggregation. Hence, we hypothesize that the reported response to mIgE binding is a result of such an Fc epsilon RI-IgE induced aggregation.

Animals↗
PubMed2005

Carbohydrate engineering of the recognition motifs in streptococcal co-aggregation receptor polysaccharides.

The cell wall polysaccharides of certain oral streptococci function as receptors for the lectin-like surface adhesins on other members of the oral biofilm community. Recognition of these receptor polysaccharides (RPS) depends on the presence of a host-like motif, either GalNAcbeta1-3Gal (Gn) or Galbeta1-3GalNAc (G), within the oligosaccharide repeating units of different RPS structural types. Type 2Gn RPS of Streptococcus gordonii 38 and type 2G RPS of Streptococcus oralis J22 are composed of heptasaccharide repeats that are identical except for their host-like motifs. In the current investigation, the genes for the glycosyltransferases that synthesize these motifs were identified by high-resolution nuclear magnetic resonance (NMR) analysis of genetically altered polysaccharides. RPS production was switched from type 2Gn to 2G by replacing wefC and wefD in the type 2Gn gene cluster of S. gordonii 38 with wefF and wefG from the type 2G cluster of S. oralis J22. Disruption of either wefC or wefF abolished cell surface RPS production. In contrast, disruption of wefD in the type 2Gn cluster or wefG in the type 2G cluster eliminated beta-GalNAc from the Gn motif or beta-Gal from the G motif, resulting in mutant polysaccharides with hexa- rather than heptasaccharide subunits. The mutant polysaccharides reacted like wild-type RPS with rabbit antibodies against type 2Gn or 2G RPS but were inactive as co-aggregation receptors. Additional mutant polysaccharides with GalNAcbeta1-3GalNAc or Galbeta1-3Gal recognition motifs were engineered by replacing wefC in the type 2Gn cluster with wefF or wefF in the type 2G cluster with wefC respectively. The reactions of these genetically modified polysaccharides as antigens and receptors provide further insight into the structural basis of RPS function.

Amino Acid Motifs↗
PubMed1985

Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields.

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.

Animals↗
PubMed1995

Randomized, Placebo-Controlled Study of Lamifiban with Thrombolytic Therapy for the Treatment of Acute Myocardial Infarction: Rationale and Design for the Platelet Aggregation Receptor Antagonist Dose Investigation and Reperfusion Gain in Myocardial Infarction (PARADIGM) Study.

The benefits of thrombolytic therapy in the treatment of acute myocardial infarction are incontrovertible. Large-scale studies combining angiographic and clinical end-points have demonstrated a perfusion-mortality relationship, with the highest survival rate among patients with early restoration of TIMI grade 3 coronary arterial flow. Despite advances in thrombolytic strategies, a substantial number of patients fail to rapidly achieve and maintain adequate coronary perfusion with thrombolysis. Conjunctive therapy with aspirin has proven useful in thrombolytic regimens, likely countering the heightened platelet activity central to acute coronary syndromes. The antiplatelet effect of aspirin is relatively weak compared with that of glycoprotein IIb/IIIa platelet receptor antagonists, which block the final common pathway of platelet aggregation. Lamifiban is a nonpeptide glycoprotein IIb/IIIa receptor antagonist. In early experimental studies, Lamifiban in combination with thrombolytic therapy has been shown to effectively restore coronary arterial patency, and phase I and phase II data have shown its use to be safe. To determine the optimal dose with regard to safety and efficacy of Lamifiban to be used with thrombolytic therapy in a large-scale trial, a phase II study is underway. The Platelet Aggregation Receptor Antagonist Dose Investigation and Reperfusion Gain in Myocardial Infarction (PARADIGM) study is a randomized, placebo-controlled study of Lamifiban in 400 patients receiving thrombolysis as treatment for acute myocardial infarction. By studying 90-minute angiography, platelet aggregation, continuous electrocardiography, and clinical outcome in PARADIGM, important insights will be obtained to determine the optimal dose of Lamifiban for phase III study. We provide the background and rationale for the study of Lamifiban in PARADIGM and other ongoing studies in acute coronary syndromes.

Journal Article↗
PubMed1975

C3-mediated cytoadherence. Formation of C3 receptor aggregates as prerequisite for cell attachment.

Inhibition of free movement of C3 receptors by either applying low temperature (3 degrees C) or fixing the cell surfce of lymphocytes with glutaraldehyde (2 times 10(-5) to 2 times 10(-1)%) results in loss of firm attachment of EAC142 3b cells to the lympocytes as demonstrated here by loss of rosette formation of Raji lymphoid cells. Under the same conditions soluble C3 can still bind but is unable to induce aggregation of C3 receptors into small patches. It is suggested that the local increase of C3 receptor density by aggregation is a prerequisite for C3-dependent cytoadherence. Microaggregation of corresponding receptor sites may be essential also in other recognition systems.

Animals↗