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PubMed1989

Reorganization and stabilization of acetylcholine receptor aggregates on rat myotubes.

Aggregation of acetylcholine receptors (AChRs) is an important early feature of the postsynaptic development of the vertebrae neuromuscular junction. At later stages of differentiation, aggregates are remodeled and stabilized. Aggregation of AChRs can be induced on rat myotubes in culture within 4 hr by treatment with embryonic pig brain extract (EBX). In this study, further sequential changes in the distribution of AChRs were followed by video-intensified fluorescence microscopy. These studies have revealed that groups of AChR aggregates that have formed after 4 hr in EBX are reorganized during the exposure to EBX for 20 additional hr to form a smaller number of larger, oval-shaped aggregates. We have named these two types of aggregates "4-hr aggregates" and "24-hr aggregates". This reorganization occurs by the expansion and merging of individual aggregates within a group, and by the incorporation of newly inserted AChRs. The 24-hr aggregates are an average of 15 times greater in area than 4-hr aggregates, and contain regions with an apparent AChR site density (fluorescence intensity) that is more than twice that of 4-hr aggregates. Electron microscopy of mapped 24-hr aggregates revealed that folded plasma membrane is associated with these regions, probably accounting for the elevated fluorescence. The 24-hr aggregates are more stable than 4-hr aggregates, as determined by their significantly slower disassembly after removal of EBX, elevation of temperature (38 degrees C), reduction of extracellular calcium levels (0.1 mM), or the addition of sodium azide (7 mM). This was determined by following disassembly both statistically (using fixed cultures) and by direct observations of living myotubes. These findings were confirmed by measuring the sequential changes in relative AChR site density over time in individual living myotubes. Thus, 24-hr aggregates form by the reorganization of 4-hr aggregates; exhibit a more regular, compact shape; and are more stable than 4-hr aggregates. These changes in AChR organization and aggregate stability resemble the changes occurring after the initial formation of junctional AChR aggregates during embryonic development, demonstrating additional similarities between this model system and the developing neuromuscular junction.

Animals↗

PubMed1998

Nitrocellulose particles adsorbed to immunoglobulins are a new and effective approach to induce cell activation dependent on receptor aggregation.

Nitrocellulose (NC) has proved to be a versatile tool for the isolation and characterization of various biomolecules. In this report we extend its scope by using antibody-coated NC particles to cross-link molecules on the surface of living cells. Ligation of receptors in Jurkat cells with NC-bound specific antibodies induced protein tyrosine phosphorylation patterns of cellular proteins comparable to conventional antibody cross-linking. In addition, the present study shows that application of NC particles coated with human IgA significantly activated monocytic cells via the Fc alpha receptor (Fc alphaR), whereas cross-linking of receptor-ligand complexes with isotype-specific antibody was less efficient. Subsequent immunoprecipitation and immunoblot analysis of aggregated Fc receptors (FcRs) complexed to Ig-adsorbed particles permits fast identification of molecules involved in the transmission of signals. Therefore, ligand-coated NC particles can be used to examine receptor-mediated cell activation events dependent upon extensive receptor aggregation.

Adsorption↗

PubMed1990

Association of cytoskeletal proteins with newly formed acetylcholine receptor aggregates induced by embryonic brain extract.

Aggregates of acetylcholine receptors (AChR) in muscle cell membranes are associated with accumulations of certain cytoskeletal and peripheral membrane proteins. We treated cultured rat myotubes briefly with embryonic brain extract (EBX) to promote AChR aggregation and determined the distribution of several of these proteins at early stages of aggregation. EBX-treated and control cultures were stained with tetramethylrhodamine-alpha-bungarotoxin to identify AChR aggregates and were then frozen and sectioned on a cryostat. These sections were stained with primary antibodies and fluoresceinated secondary antibodies to localize cytoskeletal proteins. The distributions of AChRs and cytoskeletal proteins was examined qualitatively and analyzed by a semiquantitative assay. Qualitatively, the 43K protein had a distribution that was virtually identical to that of AChR in both control and EBX-treated cultures, and it always colocalized with early AChR aggregates. The 58K protein similarly colocalized with early AChR aggregates, but it was also in aggregate-free areas of muscle membrane. The association of vinculin with the aggregates was quantitatively similar to that of the 43K and 58K proteins, but, qualitatively, its distribution did not follow that of the AChR as closely. Like the 58K protein and vinculin, alpha-actinin, filamin, and actin were concentrated in AChR aggregates and were also enriched elsewhere. However, they were less closely associated with the aggregates, both quantitatively and qualitatively. These results show that AChR aggregates induced by EBX tend to be enriched in the same cytoskeletal proteins that are present at the neuromuscular junction in vivo and at AChR clusters formed at sites of cell-substrate adhesion in vitro. Semiquantitative analysis also revealed that the fractional area of the cell surface associated with vinculin, alpha-actinin, and the 58K protein was the same in controls and EBX-treated myotubes, although the area enriched in AChR and the 43K protein increased about three-fold upon EBX treatment. These results suggest that AChR aggregates may form preferentially in membrane regions that are already enriched in these proteins.

Actinin↗

PubMed1990

Concanavalin A-induced receptor aggregation stimulates the tyrosine kinase activity of the insulin receptor in intact cells.

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.

Animals↗

PubMed1992

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1998

A minigene of neural agrin encoding the laminin-binding and acetylcholine receptor-aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres.

The extracellular matrix molecule agrin is both necessary and sufficient for inducing the formation of postsynaptic specializations at the neuromuscular junction (NMJ). At the mature NMJ, agrin is stably incorporated in synaptic basal lamina. The postsynapse-inducing activity of chick agrin, as assayed by its capability of causing aggregation of acetylcholine receptors (AChRs) on cultured muscle cells, maps to a 21 kDa, C-terminal domain. Binding of chick agrin to muscle basal lamina is mediated by the laminins and maps to a 25 kDa, N-terminal fragment of agrin. Here we show that an expression construct encoding a 'mini'-agrin, in which the laminin-binding fragment was fused to the AChR-clustering domain, is sufficient to induce postsynaptic differentiation in vivo when injected into non-synaptic sites of rat soleus muscle. As shown for ectopic postsynaptic differentiation induced by full-length neural agrin, myonuclei underneath the ectopic sites expressed the gene for the AChR epsilon-subunit. Altogether, our data show that a 'mini'-agrin construct encoding only a small fraction of the entire agrin protein is sufficient to induce postsynapse-like structures that are reminiscent of those induced by full-length neural agrin or innervation by motor neurons.

Agrin↗

PubMed1988

Control of the aggregation factor-aggregation receptor interaction in sponges by protein kinase C.

By means of immunobiochemical and immunocytological techniques it was found that the aggregation factor (AF) from the sponge Geodia cydonium is stored in vesicles of spherulous cells. During the reaggregation process of dissociated cells, the AF which is present extracellularly was determined to be bound to the cell-surface-associated aggregation receptor (AR) only during the initial phase (0-5 h after addition of the AF to the single cell suspension). At later stages (20 h), the AF colocalized with extracellular structures, e.g., collagen and glycoconjugates. Immobilized to nitrocellulose, the AR, a molecule with Mr of 43.5 kDa, displayed its binding affinity to the AF only if it was isolated from early aggregates (5 h). The transition of the AF-susceptible to the AF-deficient state of the plasma membrane was mimicked in vitro by incubation of plasma membranes from early aggregates with purified protein kinase C. This conversion to the AF-deficient state could be prevented by the protein kinase C inhibitor staurosporine. Together with earlier findings, which revealed that the AR is phosphorylated by protein kinase C, we propose that in the sponge system this enzyme controls intercellular processes involved in morphogenesis.

Animals↗

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↗

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↗