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J P Merlie

Publications and source records attributed to J P Merlie.

At least 109 records · Page 6Linked to original sources
PubMed1986

An acetylcholine receptor precursor alpha subunit that binds alpha-bungarotoxin but not d-tubocurare.

We have identified an intracellular form of the alpha subunit of the acetylcholine receptor that binds alpha-bungarotoxin with high affinity. Unlike the mature receptor complex, an alpha 2 beta gamma delta pentamer that migrates as a 9S species in velocity sedimentation analysis, the intracellular species moves as a 5S component. The kinetics of appearance of alpha subunit in the 5S component and the mature receptor complex indicate that the intracellular 5S component is a precursor of the mature receptor. The precursor species differs from 9S receptor in two critical features: (i) the precursor alpha subunit is not associated with beta subunit and (ii) alpha-bungarotoxin binding to the precursor alpha subunit is not inhibited by the cholinergic ligands decamethonium or d-tubocurarine. The properties of the precursor suggest that the acquisition of the ligand binding site by alpha subunit occurs at a distinct stage in the posttranslational development of functional acetylcholine receptor.

Animals↗
PubMed1986

DNase I-hypersensitive sites surround the mouse acetylcholine receptor delta-subunit gene.

The mouse skeletal muscle acetylcholine receptor is regulated, at least in part, by transcriptional mechanisms. As a first step toward defining the cis- and trans-acting factors involved in acetylcholine receptor transcriptional regulation, we examined mouse muscle and fibroblast cells for the presence of DNase I-hypersensitive sites in the chromatin surrounding the delta-subunit gene. We detect six DNase I-hypersensitive sites in muscle DNA blots hybridized with a mouse delta-subunit cDNA. Two hypersensitive sites lie distantly 5' to the delta gene, one lies near the 5' end of the delta gene, one lies near its 3' end, and two hypersensitive sites lie distantly 3'. The pair of distantly 3' hypersensitive sites should by analogy to chicken and human genomes lie near the gamma-subunit gene. Only the distantly 5' pair of hypersensitive sites is present in fibroblasts; the remainder is muscle specific. Two hypersensitive sites, one at the 3' end of the delta gene and one distantly 3', appear only after terminal muscle differentiation and, therefore, are developmentally regulated, similarly to receptor subunit mRNAs. All four muscle-specific hypersensitive sites are excellent candidates for regulatory loci for the delta- and gamma-subunit genes of mouse acetylcholine receptor.

Animals↗
PubMed1986

Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle.

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.

Animals↗
PubMed1984

Alpha and beta subunits of the nicotinic acetylcholine receptor contain covalently bound lipid.

Labeling of the BC3H1 muscle-like cell line with [3H] palmitate, followed by immunoprecipitation of the acetylcholine receptor, indicated that the alpha and beta subunits of the receptor contain covalently bound fatty acid. After acid hydrolysis, fatty acid methyl esters could be recovered from the isolated [3H]palmitate-labeled alpha subunit. Treatment of differentiated BC3H1 cells with cerulenin, an inhibitor of fatty acid and sterol synthesis and fatty acid acylation of proteins, resulted in a 50% inhibition in expression of the acetylcholine receptor on the cell surface under conditions where there was minimal inhibition of protein synthesis. We conclude that this previously undetected post-translational modification may play a role in assembly and/or surface expression of the acetylcholine receptor.

Animals↗
PubMed1984

Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line.

The accumulation of translatable acetylcholine receptor alpha-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of alpha-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an alpha-subunit-specific monoclonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of alpha-subunit mRNA is detectable in cells under these conditions. Furthermore, alpha-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Following growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable alpha-subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of alpha-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abundance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the level of receptor subunit mRNA accumulation.

Animals↗
PubMed1984

Denervation supersensitivity in skeletal muscle: analysis with a cloned cDNA probe.

Motor neurons regulate the acetylcholine sensitivity of the muscles they innervate: denervated muscle fiber become "supersensitive" to acetylcholine, due to insertion of newly synthesized acetylcholine receptors (AChRs) in the plasma membrane. We used hybridization analysis with a cloned cDNA specific for AChR alpha-subunit to compare the abundance of AChR mRNA in innervated and denervated adult mouse muscles. Within 3 d of denervation, levels of AChR mRNA increased 100-fold; levels of actin mRNA changed little. The increase in AChR mRNA level was sufficiently large and rapid to account for denervation supersensitivity.

Actins↗
PubMed1983

Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line.

The regulation of acetylcholine receptor synthesis, assembly, and cell surface expression in response to serum and cell growth in the BC3H1 muscle-like cell line have been examined. In the presence of media containing low concentrations of serum, BC3H1 cells withdraw from the cell cycle and express high levels of cell surface acetylcholine receptors. Exposure of quiescent, differentiated BC3H1 cells to high concentrations of serum results in re-entry of cells into the cell cycle and a loss of surface acetylcholine receptors by a first order process (t1/2 = 8.6 h) which appears to be initiated within 1 h following serum addition. Under these conditions, the rate of synthesis of acetylcholine receptor alpha-subunits is not significantly reduced; however, the efficiency of assembly of alpha-subunits into functional acetylcholine receptors is significantly less than that observed in quiescent cells. The receptors which are assembled following serum stimulation of quiescent cells are not transported to the cell surface, nor do they accumulate within the cells in a functional form (defined by the ability to bind alpha-bungarotoxin) in the cells. We conclude that reinitiation of cell division in quiescent cultures of BC3H1 cells results in alterations in several post-translational processing steps required for the surface expression of acetylcholine receptors.

Animals↗
PubMed1983

Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro.

The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor. Evidence from peptide maps and the effect of co-translational incubation with dog pancreas microsomes suggests that the in vitro product differs in two ways from native alpha subunit: 1) it is synthesized with an NH2-terminal signal peptide which is removed in vivo, and 2) the in vitro product is not glycosylated. We have characterized the alpha subunit mRNA activity by using a quantitative the membrane-bound polysome fraction. It is poly(A+) and approximately 2000 nucleotides long. Finally, we have shown that in BC3H-1 cells, alpha subunit mRNA is regulated developmentally. We detected a 10-fold increase in the relative abundance of alpha subunit mRNA in cells which had undergone the transition from log phase growth to differentiated myoblast.

Animals↗
PubMed1983

Assembly in vivo of mouse muscle acetylcholine receptor: identification of an alpha subunit species that may be an assembly intermediate.

We have studied assembly of acetylcholine receptor in vivo using subunit-specific monoclonal antibodies and immunoprecipitation with alpha-bungarotoxin and antitoxin. We have identified three distinct forms of the alpha subunit. The newly synthesized alpha subunit species has a sedimentation coefficient of 5S and is recognized only by antibody specific for SDS-denatured alpha subunit. We have called this species alpha 61. The 5S alpha Tx species is not associated with beta subunits and is probably monomeric. alpha Tx is formed from alpha 61 with a half-time of 15 min and an efficiency of approximately equal to 30%. Formation of alpha Tx involves a conformational change, and we suggest that this conformation is dependent upon or stabilized by disulfide bond formation. The assembly of alpha Tx with beta subunits (and probably gamma and delta) into a 9S complex appears to be an efficient but slow process requiring more than 90 min. Unassembled alpha 61 subunits are degraded rapidly. However, subunit degradation is a result of failure to assemble, rather than its cause.

Animals↗
PubMed1983

cDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1.

Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.

Animals↗
PubMed1983

The regulation of acetylcholine receptor expression in mammalian muscle.

The synthesis of functional AChRs can be described as a pathway leading from the translation of subunit mRNAs to the plasma membrane forms of extrajunctional and junctional receptors (Fig. 9). We have not included in this scheme pretranslational steps for the synthesis and processing of RNA coding for receptor subunits because very little is known about such processes. Several aspects of Figure 9 are worthy of note: It is now well established that polypeptide synthesis is initiated on free cytoplasmic polysomes and that once sufficient nascent subunits bearing signal peptides at the amino terminus is formed, polysomes assemble with the membranes of the rough endoplasmic reticulum via a mechanism that employs the signal recognition particle (Anderson et al. 1982). Nascent subunits undergo cotranslational insertion through the rough endoplasmic reticulum membrane, signal peptide removal, and core glycosylation (Anderson and Blobel 1981; Merlie et al. 1981; Anderson et al. 1982; Sebbane et al. 1983). Anderson and Blobel (this volume) have demonstrated that subunits synthesized in vitro and inserted into membrane vesicles do not undergo heterologous subunit-subunit associations. We have shown that alpha- and beta-subunits newly synthesized in vivo are not associated with each other. Our data indicate that the alpha-subunit is initially present in vivo in a conformation that is radically different from its native conformation in the mature receptor complex. We assume that beta-, gamma-, and delta-subunits also are synthesized as conformationally "immature" forms, but verification of this point must await the availability of new monoclonal antibody specificities. Our data indicate that only a fraction of the newly synthesized alpha-subunit undergoes conformational maturation to the 5S species which binds both alpha-bungarotoxin and anti-main immunogenic region monoclonal antibodies. alpha-Subunits synthesized during a 5-minute pulse labeling require 30 minutes for completion of this process. alpha-Subunits that do not undergo conformational maturation are degraded rapidly (t1/2 = 0.5 hr) ( Merlie et al. 1982). Assembly of alpha- and beta-subunits synthesized during a 5-minute pulse labeling lags for approximately 30 minutes and is not complete until 90 minutes. Finally, assembled receptors are transported to the surface and appear in the plasma membrane. These processes occur during expression of AChRs in differentiated myoblasts. We do not know how undifferentiated myogenic cells, in vivo or in tissue culture, differ with regard to any of these steps.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Inhibition of glycosylation with tunicamycin blocks assembly of newly synthesized acetylcholine receptor subunits in muscle cells.

We have characterized the oligosaccharide chains of the alpha subunit of acetylcholine receptor of the clonal mouse muscle cell line BC3H-1 by their sensitivity to end-beta-N-acetylglucosaminidase H and by comparison of the native glycosylated polypeptide with the nonglycosylated form made in tunicamycin-treated cells. These studies indicate that the native alpha subunit has a single N-asparagine-linked oligosaccharide chain of the "high mannose" or "simple" type. Furthermore, these results considered in light of our previous characterization of the alpha subunit synthesized in vitro suggest that the alpha subunit contains no "complex"-type N-linked oligosaccharide chains. We have investigated the role of glycosylation in the biogenesis of the acetylcholine receptor. Receptor biogenesis in normal cells involves the assembly of newly synthesized alpha subunits into a form active for binding alpha-bungarotoxin. This process is only 30% efficient and is complete by 30 min postsynthesis. When glycosylation is inhibited by tunicamycin, alpha subunit synthesis is inhibited only slightly but assembly into an alpha-bungarotoxin binding species is reduced dramatically.

Animals↗
PubMed1981

Acetylcholine receptor synthesis from membrane polysomes.

We have established conditions for the fractionation of cytoplasmic and membrane-bound polyribosomes from the clonal mouse cell line BC3H-1. Polyribosome fractions are obtained in good yield and purity. They are active in protein synthesis when incubated with nuclease-treated rabbit reticulocyte lysates, and we have demonstrated that the cytoplasmic and membrane-bound fractions direct the synthesis of distinctly different sets of proteins. Using immunoprecipitation and sodium dodecyl sulfate gel analysis, we have shown that the membrane-bound but not the cytoplasmic polyribosomes direct the synthesis of two protein species (Mr = 39,000 nd 42,000) which are homologous to the native alpha subunit of acetylcholine receptor. Peptide maps suggest that the two species synthesized in vitro may correspond to the nonglycosylated and glycosylated forms, respectively, of the alpha subunit.

Animals↗
PubMed1981

Acetylcholine receptor subunits transit a precursor pool before acquiring alpha-bungarotoxin binding activity.

We have developed methods for the rapid immunoprecipitation of acetylcholine receptor from the clonal mouse cell line BC3H-1. One antiserum, anti-alpha-bungarotoxin, precipitates receptors to which alpha-bungarotoxin is bound. A second antiserum prepared against sodium dodecyl sulfate-denatured receptor precipitates receptor polypeptides independent of toxin binding activity. We have used these sera to analyze the products of synthesis in vivo after short pulses of [35S]methionine. Acetylcholine receptor polypeptides of synthesized during a 5-min pulse require 15 min before they become fully active for alpha-bungarotoxin binding. These experiments identify an early step in the assembly of functional acetylcholine receptor and suggest a novel mechanism by which receptor synthesis may be controlled post-translationally.

Animals↗
PubMed1979

Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies.

Acetylcholine receptor located at the neuromuscular synapse of normal innervated adult muscle fibers is extremely stable metabolically. We have studied the kinetics of receptor degradation in both normal innervated and denervated rat diaphragms in organ culture. These studies show that degradation of receptor-bound 125I-alpha-bungarotoxin is a valid measure of junctional receptor degradation. Degradation of junctional receptor is similar or identical to degradation of extrajunctional receptor in many ways: 1) both require energy, 2) both are inhibited by specific lysosomal protease inhibitors, 3) both are inhibited by treatment with colchicine, and 4) both are stimulated by treatment with anti-acetylcholine receptor antibodies. The one important distinction between degradation of junctional and extrajunctional receptor is a 10-fold difference in rate constant for the process.

Acetylcholine↗
PubMed1979

Degradation of acetylcholine receptor in diaphragms of rats with experimental autoimmune myasthenia gravis.

The degradation of acetylcholine receptor observed in denervated and innervated normal rat diaphragms in organ culture is stimulated by exogenous antireceptor serum. In this paper we demonstrate that diaphragms from rats with experimental autoimmune myasthenia gravis contain reduced amounts of acetylcholine receptor. Acetylcholine receptor from myasthenic, but not from normal, rats has antibody bound to it and is degraded at an accelerated rate. We conclude that in the chronic phase of experimental autoimmune myasthenia gravis increased acetylcholine receptor degradation can be accounted for by a mechanism involving antigenic modulation, and that such a process can contribute to the clinical symptoms of impaired neuromuscular transmission.

Acetylcholine↗
PubMed1978

Skeletal muscle acetylcholine receptor. Purification, characterization, and turnover in muscle cell cultures.

Acetylcholine receptor has been purified from embryonic skeletal muscle cells grown and allowed to differentiate in tissue culture. The polypeptide composition of purified receptor has been determined by two-dimensional electrophoresis. The purest preparations are composed of a single Mr = 41,000 class of polypeptide which exhibits some charge heterogeneity. By high resolution two-dimensional electrophoresis a spot corresponding to acetylcholine receptor was localized among total proteins of muscle membrane extracts. Synthesis of this component is shown to be developmentally regulated. Quantitative analysis of receptor synthesis and degradation has led to the conclusion that receptor is one of a class of proteins whose synthesis is tightly regulated during terminal steps of myogenesis.

Acetylcholine↗