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J P Merlie

Publications and source records attributed to J P Merlie.

At least 73 records · Page 4Linked to original sources
PubMed1991

Mutagenesis of the 43-kD postsynaptic protein defines domains involved in plasma membrane targeting and AChR clustering.

The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.

Animals↗
PubMed1991

Selective expression of an acetylcholine receptor-lacZ transgene in synaptic nuclei of adult muscle fibers.

Acetylcholine receptors (AChRs) are highly concentrated in the postsynaptic membrane at the neuromuscular junction. To investigate mechanisms that lead to the formation or maintenance of this synaptic specialization, we generated transgenic mice in which regulatory elements from the AChR alpha or epsilon-subunit genes are linked to a gene for a reporter protein that is targeted to the nucleus (nlacZ). Both transgenes were selectively expressed and developmentally regulated in muscle; nuclei in both extrafusal (ordinary) and intrafusal (spindle) muscle fibers were labeled. Within individual muscle fibers from epsilon-nlacZ mice, nuclei near synaptic sites were nlacZ-positive, whereas extrasynaptic nuclei were nlacZ-negative. In contrast, nlacZ was expressed in both synaptic and extrasynaptic nuclei when under the control of regulatory elements from the AChR alpha-subunit gene; however, synaptic nuclei were somewhat more intensely stained than extrasynaptic nuclei in a minority of muscle fibers from these mice. Together, our results provide direct evidence for molecular differences between synaptic and extrasynaptic nuclei within a single cytoplasm, and suggest that the motor nerve regulates synapse formation by selectively affecting transcription in synaptic nuclei.

Animals↗
PubMed1991

Nerve-dependent modulation of acetylcholine receptor epsilon-subunit gene expression.

The relative abundance of mRNAs encoding the gamma- and epsilon-subunits of acetylcholine receptor changes in opposite directions during mouse development. Both gamma- and epsilon-mRNAs are expressed early in muscle development in vivo, and in aneural embryonic muscle and myogenic cell lines in vitro, though gamma-mRNA is at least 20-fold more abundant than epsilon-mRNA in these circumstances. While during normal development, gamma-mRNA decreases to an undetectable level by postnatal day 12, epsilon-mRNA first increases 10-fold between day 2 and day 12-15, then decreases to the level characteristic of adult muscle. We have found that the transition form gamma- to epsilon-mRNA is influenced by the levels of thyroid hormones. Indeed, high and low levels of thyroid hormones, respectively, accelerated and delayed the switch between gamma- and epsilon-mRNAs. Neither the dramatic postnatal rise in epsilon-mRNA nor its sensitivity to thyroid hormones was observed in denervated newborn animals. By contrast, denervation was without effect on epsilon-mRNA expression in adult muscle. These results suggest that, although not required for the initial activation of the epsilon-gene nor its maintenance in adult muscle, the nerve plays a major role in the perinatal regulation of epsilon-gene transcription.

Aging↗
PubMed1990

Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts.

We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha-bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha-bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.

Animals↗
PubMed1990

Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly.

The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin-treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR-expressing cells.

Animals↗
PubMed1990

S-laminin.

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Amino Acid Sequence↗
PubMed1989

Primary sequence of a motor neuron-selective adhesive site in the synaptic basal lamina protein S-laminin.

S-laminin, a novel homolog of laminin, is concentrated in a subset of basal laminae including the basal lamina that passes between motor nerve terminals and muscle fibers at the neuromuscular junction. Here we used recombinant fragments to localize a neuronal attachment site to the C-terminal 10% of s-laminin. We then used synthetic peptides spanning the active fragment to identify the primary sequence of the adhesive site as Leu-Arg-Glu (LRE): neurons attach to an immobilized LRE-containing peptide, and soluble LRE blocks attachment of neurons to the s-laminin fragment. Whereas ciliary ganglion neurons (which normally innervate muscle fibers) adhered well both to laminin and to an s-laminin fragment, sensory and central neurons and several neuronal cell lines all adhered well to laminin but poorly to the s-laminin fragment. Together, these results define a motor neuron-selective attachment site on s-laminin.

Amino Acid Sequence↗
PubMed1989

Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells.

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.

Amino Acids↗
PubMed1989

Isolation and characterization of the beta and epsilon subunit genes of mouse muscle acetylcholine receptor.

The genes coding for the beta and epsilon subunits of the mouse muscle nicotinic acetylcholine receptor (nAChR) were mapped by Southern blot analysis, and the entire loci for both genes cloned. The results indicate that they are single-copy genes. Both were sequenced to determine their size and structural organization. The beta subunit gene spans approximately 8 kilobases and is organized into 11 exons. A region containing cysteines, which are thought to form a disulfide bond and which are highly conserved, is encoded by one exon in all muscle acetylcholine receptor genes with the exception of the beta subunit gene, where it is split into two exons. The epsilon subunit gene spans 4.3 kilobases and contains 12 exons; it has the same structure as the gamma and delta nAChR genes. The intron-exon boundaries and exonic organization of the five known nAChR genes were compared. The analysis showed that the first 4 exons and the last exon of all muscle and brain nAChR subunit genes have the same boundaries, with the exception of a nAChR-related gene in Drosophila.

Amino Acid Sequence↗
PubMed1989

A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction.

A striking example of topographic specificity in synapse formation is the preferential reinnervation of original synaptic sites on denervated muscle fibres by regenerating motor axons. This specificity is mediated by the basal lamina of the synaptic cleft. A glycoprotein, s-laminin, has now been identified that is selectively associated with synaptic basal lamina and is recognized by motoneurons. Molecular cloning reveals that s-laminin is a novel homologue of laminin, a potent promoter of neurite outgrowth.

Base Sequence↗
PubMed1989

Neural regulation of gene expression by an acetylcholine receptor promoter in muscle of transgenic mice.

Motor neurons regulate the quantity and distribution of acetylcholine receptors (AChR) in the muscles they innervate. Here, we report that an AChR alpha subunit gene fragment contains cis-acting regulatory sequences that confer neural regulation as well as tissue-specific regulation of transcription. An 850 bp fragment from the 5' end of the chicken AChR alpha gene fused to the reporter gene, chloramphenicol acetyltransferase (CAT), has been introduced into the genomes of several lines of transgenic mice. Expression of CAT enzyme activity in these mice is tissue-specific; the onset of expression in embryonic muscle correlates well with that of many other muscle-specific proteins. Most importantly, CAT enzyme is down-regulated 100-fold soon after birth, an effect that can be completely reversed by denervation.

Animals↗
PubMed1989

Expression of RAPsyn (43K protein) and nicotinic acetylcholine receptor genes is not coordinately regulated in mouse muscle.

RAPsyn (also known as 43K protein), a mouse muscle protein localized to the synaptic membrane, is thought to be involved in the localization of nicotinic acetylcholine receptors at the neuromuscular junction. We have characterized the transcriptional regulation of the RAPsyn gene and the synthesis of the RAPsyn protein during muscle cell differentiation. Nuclear run-on experiments and RNAase protection analyses showed that mRNA encoding RAPsyn, but not the acetylcholine receptor subunits, is present in undifferentiated muscle cells. The RAPsyn protein present in undifferentiated and differentiated muscle cells cannot be distinguished by peptide maps, turnover rates, cellular subfractionation, or ability to incorporate myristate. Whereas the amount of acetylcholine receptor subunit mRNA is increased approximately 100-fold after denervation, the amount of RAPsyn mRNA is increased just 2- to 3-fold. We conclude that the expression of RAPsyn and the acetylcholine receptor is not coordinately regulated in mouse muscle.

Animals↗
PubMed1989

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor.

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.

Animals↗
PubMed1989

Myosin light chain enhancer activates muscle-specific, developmentally regulated gene expression in transgenic mice.

The rat myosin light chain (MLC)1/3 gene locus contains a potent muscle-specific enhancer, located downstream of the coding region, greater than 24 kilobases away from the MLC1 transcription start site. To assess the role of this enhancer in the activation of MLC expression during development, transgenic mice were generated carrying multiple copies of a MLC1 promoter-chloramphenicol acetyltransferase (CAT) transcription unit linked to a genomic fragment including the enhancer. CAT expression was detected in four mouse lines, up to 1000-fold higher in skeletal muscles than in other tissues. Activation of endogenous MLC1 transcription in these animals 4 days before birth was reflected in the onset of CAT transgene expression. This study identifies the transcriptional control elements necessary to activate the 21-kilobase MLC1/3 locus at the appropriate fetal stage and indicates that the MLC enhancer is sufficient to induce developmentally regulated expression from the MLC1 promoter exclusively in skeletal muscle cells.

Aging↗
PubMed1989

The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells.

Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes (Frail, D.E., L.S. Musil, a. Bonanno, and J.P. Merlie, 1989. Neuron. 2:1077-1086) led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with [35S]methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

Animals↗
PubMed1988

Identification of the mouse muscle 43,000-dalton acetylcholine receptor-associated protein (RAPsyn) by cDNA cloning.

The nicotinic acetylcholine receptor and a receptor-associated protein of 43 kDa are the major proteins present in postsynaptic membranes isolated from Torpedo electric organ. Immunochemical analyses indicated that a protein sharing antigenic determinants with the receptor-associated protein is also present at receptor clusters of muscle cell lines and postsynaptic membranes of vertebrate neuromuscular junctions. We now provide definitive proof that a homolog of the 43-kDa protein exists in mammals. Complimentary DNA clones encoding the complete protein sequence have been isolated from the mouse muscle cell line, BC3H1. We heretofore refer to these proteins as nicotinic receptor-associated proteins at synapses or N-RAP-syns. The deduced sequence of mouse RAPsyn has 412 amino acids and a molecular mass of 46,392 daltons. The overall identity with Torpedo RAPsyn is 70%; some regions are extremely well conserved and are therefore postulated to be functionally important. Important domains, including the amino terminus and a cAMP-dependent protein kinase phosphorylation site, are conserved between species. Several structural features are consistent with the proposal that RAPsyn is a peripheral membrane protein that associates with membranes by virtue of covalently bound myristate. Although multiple mRNAs were previously identified in Torpedo electric organ, RNA blot analysis reveals a single polyadenylated RAPsyn mRNA of approximately equal to 2.0 kilobases in newborn and 4-week-old mouse muscle. Finally, genomic DNA blot analysis indicates that a single N-RAPsyn gene is present in the mouse genome.

Amino Acid Sequence↗