Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 165-173 ◽  
Author(s):  
C.H. Barton ◽  
G. Dickson ◽  
H.J. Gower ◽  
L.H. Rowett ◽  
W. Putt ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Cell Surface ◽  
Protein Sequence ◽  
Single Copy ◽  
In Vitro Transcription ◽  
Full Length ◽  
Covalent Attachment ◽  
Size Difference

Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 × 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 × 10(3). The size difference between the 125 × 10(3). The size difference between the 125 × 10(3) Mr skeletal muscle form and the 120 × 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 × 10(3) which is processed by microsomal membranes to yield a 122 × 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 × 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.

Contraction-related stimuli regulate GLUT4 traffic in C2C12-GLUT4myc skeletal muscle cells

2010 ◽  
Vol 298 (5) ◽  
pp. E1058-E1071 ◽  
Author(s):  
Wenyan Niu ◽  
Philip J. Bilan ◽  
Shuhei Ishikura ◽  
Jonathan D. Schertzer ◽  
Ariel Contreras-Ferrat ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Nicotinic Acetylcholine Receptors ◽  
Cell Model ◽  
Cytosolic Calcium ◽  
Atpase Inhibitor ◽  
Cellular Models ◽  
Compound C

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C2C12 myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4 myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4 myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl- p-toluenesulfonamide. The gain in surface GLUT4 myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 β-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4 myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4 myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.

scholarly journals Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1971 ◽  
Vol 121 (5) ◽  
pp. 817-827 ◽  
Author(s):  
R. C. Hider ◽  
E. B. Fern ◽  
D. R. London
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Extensor Digitorum Longus ◽  
Extensor Digitorum ◽  
Longus Muscle ◽  
Kinetics Of

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.

Covalent attachment of amino acids to casein. 1. Chemical modification and rates of in vitro enzymic hydrolysis of derivatives

1979 ◽  
Vol 27 (5) ◽  
pp. 1098-1104 ◽  
Author(s):  
Antoine J. Puigserver ◽  
Lourminia C. Sen ◽  
Elvira Gonzales-Flores ◽  
Robert E. Feeney ◽  
John R. Whitaker
Keyword(s):  
Amino Acids ◽  
Chemical Modification ◽  
Covalent Attachment ◽  
Enzymic Hydrolysis ◽  
Hydrolysis Of

scholarly journals PTP1B Modulates the Association of β-Catenin with N-cadherin through Binding to an Adjacent and Partially Overlapping Target Site

2002 ◽  
Vol 277 (51) ◽  
pp. 49989-49997 ◽  
Author(s):  
Gang Xu ◽  
Carlos Arregui ◽  
Jack Lilien ◽  
Janne Balsamo
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Tyrosine Phosphatase ◽  
Target Site ◽  
Target Domain ◽  
Chick Retina ◽  
Cell Permeable Peptide ◽  
A Cell ◽  
Relationship Of

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to competein vitrobinding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function.

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

scholarly journals Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

2001 ◽  
Vol 75 (13) ◽  
pp. 6095-6106 ◽  
Author(s):  
Stephen J. Polyak ◽  
Khalid S. A. Khabar ◽  
Denise M. Paschal ◽  
Heather J. Ezelle ◽  
Gilles Duverlie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Response ◽  
Full Length ◽  
Interleukin 8 ◽  
Antiviral Resistance ◽  
Ns5a Protein ◽  
Terminal Amino

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

scholarly journals In Vitro Assembly of Sindbis Virus Core-Like Particles from Cross-Linked Dimers of Truncated and Mutant Capsid Proteins

2001 ◽  
Vol 75 (6) ◽  
pp. 2810-2817 ◽  
Author(s):  
Timothy L. Tellinghuisen ◽  
Rushika Perera ◽  
Richard J. Kuhn
Keyword(s):  
Capsid Protein ◽  
Sindbis Virus ◽  
Terminal Region ◽  
Full Length ◽  
Capsid Proteins ◽  
Size Difference ◽  
Cross Linking ◽  
Cross Link ◽  
Amino Terminal

ABSTRACT A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.

scholarly journals Identification of a talin binding site in the cytoskeletal protein vinculin.

1989 ◽  
Vol 109 (6) ◽  
pp. 2917-2927 ◽  
Author(s):  
P Jones ◽  
P Jackson ◽  
G J Price ◽  
B Patel ◽  
V Ohanion ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Cytoskeletal Protein ◽  
Translation System ◽  
Cos Cells ◽  
Cell Matrix ◽  
Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

scholarly journals In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus

1986 ◽  
Vol 83 (14) ◽  
pp. 5043-5047 ◽  
Author(s):  
T. Meshi ◽  
M. Ishikawa ◽  
F. Motoyoshi ◽  
K. Semba ◽  
Y. Okada
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
In Vitro Transcription ◽  
Full Length

scholarly journals Polymorphism in an androgen-regulated mouse gene is the result of the insertion of a B1 repetitive element into the transcription unit.

1986 ◽  
Vol 6 (1) ◽  
pp. 209-217 ◽  
Author(s):  
D King ◽  
L D Snider ◽  
J B Lingrel
Keyword(s):  
Inbred Mouse ◽  
Rna Polymerase Iii ◽  
Single Copy ◽  
Transcription Unit ◽  
Mouse Strains ◽  
Size Difference ◽  
Cdna Clones ◽  
S1 Nuclease ◽  
Nuclease Mapping

The single-copy RP2 gene in mice produces three major mRNAs, the abundances of which are significantly increased in the kidneys by the administration of testosterone. S1 nuclease analysis of the kidney mRNAs indicated that they differ in the lengths of their 3' untranslated regions as a result of the use of different polyadenylation sites. When the mRNAs from different inbred mouse strains were examined by Northern blot analysis, it was observed that the largest mRNA varies in size, whereas the sizes of the other mRNAs remain the same. In DBA/LiHa and DBA/2J mice, the largest mRNA is approximately 2,150 nucleotides long, whereas the corresponding mRNA in C57BL/6J and BALB/cJ mice is only 1,950 nucleotides in length. All of these strains also have RP2 mRNAs that are 1,450 and 1,350 nucleotides long. By S1 nuclease mapping and comparison of the sequence of cDNA clones representing these mRNAs in DBA/LiHa and C57BL/6J mice, we determined that this size difference or polymorphism observed in the largest mRNA is the result of the insertion of a member of the B1 family of repeats into the 3' untranslated region of the RP2 gene in DBA mice. This particular B1 repeat is transcribed by RNA polymerase III in vitro, and its transcriptional orientation is opposite to that of the RP2 transcript. The polymorphism described here is evidence for the mobility of B1 repetitive elements within the genome.

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