Rat Skeletal Muscle cDNA Research Articles

SRP-35 A Putative NAD(P)H Binding Protein Of Skeletal Muscle Sarcoplasmic Reticulum Membrane

SRP-35 (sarcoplasmic reticulum protein of 35 kDa) is a newly identified integral membrane protein constituent of skeletal muscle sarcoplasmic reticulum. We have deduced the primary structure of the protein from cDNA clones isolated from mouse and rat skeletal muscle cDNA libraries. Primary sequence prediction analysis indicates that the NH2- terminal sequence of SRP-35 encompasses a transmembrane spanning segment or a signal sequence. In addition, SRP-35 is homologous to proteins belonging to the short-chain dehydrogenase/reductases family. Members of this protein family has two domains: the first involved in binding the nucleotide co-factor NAD(P)H, the second responsible for the catalysis of the substrate. SRP35 contains only a putative NAD(P)H binding site. Analysis of tissue distribution of SRP-35 by western blot analysis with affinity purified Ab shows that SRP-35 expression is specific for skeletal muscle since our Ab did not stain any protein in other tissues including heart, brain, liver, kidney, lung, spleen and stomach. Immunohistochemistry of primary cultured mouse myotubes transfected with SRP-35 EGFP construct indicates that SRP-35 is distributed on sarco(endo)plasmic reticulum membranes. Staining of western blot of sarcoplasmic reticulum membrane subfractions isolated from adult mouse skeletal muscle revealed that SRP-35 is associated with heavy sarcoplasmic reticulum. In addition, we found that SRP-35 is an integral membrane protein since it was extracted neither by NaCO3 nor by high salt treatment of isolated sarcoplasmic reticulum membrane, but was solubilised by non-ionic detergent such as CHAPS, DDM and DHPC. We propose that , SRP-35 protein might provide the co-factor to enzymes involved in the generation of local reactive oxygen species within cellular subdomains

Translationally Controlled Tumor Protein Interacts with the Third Cytoplasmic Domain of Na,K-ATPase α Subunit and Inhibits the Pump Activity in HeLa Cells

Translationally controlled tumor protein (TCTP) is a growth-related protein under transcriptional as well as translational control. We screened a rat skeletal muscle cDNA library using yeast two-hybrid system and found that TCTP interacts with the third large cytoplasmic domain of alpha1 as well as alpha2 isoforms of Na,K-ATPase, believed involved in the regulation of Na,K-ATPase activity. Interaction between TCTP and Na,K-ATPase was confirmed by coimmunoprecipitation in yeast and mammalian cells. We also showed, using (86)Rb(+) uptake assay, that overexpression of TCTP inhibited Na,K-ATPase activity in HeLa cells. Northern and Western blotting studies of HeLa cells transiently transfected with GFP-tagged TCTP showed that overexpression of TCTP did not change mRNA and protein levels of Na,K-ATPase. Recombinant TCTP protein purified from an Escherichia coli expression system inhibited purified HeLa cell plasma membrane Na,K-ATPase in a dose-dependent manner. Using deletion analysis, we also found that the C-terminal 102-172-amino-acid region of rat TCTP that contains the TCTP homology region 2 is essential for its association with, and inhibition of, Na,K-ATPase.

Ferrying Fuel to the Sodium Pump

Jung et al . investigated enhancement of Na + ,K + -ATPase (adenosine triphosphatase) activity by the actin-binding protein cofilin and discovered a novel regulatory mechanism in which cofilin recruits the glycolytic enzyme triose-phosphate isomerase (TPI) to provide a ready supply of ATP. Na + ,K + -ATPase plays a crucial role in cell volume regulation and electrical signaling by using energy from the hydrolysis of ATP, preferentially derived from glycolysis, to maintain Na + and K + gradients across the cell membrane. Cofilin, which, when dephosphorylated, promotes actin filament turnover and thereby plays a role in cellular processes that depend on actin dynamics, interacts with Na + ,K + -ATPase and enhances its activity. Jung et al . used a yeast two-hybrid assay to screen a rat skeletal muscle cDNA library and discovered that cofilin interacted with TPI. The association of cofilin, TPI, and Na + ,K + -ATPase was confirmed in vivo both by immunofluorescence and by Western analysis of immunoprecipitates of HeLa cells transfected with labeled cofilin and labeled TPI. Pharmacological analysis indicated that activation of Rho signaling pathways resulted in translocation of cofilin and TPI to the cell membrane and increased association with Na + ,K + -ATPase. Activating Rho or overexpressing cofilin increased Na + ,K + -ATPase activity, as assessed by ouabain-sensitive 86 Rb + uptake. However, inhibiting the Rho pathway, or overexpressing cofilin-S3A, a mutant form that could not be phosphorylated, attenuated Na + ,K + -ATPase activity. Pharmacological analysis indicated that Rho pathway activation led to decreased [Na] i and confirmed the dependence of Na + ,K + -ATPase on glycolytic supplies of ATP, rather than ATP derived from oxidative phosphorylation. The data suggest a role for phosphorylated cofilin, and for Rho, in regulation of the Na + ,K + -ATPase. J. Jung, T. Yoon, E. C. Choi, K. Lee, Interaction of cofilin with triose-phosphate isomerase contributes glycolytic fuel for Na + ,K + -ATPase via Rho-mediated signaling pathway. J. Biol. Chem. 277 , 48931-48947 (2002). [Abstract] [Full Text]

Interaction of Cofilin with Triose-phosphate Isomerase Contributes Glycolytic Fuel for Na,K-ATPase via Rho-mediated Signaling Pathway

We reported previously that cofilin, an actin-binding protein, interacts with Na,K-ATPase and enhances its activity (Lee, K., Jung, J., Kim, M., and Guidotti, G. (2001) Biochem. J. 353, 377-385). To understand the nature of this interaction and the role of cofilin in the regulation of Na,K-ATPase activity, we searched for cofilin-binding proteins in the rat skeletal muscle cDNA library using the yeast two-hybrid system. Several cDNA clones were isolated, some of which coded for triose-phosphate isomerase, a glycolytic enzyme. The interaction of cofilin with triose-phosphate isomerase as well as Na,K-ATPase was confirmed by immunoprecipitation and confocal microscopy in HeLa cells. Cofilin was translocated to the plasma membrane along with triose-phosphate isomerase by the Rho activator lysophosphatidic acid but not by the p160 Rho-associated kinase inhibitor Y-27632, suggesting that the phosphorylated form of cofilin bound to TPI interacts with Na,K-ATPase. Ouabain-sensitive (86)Rb(+) uptake showed that Na,K-ATPase activity was increased by the overexpression of cofilin and lysophosphatidic acid treatment, but not by the overexpression of mutant cofilin S3A and Y-27632 treatment. Pretreatment with the glycolytic inhibitor iodoacetic acid caused a remarkable reduction of Na,K-ATPase activity, whereas pretreatment with the oxidative inhibitor carbonyl cyanide m-chlorophenylhydrazone caused no detectable changes, suggesting that the phosphorylated cofilin is involved in feeding glycolytic fuel for Na,K-ATPase activity. These findings provide a novel molecular mechanism for the regulation of Na,K-ATPase activity and for the nature of the functional coupling of cellular energy transduction.

Insulin regulation of a novel WD-40 repeat protein in adipocytes.

A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

Interaction of the α subunit of Na,K-ATPase with cofilin

The α1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the α subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive 86Rb+ uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

Interaction of the alpha subunit of Na,K-ATPase with cofilin.

The alpha1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the alpha subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive (86)Rb(+) uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

CDNA cloning of rat mitochondrial cyclophilin

Human cyclophilin-3 (hCyp-3) cDNA was used to screen a rat skeletal muscle cDNA library which led to the isolation of a full-length cDNA encoding the rat homologue. The derived amino acid sequence showed 89.5% identity with the human sequence, with major differences being restricted to the mitochondrial targeting sequence. The N-terminal sequence of the purified rat liver mitochondrial cyclophilin (CyP-D) corresponded to that derived from the cDNA following 30 amino acids of targeting sequence. This confirms that hCyp-3 encodes mitochondrial matrix CyP (CyP-D), which plays a crucial role in the mitochondrial permeability transition. CyP-D mRNA of a single sized (1.5 kb) was shown by Northern blotting to be present in liver, heart, skeletal muscle and brain. © 1997 Elsevier Science B.V. All rights reserved.

Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII α of cAMP-dependent protein kinase

The gene for the regulatory subunit RII α of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5′-flanking region (1.2 kb) and exon 1 of the human RII α gene. S1 nuclease mapping and primer extension experiments revealed the presence of a major transcriptional start site located 208 nucleotides upstream of start for translation. The 5′-flanking region of the RII α gene did not contain a TATA box and was highly G/C-rich. A basal promoter directing high levels of chloramphenicol acetyl transferase (CAT) activity was identified in the 5′-flanking sequence. Several potential binding sites for transcription factors were identified in this region, which may be responsible for the germ cell-specific regulation of this gene. We have previously reported that the human testis RII α cDNA contains a region (amino acids 45-75) with little or no homology to the corresponding rat skeletal muscle cDNA (Øyen, O., Myklebust, F., Scott, J.D., Cadd, G.G., McKnight, G.S., Hansson, V. and Jahnsen, T. (1990) Biol. Reprod. 43, 46–54). We examined whether this difference could arise due to organ-specific splice mechanisms or represented a species difference. We show that the low homology region of the human RII α cDNA resides entirely within exon 1, and does not originate from a tissue-specific alternate splicing of this distinct region.

Cloning and nucleotide sequence of a cDNA encoding the rat triosephosphate isomerase

A gene coding for triosephosphate isomerase (TPI) from a rat skeletal muscle cDNA library was cloned and its nucleotide sequence was determined. The 1,348-bp cDNA clone contains 24 bp 5′ noncoding region, the entire 750 bp coding region corresponding to a protein of 249 amino acids, 547 bp 3′ noncoding region and part of a poly(A) tail. It also contains a polyadenylation signal, AATAAA, starting from 17 bp upstream of the poly(A) tail. The calculated molecular weight of rat TPI is 27.8 kDa and the net charge is +4. The deduced amino acid sequence from rat TPI cDNA sequence has 93% and 94% homology with that of mouse and human clones, respectively. The amino acids at the residue of Asn12, Lys14, His96, Glu166, His96, His101, Ala177, Tyr165, Glu130, Tyr209, and Ser212 in catalytic site are completely identical, confirming that the functional residues in TPI proteins are highly conserved throughout evolution. The most profound characteristic of rat TPI enzyme, compared with other TPIs, is that there are five cysteine substitutions at the residue of 21, 27, 159, 195 and 204. A Glu123 instead of Gly was found in rabbit, rhesus, mouse and human sequences. Through the method of RT-PCR, the mRNA transcription level of TPI gene was found to be different among various tissues and was highest in muscle.

Cloning of cDNA for the gamma-subunit of mammalian translation initiation factor 2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2.

Peptide sequence data were obtained from rabbit protein synthesis initiation factor subunit eIF2B gamma. Searching the database of expressed sequence tags (dbEST) revealed nucleotide sequences potentially encoding human eIF2B gamma that contained peptides corresponding to those from the rabbit subunit. PCR primers were derived from these sequences and used to generate a probe. This was used to screen a rat skeletal muscle cDNA library, and a clone encoding rat eIF2B gamma was isolated. This cDNA gave a product in coupled transcription/translation that co-migrated with the gamma-subunit of purified eIF2B under SDS/PAGE. The sequence of this rat eIF2B gamma cDNA is reported. The protein sequence shows homology with that of yeast eIF2B gamma (the GCD1 gene product). We have also identified an open reading frame from the Caenorhabditis elegans genome project that probably encodes the gamma-subunit of C. elegans eIF2B. All these sequences show similarity to nucleotidyl- and acyltransferases, as previously reported for GCD1 [Koonin (1995) Protein Sci. 4, 1608-1617], and contain conserved motifs potentially involved in nucleotide binding. They also contain "I-patch' motifs: isoleucine-rich hexamer repeats that have been associated with the binding of acyl groups in bacterial acyltransferases. The roles of these motifs are discussed in relation to the known properties of eIF2B.

Cloning and characterization of cDNAs for novel proteins with glutamic acid-proline dipeptide tandem repeats

cDNAs with identical 3′ sequences containing a hexanucleotide repeat -(GAGCCG) 9- were isolated from rat pheochromocytoma and brain cDNA libraries. The cDNA with the longest open reading frame codes for a protein of 24.6 kDa containing a 16-fold -(Glu-Pro)-dipeptide repeat within a glutamate and proline rich region at its deduced C-terminus. cDNAs with the identical 3′ sequence and a divergent 5′ sequence were isolated from a rat skeletal muscle cDNA library. The latter are predicted to code for a protein of 15.5 kDa with a C-terminal repetitive domain identical to that in the pheochromocytoma and brain cDNAs. The cDNAs recognize a 1.8 kb mRNA species present in a variety of tissues, being particularly abundant in cardiac and skeletal muscle.

CDNA cloning of MCT1, a monocarboxylate transporter from rat skeletal muscle

PCR was used to amplify the coding region of CHO MCT1 cDNA. This was then used to screen a rat skeletal muscle cDNA library which lead to the isolation of a full length cDNA encoding MCT1 from rat. The cDNA derived amino acid sequence shows 94% and 86% identity to CHO and human MCT1, respectively.

Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase.

We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from rat skeletal muscle cDNA by the polymerase chain reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDNA from the 5'-end portion of the gene was further generated by polymerase chain reaction. These two cDNAs were used to construct a cDNA encoding the entire sequence of rat sialidase. The composite sequence encodes an open reading frame of 379 amino acids that include all sequenced peptides. Although the deduced amino acid sequence is not largely similar to those of bacterial and parasite sialidases, it contains two Asp blocks, the conserved sequence of the sialidases from these microorganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitated by antiserum against the cytosolic sialidase of rat skeletal muscle.

Nucleotide sequence of cDNA encoding subunit VIII of cytochrome c oxidase from rat heart

In this study, the complete cDNA of subunit VIII-h of rat cytochrome c oxidase is presented. A rat skeletal muscle cDNA library was screened with a 132 bp fragment of the cDNA of rat COX subunit VIII-h. Four positive clones were sequenced in both directions.

Identification of skeletal muscle protein-tyrosine phosphatases by amplification of conserved cDNA sequences

Specific protein-tyrosine phosphatase (PTPase) enzymes that regulate signal transduction by the insulin receptor in target tissues have not been identified. We evaluated the expression of PTPase homologs in skeletal muscle since this tissue is the major site of insulin-mediated glucose disposal in vivo. A rat skeletal muscle cDNA pool was prepared with a set of degenerate oligonucleotide primers and PTPase cDNA sequences were amplified using pairs of “guess-mers” that were deduced from highly conserved residues within the known catalytic domains of these enzymes. Sequences encoding three “receptor-like” transmembrane PTPases were identified and two of these (known as LAR and LRP) were confirmed to be expressed in muscle by subsequent cDNA library screening and Northern blot analysis. The expression of the LAR and LRP PTPases in skeletal muscle suggests that these enzymes might have a role in the regulation of insulin action in muscle and other insulin-sensitive tissues.

Pretranslational mechanisms determine the type of potassium channels expressed in the rat skeletal and cardiac muscles

We have cloned a cDNA (RMK2) coding for a Shaker type delayed rectifier K+ channel from a rat skeletal muscle cDNA library. The clone encodes a putative protein of 602 amino acids, identical with a rat brain K+ channel Kv1 (Swanson, R., Marshall, R., Smith, J. S., Williams, J. B., Boyle, M. B., Folander, K., Luneau, C. J., Antanavage, J., Oliva, C., Burhow, S. A., Bennet, C., Stein, R. B., and Kaczmarek, L. K. (1990) Neuron 4, 929-939). Northern blot analysis showed that RMK2 is expressed in skeletal and cardiac muscle. RNase protection analysis showed that the 3'-noncoding regions of the brain, cardiac, and skeletal muscle RMK2 transcripts are identical. Cloning of the gene confirmed that the protein is encoded by a single exon (Swanson et al. (1990) Neuron 4, 929-939). We expressed RMK2 in Xenopus oocytes and showed that it encodes noninactivating delayed rectifier K+ channels, resistant to block by external tetraethylammonium, with a small unitary conductance of 8.0 picosiemens. Coinjection of RMK2 and RCK1 (RMK1) (Baumann, A., Grupe, A., Ackermann, A., and Pongs, O. (1988) EMBO J. 7, 2457-2463; Koren, G., Liman, E. R., Logothetis, D. E., Nadal-Ginard, B., and Hess, P. (1990) Neuron 4, 39-51) into Xenopus oocytes resulted in the expression of currents that have tetraethylammonium inhibition curves that differ from the linear combination of inhibition curves of the two types expressed individually. Thus, RMK2 and RCK1 (RMK1) can form heteromultimers. RNA blot hybridization analysis revealed that the RMK2 transcript is developmentally regulated in a different manner in the rat skeletal muscle, ventricle, and atrium.

Regulation of muscle sodium channel transcripts during development and in response to denervation

We have recently described the cloning and functional expression of a new sodium channel subtype, μI, isolated from a denervated rat skeletal muscle cDNA library. In studies described here, we have used RNase protection and Northern blot analyses to examine the expression of μI mRNA in different tissues and in neonatal, adult, and adult denervated muscle. We found that μI transcripts were not expressed in brain or heart, or in the myogenic cell line L6, even after differentiation to myotubes. Transcripts for μI were present at low levels in neonatal skeletal muscle and increased to maximum levels in adult tissue, paralleling the expression of tetrodotoxin (TTX)-sensitive sodium currents. Surprisingly, denervation of adult muscle was also followed by a rise in μI mRNA, at a time when TTX-insensitive currents reappear. These results show that expression of this channel subtype is regulated by tissue type, development, and innervation.

Coordinate regulation of RNAs encoding two isoforms of the rat muscle nicotinic acetylcholine receptor beta-subunit.

The nicotinic acetylcholine receptor (nAchR) mediates communication between nerve and skeletal muscle. The properties, levels and distribution of these receptors change during development of the neuromuscular junction. These changes may be due, in part, to expression of different gene products. We are using nuclease protection experiments and cDNA cloning to identify the RNA transcripts that encode nAchRs in rat muscle. This analysis has identified two beta-subunit mRNAs. Complementary DNAs corresponding to these two RNAs have been isolated from a rat skeletal muscle cDNA library. Based on nucleotide sequence analysis, these RNAs differ by 9 bases in their 5' coding sequence. The levels of both mRNAs change similarly during muscle development and upon denervation of adult skeletal muscle. These two beta-subunit-RNAs probably result from the use of different exon/intron splice sites in the beta-subunit gene.

Regulation of glycolytic enzyme RNA transcriptional rates by oxygen availability in skeletal muscle cells.

Cytoplasmic beta-actin and five glycolytic enzyme cDNAs were isolated from a rat skeletal muscle cDNA library and together with a genomic clone of rat cytochrome c were used as probes to quantitate the respective RNA transcription rates in isolated nuclei run off transcription assays from stationary cells cultured under normal or 2% oxygen. The transcription rates of lactate dehydrogenase, pyruvate kinase, triosephosphate isomerase and aldolase increased by 2-5 fold during the 72 hr exposure to 2% oxygen. There was a small increase in actin RNA transcription while both cytochrome c and glyceraldehyde-3-phosphate dehydrogenase RNA transcription rates decreased. Since previous studies demonstrated an increase in steady state glyceraldehyde-3-phosphate dehydrogenase RNA during low O2 exposure it is concluded that the level of this RNA is regulated post transcriptionally whereas the other four glycolytic enzyme RNAs are regulated at least partially at the level of transcription by oxygen availability. The relative transcriptional rates of the RNAs in this study are related to their cellular RNA and protein concentrations.