cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

Sex-dependent and independent expression of the P-glycoprotein isoforms in Chinese hamster

P-glycoprotein (Pgp) is a small family of membrane proteins which belongs to a superfamily of energy-dependent membrane transport proteins identified in phylogenetically distant species, from bacteria to man. Among mammalian species, some of the Pgp isoforms can mediate multidrug resistance by acting as an energy-dependent drug efflux pump. However, the physiologic functions of the Pgp isoforms have not been defined. In this study we examined the expression of the three hamster Pgp isoforms in normal hamster tissues, by using isoform-specific monoclonal antibodies in a competitive immunohistochemical assay. We showed that each Pgp isoform is predominantly expressed in a small, distinct group of differentiated cells, where it is likely to function in specific secretory pathways. The expression of the Pgp isoforms appears to be tightly regulated and, at least in some cells, under complex hormonal control. Furthermore, there is a striking sex difference in Pgp content of the adrenal cortex. These findings are important for the ultimate understanding of the normal physiologic roles of the Pgp gene family members.     

Designed Ankyrin Repeat Proteins (DARPins) as Novel Isoform-Specific Intracellular Inhibitors of c-Jun N-Terminal Kinases

The c-Jun N-terminal kinases (JNKs) are involved in many biological processes such as proliferation, differentiation, apoptosis, and inflammation and occur in highly similar isoforms in eukaryotic cells. Isoform-specific functions and diseases have been reported for individual JNK isoforms mainly from gene-knockout studies in mice. There is, however, a high demand for intracellular inhibitors with high selectivity to improve the understanding of isoform-specific mechanisms and for use as therapeutic tools. The commonly used JNK inhibitors are based on small molecules or peptides that often target the conserved ATP binding site or docking sites and thus show only moderate selectivity. To target novel binding epitopes, we used designed ankyrin repeat proteins (DARPins) to generate alternative intracellular JNK inhibitors that discriminate two very similar isoforms, JNK1 and JNK2. DARPins are small binding proteins that are well expressed, stable, and cysteine-free, which makes them ideal candidates for applications in the reducing intracellular environment. We performed ribosome display selections against JNK1α1 and JNK2α1 using highly diverse combinatorial libraries of DARPins. The selected binders specifically recognize either JNK1 or JNK2 or both isoforms in vitro and in mammalian cells. All analyzed DARPins show affinities in the low nanomolar range and isoform-specific inhibition of JNK activation in vitro at physiological ATP concentrations. Importantly, DARPins that selectively inhibit JNK activation in human cells were also identified. These results emphasize the great potential of DARPins as a novel class of highly specific intracellular inhibitors of distinct enzyme isoforms for use in biological studies and as possible therapeutic leads.     

Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.     

Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes

Myosin II is a hexameric protein complex consisting of two myosin heavy chains, two myosin essential light chains and two myosin regulatory light chains. Multiple subunit isoforms exist, allowing great diversity in myosin II composition which likely impacts on its contractile properties. Little is known about the evolutionary origin, expression pattern and function of myosin regulatory light chain (MLC2) isoforms. We analysed the evolutionary relationship between smooth muscle (sm), nonmuscle (nm) and nonmuscle-like (nml) MLC2 genes, which encode three homologous proteins expressed in nonmuscle cells. The three genes arose by successive gene duplication events. The high sequence similarity between the tandemly arranged nm- and nml-MLC2 genes is best explained by gene conversion. Urea/glycerol-polyacrylamide gel electrophoresis and RNA analysis were employed to monitor expression of sm-, nm- and nml-MLC2 in human and mouse cell lines. Conspicuous differences between transformed and non-transformed cells were observed, with sm-MLC2 being suppressed in Ras-transformed cells. Our findings shed light on the evolutionary history of three homologous MLC2 proteins and point to isoform-specific cell growth-related roles in nonmuscle cell myosin II contractility.     

Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change

It is increasingly clear that alterations in Na+-K+-ATPase kinetics to fit the demands in specialized cell types is vital for the enzyme to execute its different physiological roles in diverse tissues. In addition to tissue-dependent expression of isoforms of the conventional subunits, alpha and beta, auxiliary FXYD proteins appear to be essential regulatory components. The present study identified genes belonging to this family in Atlantic salmon by analysis of expressed sequence tags. Based on the conserved domain of these small membrane proteins, eight expressed FXYD isoforms were identified. Phylogenetic analysis suggests that six isoforms are homologues to the previously identified FXYD2, FXYD5, FXYD6, FXYD7, FXYD8, and FXYD9, while two additional isoforms were found (FXYD11 and FXYD12). Using quantitative PCR, tissue-dependent expression of the different isoforms was analyzed in gill, kidney, intestine, heart, muscle, brain, and liver. Two isoforms were expressed in several tissues (FXYD5 and FXYD9), while six isoforms were distributed in a discrete manner. In excitable tissues, two isoforms were highly expressed in brain (FXYD6 and FXYD7) and one in skeletal muscle (FXYD8). In osmoregulatory tissues, one isoform was expressed predominantly in gill (FXYD11), one in kidney (FXYD2), and one equally in kidney and intestine (FXYD12). Expression of several FXYD genes in kidney and gill differed between fresh water and seawater salmon, suggesting significance during osmoregulatory adaptations. In addition to identifying novel FXYD isoforms, these studies are the first to show the tissue dependence in their expression and modulation by salinity in any teleosts.     

Wnt-16a, a novel Wnt-16 isoform, which shows differential expression in adult human tissues

The WNT genes encode a large family of secreted glycoprotein signalling molecules important from the earliest stages of development through to the adult. We have identified a novel isoform of the recently described WNT family member, Wnt16, following analysis of chromosome 7q31 genomic sequence. We find differential organisation of Wnt16 with the generation of two mRNA isoforms, Wnt16a and Wnt16b. These isoforms differ in the composition of their 5'-UTR and first exons and show evidence of differential expression. In normal human tissues, Wnt16a is expressed at significant levels only in the pancreas, whereas Wnt16b is expressed more ubiquitously with highest levels in adult kidney, placenta, brain, heart, and spleen. Wnt16 is one of a growing number of WNT genes showing evidence of distinct isoforms. We present evidence to suggest that these isoforms may be regulated from alternative promoters and discuss the potential functional differentiation afforded by these WNT isoforms. This may reveal subtle new mechanisms of regulation of WNT expression and function.