Characterization of sorCS1, an alternatively spliced receptor with completely different cytoplasmic domains that mediate different trafficking in cells

We previously isolated and sequenced murine sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich domain. We now show that human sorCS1 has three isoforms, sorCS1a-c, with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b, whereas the a and c tails have no reported counterparts. Like the Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is synthesized as a proreceptor that is converted in late Golgi compartments by furin-mediated cleavage. Mature sorCS1 bound its own propeptide with low affinity but none of the ligands previously shown to interact with sortilin and sorLA. In transfected cells, about 10% of sorCS1a was expressed on the cell surface and proved capable of rapid endocytosis in complex with specific antibody, whereas sorCS1b presented a high cell surface expression but essentially no endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with completely different cytoplasmic domains that mediate different trafficking in cells.     

Structural and functional aspects of the multiplicity of Neu differentiation factors.

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.     

Identification and characterization of the two isoforms of the vertebrate H2A.Z histone variant

Histone variants play important roles in the epigenetic regulation of genome function. The histone variant H2A.Z is evolutionarily conserved from yeast to vertebrates, and it has been reported to have multiple effects upon gene expression and insulation, and chromosome segregation. Recently two genes encoding H2A.Z were identified in the vertebrate genome. However, it is not yet clear whether the proteins transcribed from these genes are functionally distinct. To address this issue, we knocked out each gene individually in chicken DT40 cells. We found that two distinct proteins, H2A.Z-1 and H2A.Z-2, were produced from these genes, and that these proteins could be separated on a long SDS–PAGE gel. The two isoforms were deposited to a similar extent by the SRCAP chromatin-remodeling complex, suggesting redundancy to their function. However, cells lacking either one of the two isoforms exhibited distinct alterations in cell growth and gene expression, suggesting that the two isoforms have differential effects upon nucleosome stability and chromatin structure. These findings provide insight into the molecular basis of the multiple functions of the H2A.Z gene products.     

Tandem promoters and developmentally regulated 5'- and 3'-mRNA untranslated regions of the mouse Scn5a cardiac sodium channel

The SCN5A gene encodes a voltage-sensitive sodium channel expressed in cardiac and skeletal muscle. Coding region mutations cause cardiac sudden death syndromes and conduction system failure. Polymorphisms in the 5'-sequence adjacent to the SCN5A gene have been linked to cardiac arrhythmias. We identified three alternative 5'-splice variants (1A, 1B, and 1C) of the untranslated exon 1 and two 3'-variants in the murine Scn5a mRNA. Two of the exon 1 isoforms (1B and 1C) were novel when compared with the published human and rat SCN5A sequences. Quantitative real time PCR results showed that the abundance of the isoforms varied during cardiac development. The 1A, 1B, and 1C mRNA splice variants increased 7.8 +/- 1.7-fold (E1A), 6.0 +/- 1.0-fold (E1B), and 20.6 +/- 3.7-fold (E1C) from fetal to adult heart, respectively. Promoter deletion and luciferase reporter gene analysis using cardiac and skeletal muscle cell lines demonstrated a pattern of distinct cardiac-specific enhancer elements associated with exons 1A and 1C. In the case of exon 1C, the enhancer element appeared to be within the exon. A 5'-repressor preceded each cardiac enhancer element. We concluded that the murine Na(+) channel has both 5'- and 3'-untranslated region mRNA variants that are developmentally regulated and that the promoter region contains two distinct cardiac-specific enhancer regions. The presence of homologous human splicing suggests that that these regions may be fruitful new areas of study in understanding cardiac sodium channel regulation and the genetic susceptibility to sudden death.     

Isolation and expression of cDNA encoding the murine homologues of CD1.

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.     

Loss of N-terminal charged residues of mouse CD3 epsilon chains generates isoforms modulating antigen T cell receptor-mediated signals and T cell receptor-CD3 interactions

The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4(+) T lymphocytes and T cell lines express CD3 epsilon chain isoforms with different isoelectric points (pI), with important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3 epsilon chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3 epsilon chain isoforms. At a high pI, degraded CD3 epsilon isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3 epsilon. The abundance of CD3 epsilon isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3 epsilon isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3 epsilon isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3 epsilon isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3 epsilon chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3 epsilon N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection.     

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.     

Ikaros isoforms: The saga continues

Through alternate splicing, the Ikaros gene produces multiple proteins. Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity. Ikaros isoforms interact to form dimers and potentially multimeric complexes. Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions. Small dominant-negative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros. Here, we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H. Although IK-1 is described as full-length Ikaros, IK-H is the longest Ikaros isoform. IK-H, which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4), is abundant in human but not murine hematopoietic cells. Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1. Moreover, IK-H can potentiate or inhibit the ability of IK-1 to bind DNA. IK-H binds to the regulatory regions of genes that are upregulated by Ikaros, but not genes that are repressed by Ikaros. Although IK-1 localizes to pericentromeric heterochromatin, IK-H can be found in both pericentromeric and non-pericentromeric locations. Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H, but not other Ikaros isoforms. The unique features of IK-H, its influence on Ikaros activity, and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.     

Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain.

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.