The mouse Ptprr gene encodes two protein tyrosine phosphatases, PTP-SL and PTPBR7, that display distinct patterns of expression during neural development.

The protein tyrosine phosphatases PTP-SL and PTPBR7 differ only in the length of their N-terminal domain. We show here that PTP-SL and PTPBR7 are isoforms derived from a single gene (Ptprr) through developmentally regulated use of alternative promoters. Isoform-specific reverse transcriptase-polymer chain reaction (RT-PCR) and RNA in situ hybridization experiments reveal that PTPBR7 is expressed during early embryogenesis in spinal ganglia cells as well as in developing Purkinje cells. Post-natally, PTPBR7 is expressed in various regions of the adult mouse brain, but expression in Purkinje cells has ceased and is replaced by the PTP-SL-specific transcript. In transient transfection experiments it is confirmed that PTPBR7 is a type I transmembrane protein tyrosine phosphatase (PTPase). PTP-SL, however, appears to be a cytosolic membrane-associated PTPase that is located at perinuclear vesicular structures that partly belong to the endosomal compartment. Thus, during maturation of Purkinje cells, a gene-promoter switch results in the replacement of a receptor-type PTPase by a cytosolic vesicle-associated isoform.

A FERM domain governs apical confinement of PTP-BL in epithelial cells.

PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins. Here we show, by transient expression of modular protein domains of PTP-BL in epithelial MDCK cells, that the presence of a FERM domain in the protein is both necessary and sufficient for its targeting to the apical side of epithelial cells. Furthermore, immuno-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revealed that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10-15 nm from the plasma membrane. Immunofluorescence microscopy suggested colocalization of the FERM domain moiety with the membrane-cytoskeleton linker ezrin. However, at the electron microscopy level this colocalization cannot be confirmed nor can we detect a direct interaction by immunoprecipitation assays. Fluorescence recovery after photobleaching (FRAP) experiments show that PTP-BL confinement is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes. Our observations suggest that relocation is mediated via a cytosolic pool, rather than by lateral movement. Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays. Both the highly restricted subcellular compartmentalization and its specific associative properties may provide the appropriate conditions for regulating substrate specificity and catalytic activity of this member of the PTP family.

Assignment of Ptprn2, the gene encoding receptor-type protein tyrosine phosphatase IA-2beta, a major autoantigen in insulin-dependent diabetes mellitus, to mouse chromosome region 12F.

The receptor-type protein tyrosine phosphatase IA-2beta gene (mouse gene symbol Ptprn2) encodes a major autoantigen in insulin-dependent diabetes mellitus. We physically mapped Ptprn2 by fluorescence in situ hybridization to band F of mouse chromosome 12, a region that lacks diabetes susceptibility loci. The mapping confirms the proposed synteny of mouse 12F with band q36 of human chromosome 7.

Developmental expression of the cell adhesion molecule-like protein tyrosine phosphatases LAR, RPTPdelta and RPTPsigma in the mouse.

Using RNA in situ hybridization we compared the expression patterns of the cell adhesion molecule-like receptor-type protein tyrosine phosphatases LAR, RPTP sigma and RPTP sigma during mouse development. We found that LAR is expressed in basal lamina-associated epithelial tissues of (neuro)ectodermal, neural crest/ectomesenchyme and endodermal origin. RPTP sigma is found in (neuro)ectodermal, neural crest-derived systems and in mesoderm-derived tissues. The expression pattern of RPTP sigma largely parallels that of RPTP sigma, in concordance with their proposed evolutionary history

Impaired mammary gland development and function in mice lacking LAR receptor-like tyrosine phosphatase activity.

The LAR receptor-like protein tyrosine phosphatase is composed of two intracellular tyrosine phosphatase domains and a cell adhesion molecule-like extracellular region containing three immunoglubulin-like domains in combination with eight fibronectin type-III-like repeats. This architecture suggests that LAR may function in cellular signalling by the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We used gene targeting in mouse embryonic stem cells to generate mice lacking sequences encoding both LAR phosphatase domains. Northern blot analysis of various tissues revealed the presence of a truncated LAR mRNA lacking the cytoplasmic tyrosine phosphatase domains and indicated that this LAR mutation is not accompanied by obvious changes in the expression levels of one of the LAR-like receptor tyrosine phosphatases PTPdelta or PTPsigma. LAR-/- mice develop and grow normally and display no appreciable histological tissue abnormalities. However, upon breeding we observed an abnormal neonatal death rate for pups from LAR-/- females. Mammary glands of LAR-/- females were incapable of delivering milk due to an impaired terminal differentiation of alveoli at late pregnancy. As a result, the glands failed to switch to a lactational state and showed a rapid involution postpartum. In wild-type mice, LAR expression is regulated during pregnancy reaching maximum levels around Day 16 of gestation. Taken together, these findings suggest an important role for LAR-mediated signalling in mammary gland development and function.

The neuronal nitric oxide synthase PDZ motif binds to -G(D,E)XV* carboxyterminal sequences.

PDZ motifs are small protein-protein interaction modules that are thought to play a role in the clustering of submembranous signalling molecules. The specificity and functional consequences of their associative actions is still largely unknown. Using two-hybrid methodology we here demonstrate that the PDZ motif of neuronal nitric oxide synthase (nNOS) can mediate the binding to several other proteins in brain. Peptide library screening showed that proteins bearing a carboxy-terminal G(D,E)XV* sequence are preferred targets for the nNOS amino-terminal PDZ motif. Potential nNOS targets include a melanoma-associated antigen, cyclophilins and the alpha1C-adrenergic receptor.

Protein-tyrosine phosphatases expressed in mouse epidermal keratinocytes.

The importance of growth factors acting via receptor-type protein-tyrosine kinases in the continuous renewal of the epidermis from the keratinocyte stem cell population has been well established. Protein-tyrosine phosphatases (PTPases), which dephosphorylate phosphotyrosine-containing proteins, may therefore be expected to play an equally important role in the control of epidermal growth and differentiation. In this study, we have made an inventory of the various PTPases that are expressed during mouse keratinocyte proliferation and maturation. A panel of 13 different PTPases probes was obtained by combining a set of PTPase cDNAs previously cloned from mouse brain and a set of PTPase probes obtained from a normalized keratinocyte PTPase cDNA library. This PTPase cDNA panel, spanning probes for receptor-type as well as cytoplasmic-type family members, was used to monitor RNA expression levels in keratinocyte fractions isolated from murine epidermis and in keratinocyte cell cultures. No overt changes were observed in PTPase mRNA levels in all strata of mouse epidermis, but comparison of cultured cells with freshly isolated keratinocytes revealed several conspicuous differences. In the cultured Balb/MK cell line, absence of PTP delta expression and upregulation of PTP kappa and, to a lesser extent, PTP gamma mRNA ratios were observed compared to the freshly isolated cells. These results provide a basis for further research on the impact of PTPase activity on epidermal growth control.

Molecular cloning of a mouse epithelial protein-tyrosine phosphatase with similarities to submembranous proteins.

Protein-tyrosine phosphatases (PTPases) form an important class of cell regulatory proteins. We have isolated overlapping cDNA clones that together comprise an 8 kb transcript encoding a novel murine PTPase which is expressed in various organs. Sequence analysis revealed an open reading frame of 2,460 amino acid residues. The predicted protein, PTP-BL, is a large non-transmembrane PTPase that exhibits 80% homology with PTP-BAS, a recently described human PTPase. PTP-BL shares some intriguing sequence homologies with submembranous proteins. It contains a band 4.1-like motif also present in the tumor suppressors neurofibromatosis 2 and expanded, five 80 amino acid repeats also present in the discs-large tumor suppressor, and a single catalytic phosphatase domain. No obvious homologies to other proteins were found for the N-terminal region of the protein other than human PTP-BAS. RNA in situ hybridization experiments show that the PTP-BL gene is expressed in epithelial cells, predominantly in kidney, lung, and skin. These data suggest a cell cortical localization for PTP-BL in epithelial cells and a possible role in the morphology and motility of epithelial tissues.

The mouse gene Ptprf encoding the leukocyte common antigen-related molecule LAR: cloning, characterization, and chromosomal localization.

The human receptor-like protein tyrosine phosphatase leukocyte common antigen-related molecule (LAR; gene symbol PTPRF) closely resembles cell adhesion molecules, which suggests that it may be involved in the regulation of phosphotyrosine levels through cell-cell or cell-matrix interactions. To obtain a better understanding of LAR function, we have characterized the mouse Ptprf gene as a first step toward site-directed mutagenesis studies in vitro and in vivo. The cytoplasmic region of the mouse LAR (mLAR) protein is encoded by 11 exons that span only 4.5 kb of genomic DNA. Compared to the known exon-intron structures of other mammalian receptor-like protein tyrosine phosphatase genes, such as Ptpra (encoding LRP) and Ptprc (coding for Ly-5), the Ptprf gene part encoding the cytoplasmic region of mLAR contains not only smaller, but also fewer introns. Sequence analysis of both phosphatase domains of mLAR and its homologs MPTP delta and mRPTP sigma revealed a higher evolutionary conservation of the second, C-terminal domain in comparison to the first domain. Fluorescence in situ hybridization was used to map the Ptprf gene to region C6-D1 on mouse chromosome 4.

A novel receptor-type protein tyrosine phosphatase with a single catalytic domain is specifically expressed in mouse brain.

Protein tyrosine phosphatases (PTPases) are important regulatory proteins that, together with protein tyrosine kinases, determine the phosphotyrosine levels in cell signalling proteins. By PCR amplification of mouse brain cDNA fragments encoding the catalytic domains of these enzymes, we identified three novel members of the PTPase gene family. Northern-blot analysis showed that two of these novel clones represent brain-specific PTPases, whereas the third originates from a large-sized mRNA that is more ubiquitously expressed. A full-length cDNA encoding one of the brain-specific PTPases, PTP-SL, was isolated. Sequence analysis revealed a transmembrane PTPase containing a single catalytic phosphatase domain that has 45% homology to a rat cytoplasmic brain-specific PTPase named STEP. This suggests a role for PTP-SL in cell-cell signalling processes in the brain.

Rapid assessment of protein-tyrosine phosphatase expression levels by RT-PCR with degenerate primers.

Using degenerate oligodeoxynucleotide primers we previously obtained cDNA fragments from ten different murine protein-tyrosine phosphatases (PTPases). Employing this same primer set, a method was developed to assess the expression levels of these PTPase family members in a fast and simple way. RT-PCR products of several cell types and tissue samples were used as probes on dot-blots containing the ten different PTPase fragments in equimolar amounts. Hybridization intensities at the various dots reflect the relative expression levels of the corresponding PTPases in the starting material. In this way expression of PTPases during mouse brain development could be monitored. Expression of PTP delta was found to be absent in embryonic stem cells but high in fetal and adult brain. PTP epsilon expression is shown to gradually increase in brain during maturation. Our method is generally applicable to gene families of which the transcripts can be detected with a single degenerate primer pair and is especially useful in situations where only limited amounts of RNA can be obtained.

Identification and typing of members of the protein-tyrosine phosphatase gene family expressed in mouse brain.

Protein-tyrosine phosphatases (PTPases) form a novel and important class of cell regulatory proteins. We evaluated the expression of PTPases in mouse brain by polymerase chain amplification of cDNA segments that encode the catalytic domains of these enzymes. Degenerate primer pairs devised on the basis of conserved protein motifs were used to generate a series of distinct PCR-derived clones. In this way, murine homologues of the human PTPases LRP, PTP beta, PTP delta, PTP epsilon and LAR were obtained. Corresponding regions in their catalytic domains were used to reveal the evolutionary relationships between all currently known mammalian PTPase protein family members. Phylogenetic reconstruction displayed considerable differences in mutation rates for closely related PTPases.

Genetic variability of the murine creatine kinase B gene locus and related pseudogenes in different inbred strains of mice.

The role of genetic variation in isoenzyme gene families is often poorly appreciated. We report here on the determination of DNA sequences and typing of genetic variability in four creatine kinase B (CKB) gene loci in different inbred strains of mice. The unique functional murine CKB gene was found to be nearly identical to the previously characterised rat and human sequences in both size and exon-intron structure. In this gene, approximately 0.5% allelic nucleotide positions as well as the lengths of simple A-rich and [TG]n repetitive elements located at the 5' and 3' sides of the transcribed segment, differed between inbred strains of mice. Preliminary experiments suggest that this sequence divergence is of importance for design of gene targeting strategies involving homologous DNA recombination. The three additional CKB-like gene loci in mice all had the characteristics of processed pseudogenes. By Southern blot analysis we could demonstrate that both the type and number of pseudogenes differed between inbred strains. Analysis of the CKB gene sequences enabled us to speculate about the evolutionary history of this highly polymorphic subfamily of genes.

Modulation of gene activity by consecutive gene targeting of one creatine kinase M allele in mouse embryonic stem cells.

The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptionally inactive during early embryogenesis. Using repeated rounds of gene targeting in mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several independent doubly targeted clones were isolated which all had insertions in the previously neo-disrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted in gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels in ES cells, led to the formation of the BM isoform of CK in these clones.

A long-range restriction map of the human chromosome 19q13 region: close physical linkage between CKMM and the ERCC1 and ERCC2 genes.

We report on the physical ordering of genes in a relatively small area of chromosome 19, segment q13, containing the locus for myotonic dystrophy (DM), the most frequent heritable muscular dystrophy of adulthood in man. DNAs from somatic cell hybrids with der 19q products that carry a breakpoint across the muscle-specific creatine kinase (CKMM) gene were analyzed by Southern blotting using probes for CKMM, APOC2, and the repair genes ERCC1 and ERCC2. Results were combined with data from CHEF and field inversion-gel-electrophoresis separation of large-sized DNA restriction fragments to establish a map localizing both DNA-repair genes and the CKMM gene within the same 250 kb of DNA, the order being cen-CKMM-ERCC2-ERCC1-ter, with APOC2 being at more than 260 kb proximal to CKMM. Transcriptional start sites of the CKMM and DNA-repair genes are all on the telomeric side of the genes. Our results provide a framework for the construction of a larger physical map of the area, which will facilitate the search for the DM gene.