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Genes to Cells (2004) 9, 919-933. doi:10.1111/j.1365-2443.2004.00773.x
© 2004 Blackwell Publishing or its licensors
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Characterization of multiple transcripts and isoforms derived from the mouse protein tyrosine phosphatase gene Ptprr

Renato G. S Chirivi, Gönül Dilaver, Rinske van de Vorstenbosch, Bas Wanschers, Jan Schepens, Huib Croes, Jack Fransen and Wiljan Hendriks*

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, University of Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The use of alternative splice sites, promoters and translation start sites considerably adds to the complexity of organisms. Four mouse cDNAs (PTPBR7, PTP-SL, PTPPBS{gamma}+ and PTPPBS{gamma}–) have been cloned that contain different 5' parts but encode identical protein tyrosine phosphatase PTPRR catalytic domains. We investigated the genomic origin and coding potential of these transcripts to elucidate their interrelationship. Mouse gene Ptprr exons were identified within a 260 kbp segment on chromosome 10, revealing PTP-SL- and PTPPBS{gamma}-specific transcription start sites within introns two and four, respectively, relative to the 14 PTPBR7 exons. Northern and RT-PCR analyses demonstrated differential expression patterns for these promoters. Furthermore, transfection studies and AUG codon mutagenesis demonstrated that in PTP-SL and PTPPBS{gamma} messengers multiple translation initiation sites are being used. Resulting 72, 60, 42 and 37 kDa PTPRR protein isoforms differ not only in the length of their N-terminal part but also in their subcellular localization, covering all major PTP subtypes; receptor-like, membrane associated and cytosolic. In summary, mouse gene Ptprr gives rise to multiple isoforms through the use of distinct promoters, alternative splicing and differential translation starts. These results set the stage for further investigations on the physiological roles of PTPRR proteins.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Reversible phosphorylation of specific tyrosine residues within proteins is an essential intracellular signal transduction mechanism. Cellular responses like differentiation, proliferation, survival, metabolic activity and more specifically for neuronal cells, migration, axonal guidance, synapse formation and activity are all regulated by modulating the phosphorylation state of target proteins (Gurd 1997; Hunter 1998; Purcell & Carew 2003). Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) are the enzymes that are instrumental in determining the spatial and temporal balance between the tyrosine phosphorylated and non-phosphorylated targets, and thus coordinately regulate these cellular responses to extracellular cues.

PTPs have been categorized into two distinct subgroups (Andersen et al. 2001). The first group possesses a transmembrane domain that follows a receptor-like extracellular region; the second group is cytoplasmic in location, and can in turn be subdivided in membrane-associated and true cytosolic PTPs. Interestingly, some PTP genes encode multiple isoforms that belong to several of these PTP subgroups. For instance, the STEP subfamily of PTPs consists of multiple transmembrane and cytosolic isoforms that are suggested to result from alternative splicing (Bult et al. 1996, 1997). Likewise, transmembrane, cytosolic and membrane-associated PTP{varepsilon} isoforms exist, but these result from the use of alternative promoters, proteolytic processing and differential initiation of translation (Gil-Henn et al. 2000). It is commonly believed that the generation of multiple isoforms from a single PTP gene through (a combination of) such mechanisms warrants fine-tuning of cellular signalling programs, both in time and space dimensions, thus adding to the organismal complexity of higher eukaryotes. Consequently, when studying the functional potential of a given gene, it is imperative to delineate its catalogue of transcripts and the exact nature of the corresponding protein products.

Several mouse cDNA clones, encoding PTPBR7, PTP-SL and PTPPBS{gamma}, respectively, have been reported that contain an identical, PTP-encoding-3' segment but differ completely in their 5' parts (Augustine et al. 2000a, 2000b; Hendriks et al. 1995; Ogata et al. 1995). We have recently proposed that the messengers that gave rise to the PTPBR7 and PTP-SL cDNAs are derived from a single mouse gene, Ptprr, through the use of developmentally regulated alternative promoters (van den Maagdenberg et al. 1999a). The origin of two other transcript variants, both encoding PTPPBS{gamma} (Augustine et al. 2000a), is as yet undefined. Moreover, uncertainties as to the translation initiation sites used in PTP-SL and PTPPBS{gamma}-encoding mRNAs has led to confusion and, occasionally, discrepancies in amino acid residue numberings (Pulido et al. 1998; van den Maagdenberg et al. 1999a).

To clarify the above issues we performed a careful annotation of transcript and protein products for mouse gene Ptprr, and investigated protein expression both in situ and at the subcellular level. Results reveal a unique gene system in which alternative use of promoters and AUG start codons accounts for the generation of distinct protein isoforms. These PTPRR proteins differ in their subcellular localization and span all major PTP subtypes; receptor-like, membrane-associated and cytosolic. This work gives important insight in the Ptprr gene structure and sets the stage for further investigations on the physiological role of the three mouse PTPRR protein isoforms, PTPBR7, PTP-SL and PTPPBS{gamma}.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Genomic structure of Ptprr

In order to make a full annotation of exonic sequences contained within the mouse gene Ptprr, we performed Blast searches using the PTPBR7, PTP-SL and PTPPBS{gamma} cDNA entries as queries (Fig. 1). Gene contigs were encountered in commercial (Celera) and public databases. In the EnsEMBL Mouse Genome database (release 20.32b.1; mouse species C57BL/6J) a single 257 251 bp stretch was identified that comprised the full annotation of all 14 PTPBR7 exon sequences (Gene ID number ENSMUSG00000020151; Unigene cluster Mm.3771). Searches using PTP-SL and PTPPBS{gamma} transcripts exclusively yielded hits within this very same genomic region, underscoring that the above transcripts all originate from the single-copy gene Ptprr. As proposed earlier (van den Maagdenberg et al. 1999a), there is a single PTP-SL-specific exon in between PTPBR7 exons two and three, defining the PTP-SL-specific promoter to reside within the largest intron, intron 2, of Ptprr. For generating the PTP-SL specific transcript, this first PTP-SL exon is joined to PTPBR7 sequences in exons 3–14 through splicing. Thus, PTP-SL transcripts differ from PTPBR7 mRNAs in that sequences contained within Ptprr exons 1 and 2 (709 bases in total) are replaced by the 135 nucleotide PTP-SL-specific exon-derived sequence.



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  		Figure 1  Schematic representation of the Ptprr exon-intron structure and its major mRNA products. In (A) mouse genomic DNA is represented by the grey horizontal bar. Exons are depicted as vertical bars with exon number or name indicated above. Arrows symbolize the relative positions of the three distinct transcription start sites. The black horizontal bar, depicted on the right, indicates 25 kbp of DNA. Resulting Ptprr transcripts are depicted in panel (B) as thick horizontal boxes, with mRNA names indicated on the right. Transcript specific nucleotide sequences are indicated in light grey at the left end of the transcript. ORFs are depicted as dark grey horizontal bars. The numbers indicate the first nucleotide of potential start codons that were included in mutational analyses experiments (Fig. 4). Nucleotide numbers are based on PTPBR7, PTP-SL and PTPPBS{gamma}+ cDNA sequences (Acc. nos. D31898 [GenBank] , BN000437 [GenBank] and BN000438 [GenBank] , respectively). Bar on the left indicates 500 bp.

 
The unique 5' segment in the PTPPBS{gamma} cDNAs (Augustine et al. 2000a) could be mapped in a 292 bp sequence stretch immediately upstream of Ptprr exon 5. Thus, Ptprr intron 4 also harbours an alternative promoter, this time giving rise to either PTPPBS{gamma}+ or PTPPBS{gamma}– mRNAs depending on whether a small (117 nucleotide) intron is excised from the unique PTPPBS{gamma}+ leader sequence. To generate PTPPBS{gamma}– transcripts therefore 175 nucleotides, derived from exonic sequences unique for both PTPPBS{gamma} mRNA variants, are joined through splicing to sequences delivered by Ptprr exons 5–14. The complete Ptprr gene structure (schematically depicted in Fig. 1A) is summarized in Table 1, where reference is made to positions in the four different cDNAs. This gene annotation has also been deposited to the mouse genome database under TPA accession numbers BN000439 to BN000454.


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  		Table 1  Ptprr exon-intron boundaries
 
Ptprr transcript expression patterns

Data supporting the differential use of the three distinct transcription start sites within mouse Ptprr have been obtained mainly by RNA in situ hybridization studies. The use of transcript-specific probes has revealed that PTPBR7 messengers appear during early embryogenesis in spinal ganglia and developing Purkinje cells. Postnatally, PTPBR7 is expressed throughout the brain but expression gradually ceases in maturing Purkinje cells and PTP-SL type transcripts take over (van den Maagdenberg et al. 1999a). PTPPBS{gamma} RNAs are quite prominent in the hippocampal region, cerebellar granular layer and in the gastrointestinal tract (Augustine et al. 2000b). Furthermore, during mouse embryo-foetal development of skeletal and intestinal systems the detected hybridization signals are believed to reflect PTPPBS{gamma}– mRNA (Augustine et al. 2000a). Northern blot analyses, on the other hand, have thus far only been performed with probes that hybridize to all Ptprr transcript variants (Hendriks et al. 1995; Ogata et al. 1995; Augustine et al. 2000b). Using such a pan-PTPRR cDNA probe on Northern blots of cerebral and cerebellar RNA samples, the large (4.1 kb) PTPBR7 transcript is detected in cerebellum and to a lesser extent in other brain areas, whereas the shorter PTP-SL (3.2 kb) or perhaps PTPPBS{gamma} (predicted to be ~3 kb) RNAs appear to be exclusively expressed in cerebellum (Fig. 2A, upper panel).



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  		Figure 2  Expression of Ptprr transcript variants in specific brain regions. (A) Northern blot analysis on RNA from different brain regions using full-length PTPBR7 cDNA (upper panel) or the unique PTP-SL 5’ untranslated region (lower panel) as a probe. PTPBR7 mRNA is expressed in cerebellum and, to a lesser extent, in the other brain areas. PTP-SL messengers on the other hand are only detectable in cerebellum. PTPPBS{gamma} type transcripts are below detection levels (data not shown). (B) PTP-SL and PTPPBS{gamma} mRNAs are detectable in total RNA isolated from several cerebral brain parts when using a sensitive transcript-specific RT-PCR assay. PTPBR7 transcripts were detected in all brain regions. Isoform-specific primer sets used were negative on mouse genomic DNA, and ß-actin primers were included as positive controls.

 
To ascertain the identity of the obtained signals we analysed RNA samples isolated from various mouse brain regions by Northern blot analysis, using PCR-generated probes that are specific for the unique 5' mRNA parts in the PTPBR7, PTP-SL and PTPPBS{gamma} type transcripts. Indeed, weak hybridization signals that corresponded to the 4.1 kb transcript mentioned above were obtained in all samples with the PTPBR7-specific probe (data not shown). Use of the PTP-SL-specific probe revealed this shorter transcript to be exclusively expressed in cerebellum (Fig. 2A, lower panel). Although PTPPBS{gamma} messengers are detectable in the brain by in situ hybridization (Augustine et al. 2000b), no specific signal resulted using the PTPPBS{gamma}-specific probe in Northern blot analysis (data not shown), indicating very low expression levels for these transcript variants.

To perform a more sensitive survey of the isoform-specific expression patterns in brain we used the same RNA samples in RT-PCR experiments (Fig. 2B). Primer sets were chosen in such a way that genomic DNA contaminations would not yield amplicons of the appropriate size. ß-actin messenger amplification was used as control for RNA integrity and reverse transcriptase activity. Except for the olfactory bulb, PTPPBS{gamma} transcripts were indeed now detectable in the various brain regions. PTPBR7 mRNA was present in all brain tissues, including olfactory bulb (Fig. 2B), but was not detected in RNA samples from other mouse tissues (i.e. heart, lung, liver; data not shown). PTP-SL transcripts thus far had only been detected in the cerebellum by in situ hybridization and Northern blot analysis (Hendriks et al. 1995; Augustine et al. 2000b). RT-PCR, however, also reveals PTP-SL messengers in the midbrain, brainstem and cortex (Fig. 2B). Taken together, these RNA expression data demonstrate that in the mouse cerebrum the PTPBR7 promoter is responsible for the vast majority of Ptprr transcripts. In the cerebellum about two-third of the transcripts arise from the PTP-SL promoter and the remaining one third is mainly of the PTPBR7 type (Fig. 2A, upper panel). Only trace amounts of PTPPBS{gamma} mRNAs, not detectable on Northern blots, are present throughout the brain.

Mapping of translation initiation sites in the Ptprr-derived mRNAs

Open reading frames encompassing 656, 549 and 412 codons in the PTPBR7, PTP-SL and PTPPBS{gamma} cDNAs predict the synthesis of 74, 62 and 47 kDa mature proteins, respectively. For PTP-SL it has been suggested that not the first AUG of the major predicted open reading frame (ORF), but the following in-frame AUG codon is used for initiation of translation (Hendriks et al. 1995) leaving the exact size of the protein as yet unclear. Likewise, the translation start site used in both PTPPBS{gamma} messenger isoforms (Augustine et al. 2000b) is enigmatic. To aid in the determination of the start codons, a pSG5-based full-length PTPPBS{gamma} cDNA expression construct was generated and used, together with similar, existing constructs encoding PTPBR7 and PTP-SL (van den Maagdenberg et al. 1999a), in transfection experiments. All three constructs resulted in multiple immunoreactive products in protein lysates from transiently transfected Neuro-2a cells upon Western blot analysis using {alpha}-SL antiserum (Fig. 3, left three lanes), whereas no signal was obtained for lysates of mock transfected cells (data not shown). For PTPBR7, two immunoreactive bands, around 72 and 65 kDa, respectively, are observed. Expression of PTP-SL cDNA revealed a doublet band around 60 kDa and two additional signals at 42 and 37 kDa. Surprisingly, the latter two immunoreactive bands also appear as the main products using the PTPPBS{gamma} expression construct. These data imply post-translational modifications, including processing through protease cleavage, and/or the use of alternative AUG start codons.



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  		Figure 3  The three different Ptprr transcripts give rise to multiple PTPRR isoforms. Left panel: Neuro-2a cells were transiently transfected with pSG5/PTPBR7-FL, pSG5/PTP-SL-FL or pSG5/PTPPBS{gamma}-FL expression plasmids. Lysates were used for Western blot analysis exploiting the rabbit {alpha}-SL antiserum that is immunoreactive against the catalytic portion in PTPBR7, PTP-SL and PTPPBS{gamma}. Middle panel: Western blot analysis of {alpha}-SL immunoreactive proteins detected in mouse total brain lysates. The rightmost panel shows monoclonal antibody 6A6 immunoprecipitates from different brain regions, visualized on blot using STEP-absorbed {alpha}-SL that is immunoreactive towards all PTPRR proteins. Please note that the polyvalent antiserum may cross-react with the nearest homologue, STEP, on immunoblots but not in immunoprecipitation experiments. Molecular mass markers (in kDa) are indicated on the left.

 
To allow unequivocal mapping of the AUG start codons used in the various transcripts, we performed site directed mutagenesis on the PTPBR7, PTP-SL and PTPPBS{gamma} expression constructs. Mutation of the ATG triplet in PTPBR7 cDNA, at nucleotide position 356–358 (BR7-356), into an isoleucine-encoding one led to a complete absence of PTPBR7 expression in transiently transfected Neuro-2a cells (Fig. 4A). A similar mutation at the next in-frame ATG triplet (BR7-719) had no effect at all, indicating that in the PTPBR7 messenger only the start codon at position 356–358 is used and showing that the 65 kDa species must be derived from a 72 kDa precursor through post-translational processing. In the PTP-SL expression construct mutations were incorporated such that either one of the suggested two candidate translation start codons (position 103–105 and 145–147 in PTP-SL cDNA; mutants SL-103 and SL-145, respectively) is changed into an isoleucine codon. Upon transfection, these mutants show that not the first but indeed the second AUG codon in the PTP-SL encoding ORF is used for translation initiation (Fig. 4B). The resulting protein product subsequently undergoes an, as yet, uncharacterized post-translational modification resulting in a doublet immunoreactive band at 60 kDa. Remarkably, the production of the immunoreactive 42 and 37 kDa proteins are not influenced by these mutations.



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  		Figure 4  Determination of the sites of translation initiation in the Ptprr-derived transcripts. Neuro-2a cells were transiently transfected with plasmids containing wild-type or mutated full-length PTPBR7 (BR7), PTP-SL (SL) and PTPPBS{gamma} ({gamma}) cDNAs. Nomenclature of mutants (indicated above the lane) corresponds to the position of the first nucleotide in the ATG codon that was altered by site-directed mutagenesis within the respective isoform cDNA (Table 2). Proteins produced were immunoprecipitated using {alpha}-PTPRR monoclonal antibody 6A6 and analysed on Western blots using the {alpha}-SL antiserum. (A) Mutating the ATG triplet at 356 in PTPBR7 cDNA results in complete loss of protein expression (BR7-356), whereas the BR7-719 mutant displays unaltered synthesis. Mock transfected cells revealed a 50 kDa background signal. Note the doublet band for PTPBR7, indicative for post-translational modifications. Molecular mass markers are indicated (in kDa) on the right. (B) Similar experiments on PTP-SL cDNA show that mutations at positions 145 643 and 760 interfere with synthesis of the 60, 42 and 37 kDa immunoreactive bands, respectively. Note the doublet band for PTP-SL around 60 kDa that is reminiscent of post-translational modification. (C) Production of the 42 and 37 kDa PTPPBS{gamma} isoforms is abrogated when ATG triplets at positions {gamma}-530 and {gamma}-647 are mutated, respectively. When these AUG mutations are combined ({gamma}-530/647 mutant) both prominent PTPPBS{gamma} bands disappear. The {gamma}-401 and {gamma}-704 mutants have no effects. (D) Schematic diagrams of the Ptprr protein isoforms that result from translation of the corresponding full length messengers in mouse Neuro-2a cells as determined in this study. Signal peptide (SP), hydrophobic region (HR), transmembrane segment (TM), kinase interacting motif (KIM) and catalytic phosphotyrosine phosphatase domain (PTP) are indicated in the appropriate isoforms.

 
Site directed mutagenesis of ATG triplets within the PTPPBS{gamma} expression construct clearly showed that in the PTP reading frame not the first (position 401–403; {gamma}-401) but rather the second (pos. 530–532; {gamma}-530) as well as the third (pos. 647–649; {gamma}-647) AUG codon is used efficiently for translation initiation (Fig. 4C), thus resulting in two PTPPBS{gamma} variants that differ in their N-termini. Elimination of both of these AUG codons (mutant {gamma}-530/647) totally blocked the synthesis of the PTPPBS{gamma} isoforms. In view of the similarity in electrophoretic mobility of the two 42 and 37 kDa PTPPBS{gamma} species and the two low-molecular weight entities in PTP-SL expressing lysates, we wondered whether the downstream AUG codons that correspond with the PTPPBS{gamma} translation start sites are also being used in PTP-SL messengers for initiation of protein synthesis. Mutation of these ATG codons (at positions 643–645 and 760–762) in PTP-SL cDNA indeed resulted in the elimination of the 42 and 37 kDa protein bands without affecting the production of the 60 kDa doublet (Fig. 4B). These data demonstrate that two downstream AUG codons in PTP-SL mRNA serve as alternative translation initiation sites and result in protein isoforms indistinguishable from the 42 and 37 kDa PTPPBS{gamma} isoforms.

The resulting four different PTPRR isoforms that are encoded by the gene Ptprr are schematically depicted in Fig. 4D. To investigate whether this catalogue of PTPRR isoforms can actually be detected in brain tissue lysates, we performed Western blot analysis of mouse total brain lysate. Three major immunoreactive proteins, with sizes of approximately 70, 60 and 40 kDa, are apparent using the rabbit {alpha}-SL serum (Fig. 3, middle panel). The two upper major bands correspond with the ones seen for PTPBR7 and PTP-SL in the in vitro studies, especially when taking into account possible in vivo post-translational modifications as witnessed by the occurrence of double bands for these isoforms in Fig. 4 (e.g. BR7-719 and SL-643/760). The smaller, 40 kDa band in brain may correspond to a PTPPBS{gamma} isoform.

PTPRR protein localization in mouse brain

To aid in the study of the in situ localization of PTPRR proteins in mouse brain tissue, monoclonal antibodies against the common part in all four isoforms were raised. To this end, the GST-SL fusion protein encompassing the PTP domain-containing carboxyl terminal 294 residues (Hendriks et al. 1995) was used to immunize mice. Absence of unique amino acid sequences in PTP-SL and PTPPBS{gamma} precluded the generation of PTPRR antibodies that would have been specific for either one of these isoforms. Obtained hybridomas were screened by ELISA using a His-tagged PTP-SL fragment as antigen to eliminate GST-reactive hybridomas. Eight different clones were obtained that secreted antibodies reacting with Neuro-2a cells ectopically expressing PTP-SL in Western analysis (Fig. 5A) and immunofluorescence assays, and remaining negative on mock-transfected cells. Various deletion mutants of the GST-SL fusion protein were exploited to map the epitope-containing region for these monoclonal antibodies. All were found to be immunoreactive towards amino acids 227–292 in PTP-SL (aa numbering according to TPA accession number BN000437), spanning the region just downstream of the KIM domain present in all four PTPRR isoforms.



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  		Figure 5  Immunoreactivity of monoclonal antibodies and polyvalent antiserum directed against the common part in PTPRR protein isoforms. Neuro-2a cells were transiently transfected with PTP-SL or STEP expression plasmids, as indicated above the lanes. Mock transfected cells were included as negative controls. Lysates were directly subjected to Western blot analysis using: (A) monoclonal antibodies (6A6, 5E4), or (C) polyvalent antiserum ({alpha}-SL, STEP-absorbed SA-{alpha}-SL) directed against the PTPRR PTP moiety. The other six PTPRR monoclonal antibodies that were obtained displayed reactivities similar to 6A6. Proteins were also immunoprecipitated from transfected cell lysates using the different antibodies, and analysed on Western blots. All eight monoclonal antibodies demonstrated specificities in immunoprecipitation applications that are identical to that of the polyvalent {alpha}-SL antiserum shown in (B) Molecular mass markers (kDa) are indicated on the left.

 
On the basis of signal intensities on mouse tissue sections three monoclonal antibodies (designated 6A6, 1E3 and 3E11) were selected for use in immunohistochemical assays. Mouse brain horizontal and coronal sections were incubated with a mixture of these anti-PTPRR monoclonal antibodies and specific brain regions revealed immunoreactivity. Clear staining was observed in the Purkinje cells of the cerebellum (Fig. 6A), in both the neuropil and the neurones of the striatum (Fig. 6B). Close to the striatum the major Islands of Calleja and the ventral pallidum also stain positive, and furthermore the hippocampal CA2/CA3 region, subthalamic nucleus, substantia nigra area, and large neurones between the medial and dorsal lateral septum stain very bright (data not shown). Of the five remaining monoclonal antibodies all except one (5E4) gave similar but much weaker staining patterns (data not shown). To our surprise monoclonal antibody 5E4 firmly stained Purkinje cells of the cerebellum (Fig. 6C), but remained negative in all other brain regions, most notably the striatum (Fig. 6D). This suggested to us a possible cross-reactivity of all other antibodies with striatum-enriched phosphatase (STEP), which is 45% identical in the PTP domain. Indeed, exploiting bacterial and mammalian cells ectopically expressing PTP-SL or STEP proteins, we found that all monoclonal antibodies, except 5E4, as well as the previously used polyvalent {alpha}-SL antiserum recognized STEP, both on immunoblots (Fig. 5A) and in immunoflorescence assays (data not shown), while remaining negative on mock-transfected cells. In immunoprecipitation experiments, however, all antibodies were specific for PTP-SL (Fig. 5B). We could successfully remove the STEP cross-reactivity from the polyvalent {alpha}-SL serum by passing it over glutathion-sepharose-bound GST-STEP fusion proteins (Fig. 5C), and the resulting PTPRR-specific rabbit serum performed comparable to 5E4 on brain sections (Fig. 6E–F). Taken together these data show that PTPRR protein isoforms are only present at detectable levels in the cerebellar Purkinje cells, and that previously observed staining patterns in other brain regions (Ogata et al. 1999; van den Maagdenberg et al. 1999a; Dilaver et al. 2003) may reflect cross-reactivity with STEP proteins.



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  		Figure 6  Immunolocalization of PTPRR protein in mouse brain. Brain cryosections were stained using a mixture of three different monoclonal antibodies (1E3, 3E11 and 6A6) immunoreactive towards the common part in PTPRR isoforms and that cross-react with STEP. Positive staining is observed in the Purkinje cells of the cerebellum (A), both the neurones and neuropil of the striatum (B). Similar sections were incubated with monoclonal antibody 5E4, which does not cross-react with STEP. Exclusively Purkinje cell staining was observed (C), and no signal could be detected in the striatum (D). The STEP-absorbed {alpha}-SL serum also shows a clear Purkinje cell staining (E) and does not react with striatum (F), suggesting that staining of the striatum (B) reflects STEP immunoreactivity.

 
The possible cross-reactivity of the {alpha}-SL serum with the STEP family of phosphatases made us re-evaluate the immunoblot analysis of PTPRR protein expression in different brain areas. To maximize sensitivity and to eliminate STEP cross-reactivity, we performed immunoprecipitations followed by Western blot analysis on protein lysates of mouse olfactory bulb, hippocampus, midbrain, brainstem, cerebellum and cortex brain areas using monoclonal antibodies and STEP-absorbed {alpha}-SL serum. One major immunoreactive band of approximately 65 kDa was seen in all lysates tested (Fig. 3, right panel), which corresponds well with the lower protein band seen for PTPBR7 in the in vitro studies. The minor bands at 70 and 75 kDa detected in most brain parts probably represent a processing intermediate and the full-length PTPBR7 protein, respectively. No other potential in-frame AUG start sites could account for these alternative sizes. Reflecting the lower and more restricted expression of PTP-SL and PTPPBS{gamma} mRNAs, specific signals for these protein isoforms are not apparent in the immunoprecipitation experiment, although the lower band may correspond with PTPPBS{gamma}-37.

PTPRR isoforms comprise cell surface, vesicle-associated and cytosolic variants

The presence of hydrophobic protein domains in the PTPBR7 and PTP-SL isoforms has led to the prediction that they may represent receptor-type PTPs (Fig. 4D). Indeed, for PTPBR7 a functional signal peptide and a transmembrane segment have been mapped (Ogata et al. 1995; van den Maagdenberg et al. 1999a). The N-terminal hydrophobic segment in PTP-SL, however, is unable to exert a signal peptide ‘start transfer’ effect (van den Maagdenberg et al. 1999a) and, consequently, the membrane topology of PTP-SL remains elusive. The construction of the PTPPBS{gamma} expression plasmid now enabled us for the first time to make a comparison of the subcellular localizations for the PTPBR7, PTP-SL and PTPPBS{gamma} PTPRR protein isoforms. Transiently transfected Neuro-2a cells were immunostained using the rabbit {alpha}-SL antiserum and analysed by confocal laser scanning microscopy (Fig. 7). The three different PTPRR expression constructs resulted in markedly different subcellular localization patterns. As described before, PTPBR7 and PTP-SL are localized at the Golgi apparatus and at late endosomal vesicles, additionally PTPBR7 is also found at the cell membrane (Ogata et al. 1999; van den Maagdenberg et al. 1999a; Dilaver et al. 2003). In contrast, the 42 and 37 kDa PTPPBS{gamma} variants are genuine cytosolic proteins that are excluded from the nucleus. Similar results were obtained when other cell types, e.g. COS-1 cells, were used (data not shown).



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  		Figure 7  PTPRR isoforms differ in their subcellular localization. Neuro-2a cells, transiently transfected with expression plasmids encoding PTPBR7 (A), PTP-SL (B) or PTPPBS{gamma} (C), were stained using {alpha}-SL antiserum and analysed by confocal laser scanning microscopy. Distinct localization patterns for each isoform type can be discerned. PTPBR7 displays a predominant cell surface staining, but also at the Golgi and vesicles throughout the cytoplasm (A). PTP-SL resides at the Golgi apparatus and on vesicles (B). The PTPPBS{gamma} 42 and 37 kDa proteins diffusely stain the cytosol (C). Bar indicates 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Here we have characterized the full collection of transcript and protein isoforms that are derived from the mouse gene Ptprr. Three different alternative promoters (Fig. 1) were found to give rise to messengers encoding PTPBR7, PTP-SL and two PTPPBS{gamma} protein variants (Figs 3 and 4) that differ in their brain expression (Fig. 2) and subcellular localization (Fig. 7) patterns. Results from the human genome project have demonstrated that the phenotypic complexity of higher organisms is achieved only in part by higher gene numbers and that mechanisms that result in the production of multiple protein isoforms from a single gene locus contribute substantially to this (Lander et al. 2001). A well-studied mechanism to create diversity at the RNA level is alternative splicing, now thought of to occur in some two-third of the mammalian primary transcripts (Boue et al. 2003). Indeed, two Ptprr transcript variants arise through an alternative splicing event: PTPPBS{gamma}+ and PTPPBS{gamma}– (Augustine et al. 2000a). However, they contain identical open reading frames and thus do not contribute differentially to the collection of PTPRR protein isoforms. The PTPPBS{gamma} splice variants only differ in their 5' non-translated region due to alternative removal of a 117 nucleotide intronic sequence that according to our genomic analysis spans positions 176–292, rather than 174–290 as reported earlier (Augustine et al. 2000b), in the PTPPBS{gamma}+ cDNA.

An alternative, well-studied means to produce multiple protein isoforms from a single gene is through the use of alternative translation initiation sites (Kozak 1999). Also this strategy is used in mouse to create isoform diversity within the Ptprr protein products. From the large open reading frame present in PTPPBS{gamma} mRNA variants it is not the first AUG codon that is used as translation start, but instead the second and the third methionine codon (Fig. 4C). Likewise, the first AUG codon in the PTP-SL reading frame is ignored by the ribosome, and rather the one at position 145–147 and to a lesser extend those at 643–645 and 760–762 are used to generate 60, 42 and 37 kDa isoforms, respectively. This translation initiation site choice appears to be depending on the expression system used. COS1 cells transiently transfected with the PTP-SL expression construct show predominance of the start at position 145–147, whereas PC12 cells mainly produce the isoforms known as PTPPBS{gamma} by using the alternative start codons at positions 643–645 and 760–762 (unpublished data).

The pioneering work of Marylin Kozak (Kozak 2002) has provided detailed knowledge on the translation initiation behaviour of mammalian ribosomes. Although surrounding RNA secondary structure elements are certainly of influence as well, the optimal context for start codon recognition has been determined as GCCRGaugG, with the most important nucleotide types in bold. Less optimal start codons may cause ‘leaky scanning’ resulting in translation initiation on further downstream AUGs. Alternatively, direct entry of ribosomes at internal AUG sites in the mRNA might occur (Kozak 2003). Finally, ‘re-initiation’ by ribosomes after translation of small upstream ORFs could explain alternative start site choices (Kozak 2002). Since certain initiation factors dissociate only gradually from the ribosome during the elongation phase, they enable the 40S subunit to resume scanning for AUG start sites after terminating from such very small ORFs. In the case of PTP-SL and PTPPBS{gamma} mRNAs indeed very small upstream ORFs near or overlapping the predicted start sites are present. We exclude cryptic promoters or splice sites in the pSG5-based expression constructs as the cause for the synthesis of 60, 42 and 37 kDa PTPRR proteins since identical staining patterns were obtained using pRK5 as vector (data not shown) or when performing in vitro translation experiments (Hendriks et al. 1995; Augustine et al. 2000b).

Yet another key mechanism for generating organismal complexity is alternative use of promoters (Landry et al. 2003). Alternative promoters may display differences in expression level, tissue specificity or developmental activity. Furthermore, the resulting alternative 5' mRNA ends may impose differences in mRNA stability and translatability or, most importantly, in the encoded proteins. The three alternative promoters present in the Ptprr gene create differences both in expression pattern as well as in coding potential of the distinct mRNA types. PTPBR7 is a receptor-type PTP that is expressed throughout the brain (Ogata et al. 1995). PTP-SL is located at perinuclear vesicular structures (Dilaver et al. 2003) and during postnatal Purkinje cell maturation PTP-SL specific transcripts replace the PTPBR7 messenger (van den Maagdenberg et al. 1999a), a developmental expression pattern that is conserved in rat (Watanabe et al. 1998). The third, cytosolic protein isoform PTPPBS{gamma} is, in contrast to the other two, not only expressed in brain but also during cartilaginous skeleton development and in the gastrointestinal tract (Augustine et al. 2000a).

The orthologous human gene, PTPRR, has a very similar exon-intron build-up (Bektas et al. 2001) and also encodes multiple isoforms (Augustine et al. 2000b). Human PTPPBS{alpha} and PTPPBS{gamma} correspond to mouse PTPBR7 and PTPPBS{gamma}, respectively, and expressed sequence tag BQ957212 [GenBank] may represent human PTP-SL. Interestingly, in human tissues yet another isoform has been identified. This PTPPBS{delta} cDNA has a unique 5' stretch of 23 nucleotides, containing an ATG sequence, which replaces the first 799 basepairs of PTPPBS{alpha} and will result in a protein with 3 unique amino acids (Augustine et al. 2000b). We could map the human PTPPBS{delta}-specific stretch in PTPRR just two nucleotides upstream of the PTPPBS{gamma}-specific exon, meaning that the PTPPBS{delta} and PTPPBS{gamma} promoters are arranged in tandem. In mouse, no PTPPBS{delta}-like isoform has been described so far. We could locate a putative PTPPBS{delta}-like 5' leader sequence (sharing 83% sequence homology) just in front of, and partly overlapping with, the first mouse PTPPBS{gamma} exon, nicely coinciding with the position of the human PTPPBS{delta} promoter. However, cDNAs containing the implicated mouse genomic segment could not be identified in the databases and even if a mouse PTPPBS{delta}-type transcript is generated this will not give rise to a mouse PTPPBS{delta} protein since the human PTPPBS{delta} ATG start codon reads GTG in mouse.

To monitor PTPRR protein expression in situ, we raised monoclonal antibodies against the PTP part that is present in all isoforms. Delineation of protein expression patterns for individual PTPRR isoforms is hampered by the absence of unique sequence stretches in the PTP-SL and 42 and 37 kDa PTPPBS{gamma} proteins. Furthermore, we stumbled upon the finding that a polyvalent antiserum and seven out of eight monoclonal antibodies directed against the common part in the PTPRR isoforms cross-reacted with STEP, that is only 45% identical in its catalytic domain (Fig. 5). Therefore, our previous immunohistochemical data, showing expression in Purkinje cells of the cerebellum, striatal neurones, hippocampal CA2/CA3 region and brain cortex (Dilaver et al. 2003; van den Maagdenberg et al. 1999a) should be reconciled. Our present survey by immunoprecipitation and Western blot analysis (Fig. 3) now reveals that PTPRR isoforms, most notably of the PTPBR7 type, are present at low levels in various brain regions but by immunohistochemistry are only detectable in cerebellar Purkinje cells (Fig. 6). The apparent discrepancy between immunoprecipitation and Western blot experiments may be explained in several ways but most likely is due to low sensitivity and limited antigen accessibility. Furthermore, mRNA and protein stabilities are crucial parameters for the ultimate detection levels.

On the basis of the initial mapping of Ptprr on chromosome 8 (van den Maagdenberg et al. 1999b) and the conspicuous PTPBR7 to PTP-SL isoform switch during postnatal Purkinje cell development we have considered Ptprr a candidate gene for the recessive neurological mouse mutation nr (nervous) (van den Maagdenberg et al. 1999a). During the fourth postnatal week 90% of the cerebellar Purkinje cells in nr mutant mice start to degenerate and, as a consequence, adult nr mice display ataxia and poor motherhood (Sidman & Green 1970). We have PCR amplified and sequenced all Ptprr exonic regions in nr mice but no differences were found when compared to wild-type controls (data not shown). Furthermore, normal levels and sizes of PTPRR transcripts and proteins were detectable in nr mice. Meanwhile, the Mouse Genome Project has revealed that Ptprr is located on chromosome 10 rather than 8 (Waterston et al. 2002). Indeed, using a Ptprr containing genomic clone from the consortium, we could confirm the chromosome 10 location by fluorescence in situ hybridization (data not shown). Obviously, a chimeric genomic clone compromised our original mapping result, and the above eliminates Ptprr as nr candidate.

Taken together, through the use of three alternative promoters, a single alternative splicing event and alternative use of AUG start codons, the mouse gene Ptprr produces four different PTPRR mRNA and protein isoforms. Although the PTPRR isoforms occupy different niches in the cell (Fig. 7), they all have a C-terminal part in common that contains in addition to the catalytic PTP domain a so-called KIM (Kinase Interaction Motif) sequence that allows them to interact with MAP kinases (Pulido et al. 1998; Zuniga et al. 1999). Intriguingly, the spatio-temporal regulation of MAP kinase activation is a crucial determinant in cell signalling as demonstrated in the paradigm example of PC12 cell response to growth factor stimulation (Marshall 1995). It is tempting to speculate that the various PTPRR isoforms perform distinct tasks in determining signalling specificity, having PTPBR7 counteracting MAP kinases at the cell surface, with PTP-SL being active during growth factor receptor endocytosis, and PTPPBS{gamma} variants in keeping cytosolic MAP kinases unphosphorylated. Clearly, elucidation of the cell biological impact of the multiple Ptprr gene products awaits further experiments.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Expression plasmid constructs and site directed mutagenesis

Plasmids pSG5/PTP-SL-FL and pSG5/PTPBR7-FL have been described elsewhere (van den Maagdenberg et al. 1999a). To obtain a full-length mouse PTPPBS{gamma} expression construct (pSG5/PTPPBS{gamma}-FL) PCR was performed on mouse genomic DNA using oligonucleotides 5'-GGACTAGTCCGTGAACCAGGTAGTTTCCAG-3' (SpeI restriction site is underlined) and 5'-TCCTTCTTTGCTCCAGAT-3', which resulted in a 362-bp fragment encompassing the nucleotides (pos. 1–292) that are unique for PTPPBS{gamma}+ cDNA. The PCR product was cloned into the pGEM-T vector (Promega, Madison, WI, USA) and analysed by sequencing to verify absence of mutations. The resulting plasmid was treated with endonucleases BglII and SpeI, and the obtained fragment was inserted into pSG5/PTP-SL-FL that had been first partially digested with BglII (position 441 in the PTP-SL cDNA) and digested to completion with SpeI.

AUG mutants of pSG5/PTPBR7-FL, pSG5/PTP-SL-FL and pSG5/PTPPBS{gamma} plasmids were generated using the QuickChange Mutagenesis protocol according to manufacturers specifications (Strategene Inc., La Jolla, CA, USA). In Table 2 the primers and nomenclature used is listed. Obtained mutant constructs were checked by sequence analysis.


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  		Table 2  Mutant nomenclature and primers used for site directed mutagenesis
 
The bacterial expression plasmid pGEX-SL has been decribed before (Hendriks et al. 1995). Deletion mutants pGEX-2T-SL1060, pGEX-2T-SL1213 and pGEX-2T-SL1582 were created by PCR using specific primers (sequences available upon request) and mPTP13-6 (Hendriks et al. 1995) as template. Resulting PCR products were cloned into the BamHI site of pGEX-2T. To enable production of His-tagged PTP-SL recombinant protein, an NcoI-XhoI cDNA fragment was excised from mPTP13-6 (Hendriks et al. 1995), blunted with Klenow and cloned into XhoI-digested and Klenow-treated pET-15b vector, rendering pET-15b-PTP-SL.

Antibodies

Rabbit polyclonal antiserum against the PTP-containing part of PTP-SL ({alpha}-SL) has been described (van den Maagdenberg et al. 1999a). In some experiments STEP-absorbed {alpha}-SL was used. Briefly, STEP cross-reactivity was removed from the polyvalent {alpha}-SL serum by passing it over glutathion-sepharose-bound GST-STEP fusion protein (Pulido et al. 1998). To generate monoclonal antibodies against PTPRR protein isoforms, the pGEX-SL construct was used for bacterial production of GST-SL fusion protein as previously described (van den Maagdenberg et al. 1999a). Immunization with purified GST-SL protein and later on fusion of mouse spleen B-cells with myeloma line Sp2/0-Ag14 was performed according to established protocols. Resulting hybridomas were screened by ELISA using 96-well plates coated with His-tagged PTP-SL protein. The latter was obtained essentially as described (Walma et al. 2002). Eight clones (6A6, 1E3, 3E11, 8F10, 5E4, 6H11, 11H8 and 6D6) were selected on the basis of their immunoreactivity and grown for antibody production and further analyses.

Bacterial expression plasmids pGEX-PTP-SL, pGEX-2T-SL1060, pGEX-2T-SL1213 and pGEX-2T-SL1582 were used for epitope mapping. In brief, expression of the various GST-PTP-SL fusion proteins was induced using 1 mM IPTG applied to DH5{alpha} bacterial cells containing the respective pGEX-based plasmid. Protein lysates were analysed by Western blot analysis essentially as described below.

Protein isolation, immunoprecipitation and Western blotting

Neuro-2a cells (ATCC nr. CCL-131) were cultured in 9-cm dishes using DMEM supplemented with 5% Foetal Calf Serum. Upon reaching 60% confluency cells were washed with Optimem (Gibco/BRL, Gaithersburg, MD, USA) and transiently transfected with appropriate plasmid DNA using Lipofectamine-Plus (Invitrogen Life Technologies, Breda, the Netherlands). Twenty-four hours after transfection, cells were washed with phosphate buffered saline (PBS) and taken up in lysis buffer (100 mM Na2HPO4, 1% Triton-X-100, 0.2% BSA, pH 8.0), containing a complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany).

For immunoprecipitation purposes, 30 µL mono {alpha}-SL (6A6) together with 30 µL of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech AB, Uppsla, Sweden) was added to 500 µL cell lysate and incubated by overnight rotation at 4 °C. The Sepharose beads with immunobound proteins were subsequently washed four times in lysis buffer, once in 0.1 M Na2HPO4, pH 8.1, and twice in 0.01 mM Na2HPO4. 2 x sample buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added to the beads, and proteins were subjected to 10% SDS-PAGE.

Mouse C57BL/6 tissue lysates were made by mechanical disintegration of whole brain or separate brain regions (olfactory bulb, hippocampus, midbrain, brainstem, cerebellum and cortex) in 1.5 mL lysis buffer. The lysates were incubated for 1 h at 4 °C and insoluble components were pelleted by centrifugation (14 000 r.p.m.) for 30 min at 4 °C. Protein concentration in the resulting supernatant was determined according to Lowry (Peterson 1977).

Protein samples were subjected to 10% SDS-PAGE gels and transferred to nitrocellulose membranes by Western blotting or alternatively first immunoprecipitated with mono {alpha}-SL (6A6) together with protein A-Sepharose CL-4B as described for cell lysates and than subjected to 10% SDS-page gel and Western blotting. Resulting membranes were blocked for 30 min using 3% non-fat dry milk in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) and incubated overnight at room temperature with a 1 : 1000 dilution of rabbit {alpha}-GST-SL in TBST. Blots were washed three times with TBST and incubated for 1 h with a 1 : 10 000 dilution of peroxidase-conjugated Goat anti-Rabbit IgG (Pierce Biotechnology Inc., Rockford, IL, USA) in TBST. Subsequent washes were done with TBST, followed by a final rinse with PBS. For the epitope mapping purposes, blots were incubated with diluted hybridoma culture supernatant (1 : 2000) and peroxidase-conjugated Goat anti-mouse IgG (1 : 20 000) (Pierce) as first and second antibody, respectively. Immunoreactive bands were visualized using freshly prepared chemiluminescent substrate (100 mM Tris-HCl, pH 8.5, 1.25 mM p-coumaric acid (Sigma Chemical Co., St Louis, MO, USA), 0.2 mM luminol (Sigma), and 0.009% H2O2) and exposed to Kodak X-omat autoradiography films (Eastman Kodak Company, Rochester, NY, USA).

RNA expression analyses

Total RNA from different brain regions (olfactory bulb, hippocampus, midbrain, brainstem, cerebellum and cortex) was purified using RNazol B (Campro Scientific, Veenendaal, the Netherlands). Ten µg of RNA was loaded on a 1% formamide agarose gel and after electrophoretical size separation, the RNA was transferred on to nylon membrane (Amersham Pharmacia Biotech BA, Uppsla, Sweden) according to standard procedures (Sambrook et al. 1989). The pSG5/PTPBR7-FL construct was 32P-labelled and used as a probe. Hybridization was carried out overnight at 65 °C (Church & Gilbert 1984), and blots were washed several times with 40 mM phosphate buffer containing 0.2% SDS at 65 °C and exposed to Kodak X-Omat S1 films. Probes specific for PTPBR7 (Acc. No. D31898 [GenBank] : pos. 414–709), PTP-SL (TPA Acc. no. BN000437 [GenBank] : bp 1–135) and PTPPBS{gamma} (TPA Acc. no. BN000438 [GenBank] : bp 1–346) were obtained by PCR on mouse genomic DNA, radioactively labelled, and used for hybridization as described above.

For RT-PCR analyses cDNA was synthesized from 2 µg total RNA by random hexamer priming using SuperScriptTM II RNase H– Reverse Transcriptase (Invitrogen Life Technologies, Breda, the Netherlands). PTPBR7, PTP-SL, PTPPBS{gamma} and ß-actin cDNA fragments were amplified by PCR using the following specific primers: BR7-forward, 5'-CCTCAATGCACACACTATGAGG-3'; BR7-reverse, 5'-TCCTTCTTTGCTCCAGAT-3'; SL-forward, 5'-TCCAGGTGACTAAACGAGG-3'; SL-reverse, 5'-TCCTTCTTTGCTCCAGAT-3'; PBS{gamma}-forward, 5'-GTGAACCAGGTAGTTCCAG-3'; PBS{gamma}-reverse, 5'-AGGGTCCACAACCACGTTCA-3'; ß-actin-forward, 5'-GCTAGAGCTGCCTGACGG-3'; ß-actin-reverse, 5'-GAGGCCAGGATGGAGCC-3'. Resulting products were analysed by electrophoresis using a 2% agarose gel for size-separation and ethidium bromide for fluorescent detection.

Immunohistochemistry

C57BL/6 mice were anaesthetized with hypnorm/dormicum and perfused with 15 mL 0.9% NaCl (saline). Brain tissue was removed, snap-frozen in liquid nitrogen and stored at –80 °C until further use. Brains were either horizontally or coronally cryo-sectioned in 8 µm slices and mounted on Superfrost/Plus glass slides (Menzel-Gläser, Braunschweig, Germany). Selected slides that were representative for the different brain regions were used for immunohistochemical analysis. Sections were treated for 10 min with ice-cold acetone, dried, preincubated in horse serum for 10 min, and subsequently incubated for 60 min with a mixture of three {alpha}-PTPRR monoclonal antibodies (diluted culture supernatant of hybridoma clones 1E3 (1 : 4), 3E11 (1 : 4) and 6A6 (1 : 8)) or with {alpha}-PTPRR monoclonal antibody 5E4 (1 : 16) in PBS with 0.1% BSA. Similar sections were also incubated with STEP-absorbed {alpha}-SL serum. After several washes with PBS and incubation for 30 min with biotin-conjugated secondary antibody (Pierce) 1 : 250 in PBS, immunoreactivity was visualized using the Vectastain protocol (Vector laboratories, Burlingame, CA, USA). 3,3'-diaminobenzidine tetrahydrochloride (brown stain; Fig. 6A–B) or 3-amino-9-ethyl-carbazole (red stain; Fig. 6C–F) were used as substrate. Subsequently, sections were rinsed with water, incubated in 0.9% NaCl and 0.5% CuSO4 for 5 min, and counterstained with haematoxylin for 3 min. Finally, sections were dehydrated with ethanol and xylene, embedded in Eukitt (Electron Microscopy Sciences, Fort Washington, PA, USA) and examined by light microscopy.

Immunofluorescence assay

Neuro-2a cells cultured on glass coverslips were transiently transfected with appropriate plasmid constructs as described above. After 24 h the cells were washed with PBS, fixed for 10 min with 1% paraformaldehyde in PHEM buffer (60 mM Pipes, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9), and permeabilized using 0.1% saponin and 20 mM Glycine in PBS for 30 min. Subsequently, cells were incubated for 1 h with a 1 : 200 dilution of {alpha}.5-SL antiserum in SPBS (PBS + 0.1% saponine) at room temperature. Following three washes with SPBS, cells were incubated with FITC-conjugated goat-anti-rabbit IgG (10 mg/mL; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) (1 : 100 dilution in SPBS) at room temperature for 1 h. Finally, after three washes with SPBS and methanol dehydration, cells were mounted on glass slides using Mowiol (Sigma) containing 2.5% sodium azide as anti-fading reagent. Images were examined and collected using confocal laser scanning microscopy (MRC 1024, Bio-Rad).


   Acknowledgements
 
We thank Ad Geurts van Kessel and Gerard Merkx for performing FISH experiments, C.E.E.M. van der Zee for help with mouse brain histological analyses, and Rafael Pulido (Valencia, Spain) for providing STEP expression constructs. R.G.S.C. and W.H. are members of the European TMR Network on Neuronal Protein Tyrosine Phosphatases. This work is financed by grants from the Nijmegen University Medical Centre (grant number V.089942 to J.F. and W.H.) and the European Community Research Fund (Research Training Network grant number CT2000-00085).


   Footnotes
 
Communicated by: Carl-Henrik Heldin

R. G. S. Chirivi and G. Dilaver contributed equally to this work.

* Correspondence: E-mail: w.hendriks{at}ncmls.kun.nl


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 18 May 2004
Accepted: 6 July 2004




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