(Received for publication, July 25, 1994; and in revised form, October 11, 1994)
From the
Protein tyrosine phosphatase has the potential to control various cellular events by negatively regulating the extent of tyrosine phosphorylation. Here, we report the isolation of a murine receptor protein tyrosine phosphatase, PTPBR7, which is expressed almost exclusively in the brain. Though the cytoplasmic portion of PTPBR7 reveals high similarity to HePTP/LC-PTP and STEP, these are, unlike PTPBR7, non-receptor protein tyrosine phosphatases. Unlike most receptor protein tyrosine phosphatases, PTPBR7 has only one cytoplasmic phosphatase domain, and its extracellular domain reveals no obvious structural similarity to known molecules. Thus, PTPBR7 defines a new subfamily of receptor-type protein tyrosine phosphatases. The putative extracellular domain of PTPBR7 was expressed in COS-7 cells as a chimeric fusion protein with an immunoglobulin Fc portion (PTPBR7-Fc). PTPBR7-Fc was secreted in the culture supernatant, confirming the capability of the extracellular domain of PTPBR7 to translocate across the cytoplasmic membrane. The cytoplasmic portion of PTPBR7 was expressed as a fusion protein in bacteria and was demonstrated to have catalytic activity. The expression of PTPBR7 was detectable in brain and especially in cerebellum but undetectable in liver, lung, heart, kidney, thymus, bone marrow, and spleen. In situ hybridization analysis revealed the most prominent signal in Purkinje cells. The predominant expression of PTPBR7 in the brain suggests that PTPBR7 may have role(s) in neuronal cells.
Protein tyrosine phosphorylation plays a crucial regulatory role
in various cellular events, including growth and differentiation.
Compared with PTK ()(protein tyrosine kinase), much less is
known about PTP (protein tyrosine phosphatase), although both enzymes
can regulate the tyrosine phosphorylation level.
There are many examples of the importance of PTK in the nervous systems. The trk family of PTKs is expressed almost exclusively in neurons and constitutes receptors of the nerve growth factor family of growth factors that regulate survival, neurite growth, and neurotransmitter production (reviewed in (1) ). The Drosophila homologue of abl, a non-receptor tyrosine kinase, is expressed in axons and can cause defects in the axon pathway formation if it is mutated in combination with the fasciclin I gene(2) . It has been reported that the rat brain contains high levels of PTK activity, and one of the highest levels of activity was found in the cerebellum(3, 4) .
Considering the importance of tyrosine phosphorylation, it is conceivable that PTPs are also involved in the biological processes of the nervous system. The family of PTPs has been growing rapidly, and there is a great diversity in the structure of PTPs (reviewed in (5, 6, 7) ). Except for the PTP domain(s), the rest of each PTP molecule is composed of diverse structural motifs that might be essential for a specific function of the PTP. Like PTKs, there are two forms of PTPs, namely, a receptor and a non-receptor form. Receptor PTPs are of special interest in the nervous system, where, as in the immune system, cell growth, survival, and differentiation are regulated by extracellular signals, and specific interaction or recognition of cells is required.
Receptor PTPs of various types are expressed in the nervous system.
Receptor PTPs can be classified into five types based on the structure
of the extracellular
domain(5, 6, 7, 8, 9) .
Types II and III receptor PTPs have an extracellular domain composed of
FN-III repeats. Type II receptor PTPs have additional Ig-like domains
and, thus, resemble N-CAM, a cell adhesion molecule. In fact, two type
II receptor PTPs, mRPTPµ and R-PTP-, have been shown to
mediate homophilic cell adhesion (10, 11, 12) . DPTP99A, DPTP10D, and DLAR are
nervous system-specific types II and III receptor PTPs in Drosophila. These PTPs are expressed in developing axons or
pioneer neurons in the embryonic central nervous system, and their
involvement in axon outgrowth and guidance has been
proposed(13, 14) . LAR(15, 16) ,
PTP
(17, 18) , and PTP
NE-3/PTP-
/PTP-P1/CPTP1 (19, 20, 21, 22) are type II
receptor PTPs in mammals, and these are expressed in neuronal tissues
as well as in other tissues. Type IV receptor PTPs have a short
extracellular domain of unknown function. LRP/RPTP
(17, 23, 24, 25, 26) is a
type IV receptor PTP that is distributed in a wide variety of tissues,
including brain. Type V receptor PTPs have a carbonic anhydrase-like
domain in addition to an FN-III
domain(8, 9, 27) . Recently, it has been
reported that an extracellular variant of a type V receptor PTP
interacts with an extracellular matrix protein and
N-CAM(28, 29) . RPTP
/PTP
and RPTP
are
type V receptor PTPs, and these are expressed in the brain in a highly
specific or a less specific manner, respectively. The physiological
role of these receptor PTPs remains to be elucidated.
Here, we report the cDNA cloning and characterization of a novel murine PTP, which we called PTPBR7. PTPBR7 is predominantly expressed in the nervous system, and the most prominent signal was detected in the Purkinje cell. Unlike most receptor PTPs, PTPBR7 has only one cytoplasmic PTP domain and reveals no obvious similarity to known PTPs in the extracellular domain. Therefore, PTPBR7 defines a new subfamily of receptor PTP, which is expressed almost exclusively in the nervous system.
cDNA was amplified with 40 amplification cycles (94 °C for 1 min, 37 °C for 2 min, and 72 °C for 3 min). PCR fragments of expected size (about 300 base pairs) were purified on 6% native polyacrylamide gel, reamplified, and cloned into SmaI/HindIII sites of pBluescript SK- plasmid (Stratagene).
Figure 1: Overlapping cDNA clones of PTPBR7. A, four overlapping clones isolated from a murine brain cDNA library, five independent clones isolated by SLIC-PCR, and the schematic structure of PTPBR7 are shown. B, a schematic drawing of the strategy of SLIC-PCR is shown in the upper part (see also ``Materials and methods'' and text). PBR7-B1 and PBR7-B3 are specific antisense primers and cover nucleotides 139-163 and 177-202, respectively. In the lower part, arrows below the 5`-end sequence of PTPBR7 indicate the starting position of the five independent SLIC-PCR clones. The numberabove the sequence indicates the nucleotide position. An MaeI site is underlined.
To obtain the 5`-end cDNA fragment of PTPBR7, the cDNA of murine whole brain was amplified, using SLIC (single strand ligation to single-stranded cDNA (ss-cDNA))-PCR as described(33) . The strategy is summarized in Fig. 1B. Briefly, a specific primer (PBR7-B3, 5`-TACAGAACCGAGCACCTGCTTCCTCT-3`) was used to synthesize ss-cDNA. Then, an anchor-oligonucleotide (SLIC-1, 5`-ACTTAACCAGGCTGAACTTGCTACCCTGGAAGAAATACTCAT-3`) was ligated to the 3` end of ss-cDNA using T4 RNA ligase. ss-cDNA was amplified by PCR (40 cycles; 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min) using a second specific primer (PBR7-B1, 5`-AGAGGAAGCAGGTGCTCGGTTCTGTA-3`) and an anchor-primer (5`-GAGAATTCAGCAAGTTCAGCCTGGTTAAGT-3`). Amplified cDNA was cloned into a plasmid vector. Clones were screened by colony hybridization using the 5` end of PBR7-47 (nucleotides 44-618) as a probe, and about 50 out of 2000 colonies showed positive signals.
To analyze the expression of PTPBR7 in various tissues and
cell lines, RNase protection assay was performed as described (37) with minor modifications. Briefly, RNA probes were
synthesized using [-
P]UTP and purified by
6% polyacrylamide gel electrophoresis. Then, the probe (1-2
10
cpm) was hybridized to total RNA samples (10
µg) in 15 µl of hybridization buffer. After hybridization for
12-20 h at 50 °C, samples were digested with RNase A and T1
at 37 °C for 60 min and separated on a 6% polyacrylamide/7 M urea gel.
For Northern hybridization analysis, 4 µg of
poly(A) RNA was separated on a 1% agarose gel and
transferred to a nylon filter (Hybond-N, Amersham). RNA on the filter
was stained by methylene blue to confirm even transfer. The filter was
then hybridized to probes at 45 °C for 18 h in 5
SSPE, 50%
formamide, 5
Denhardt's reagent, 0.5% SDS, 20 µg/ml
of denatured salmon sperm DNA, and 5
10
cpm/ml of
probes radiolabeled by random priming method.
Figure 4:
Expression and secretion of PTPBR7-Fc in
COS-7 cells. A, a schematic drawing of a chimeric gene
encoding PTPBR7-Fc is shown. The nucleotide and amino acid sequences of
the junction connecting the extracellular domain of PTPBR7 (nucleotides
346-1029) and the hinge region of human IgG are shown below. Added nucleotides or amino acids to connect the
sequences are indicated by smallletters or an asterisk, respectively. B, the chimeric genes were
transiently expressed in COS-7 cells. The fusion protein was prepared
from cell lysate (lanes 1-3) or culture supernatant (lanes 4-6) and analyzed by SDS-PAGE under reducing
conditions. Lanes1 and 4, mock transfection
(negative control); lanes2 and 5,
transfection with a construct encoding a mRPTP-
-Fc, in which the
extracellular portion of the receptor PTP (amino acids 1-1256) (53) was fused to the Fc portion of IgG
(positive
control,
190 kDa); lanes3 and 6,
transfection with a construct encoding PTPBR7-Fc; lane7, marker. The size (kDa) of the marker is shown on the right.
For the
construction of a plasmid encoding GST-PTPBR7, the pGEX-3X vector
(Pharmacia LKB Biotechnology AB, Uppsala, Sweden) was digested with BamHI, filled with Klenow fragment, and digested with EcoRI. The cDNA insert of PBR7-47, subcloned in
pBluescript SK plasmid, was digested with SmaI and NotI. Then, the cDNA fragment encompassing nucleotides
1440-2844 of PTPBR7 was separated by agarose gel electrophoresis.
This cDNA fragment was ligated to the vector treated as described above
with a NotI/EcoRI fragment as a filler. Escherichia coli (XL1-Blue) transformed with the plasmid was
cultured at 29 °C in 200 ml of LB medium, and expression of the
chimeric gene was induced by adding 0.1 mM of
isopropyl-1-thio--D-galactopyranoside when absorbance at
600 nm of the culture reached 0.40. After a 3-h incubation, cells were
centrifuged, and the pellet was resuspended in 4 ml of 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride. The bacteria
was then lysed by sonication and centrifuged at 10,000
g for 10 min at 4 °C. The supernatant was mixed with 200 µl
(pellet volume) of glutathione-Sepharose beads suspended in
phosphate-buffered saline (1:1, v/v) and rocked for 1 h at 4 °C.
The beads were washed five times with 1 ml of 50 mM Hepes pH
7.6, 150 mM NaCl, 0.1% Triton X-100 and once with the
phosphatase assay buffer described below. Finally, the beads were
suspended in the phosphatase assay buffer. An aliquot was mixed with an
equal volume of 2
SDS-PAGE sample buffer containing
-mercaptoethanol, boiled for 3 min, electrophoresed on a 10%
polyacrylamide gel, and stained with Coomassie Brilliant Blue. The
amount of GST-PTPBR7 protein captured on the beads was estimated by
comparing the stained band with that of a known amount of purified
glutathione S-transferase protein.
Four overlapping cDNA clones (PBR7-7, -17, -26, and -47) were isolated from a murine brain cDNA library (Fig. 1A). The length of the consensus sequence generated from these clones agreed with the size of the longest transcript detected by Northern hybridization analysis (see Fig. 6B). However, there was no in-frame stop codon preceding the first ATG in the longest open reading frame, and it was possible that the coding region was extending further in the 5` direction. Therefore, we screened more than one million clones from a brain cDNA library (primed with oligo(dT)) and an additional one million clones from another brain cDNA library (primed with oligo(dT) plus random hexamer), but we could not isolate a clone containing more upstream sequence.
Figure 6:
RNase protection analysis of the
expression of PTPBR7 in various tissue and cell lines. A, 10
µg of total RNA from indicated organs or cell lines was hybridized
to the P-labeled antisense RNA probe, coding for a part of
the PTP domain (nucleotides 1755-2122). The size of the
undigested probe is 426 nucleotides, and protection of PTPBR7
transcripts yields a 348-nucleotide fragment. For each RNA sample, an
aliquot was electrophoresed on a 1% agarose gel, and intactness of the
RNA was confirmed by visualizing 18 and 28 S rRNA bands (data not
shown). 200 cpm of undigested probe (lane1) and
yeast tRNA (lane2) were included as controls. The
positions of the markers are indicated on the left, and the numbers indicate the size (in nucleotides). B, 4
µg of poly(A)
RNA immobilized on filters was
probed with
P-labeled cDNA fragments covering nucleotides
122-1538 of PTPBR7. Positions of murine 28 S (4.7 kb) and 18 S (1.9 kb)
rRNAs are indicated on the right.
Finally, to obtain the 5`-end cDNA fragment of PTPBR7, we employed SLIC-PCR (33) (Fig. 1B) using the whole brain RNA. The 5`-end fragment of the cDNA was specifically synthesized by reverse transcription with a specific primer (PBR7-B3), which was complementary to the sequence near the 5` end of PBR7-47. Because the amount of the cDNA generated was very small, the cDNA was amplified by PCR, using an anchor primer and the second specific primer (PBR7-B1). After cloning into a plasmid vector, clones containing the 5`-end sequence of PBR7-47 were determined by colony hybridization. We randomly picked up 39 positive clones and mapped the XhoI and MaeI sites in each insert. The XhoI site was present at nucleotide 122, and a preliminary sequencing experiment of one of the PCR clones showed that there was an MaeI site (CTAG) at nucleotide 36. Twenty-two of 39 clones had expected MaeI and XhoI sites, showing that these clones contained the 5` sequence at least up to nucleotide 36. The size of the insert was then compared, and five clones that had inserts longer than or equal to others were analyzed by sequencing. The result is summarized in Fig. 1B. We found two independent clones (SLICB1-110 and -117) starting from nucleotide 1. The other three clones started at nucleotide 20 or 21.
Figure 2: Nucleotide and predicted amino acid sequence of PTPBR7. The deduced amino acid sequence of PTPBR7 is shown below the nucleotide sequence. The putative signal peptide sequence is underlined, the transmembrane domain is doubleunderlined, and the approximate location of the putative PTP domain is shaded. The cleavage site of the signal peptide was estimated using a scoring method as described (43) . Equally high scores (8.68 and 8.13) were observed when the C at position 21 and the S at position 23 were assigned the -1 position of the cleavage site. The C at position 21 showed a much lower score in the c-region than in the h-region (1.66 versus 7.02), whereas the S at position 23 showed a score in the c-region comparable with that in the h-region (4.86 versus 3.27). Thus, we estimated the -1 position of the cleavage site to be the S at position 23.
Figure 3: Comparison of the amino acid sequence of PTPBR7 with that of HePTP and STEP. A, an alignment of PTPBR7 with two cytoplasmic PTPs, HePTP and STEP, is shown. The amino acid residues conserved in at least two of the three PTPs are shaded. The stars beneath the amino acid sequence indicate an amino acid residue identical in all three proteins. The numbers in parentheses indicate the amino acid position in the deduced protein sequences. B, schematic representations of PTPBR7, HePTP, and STEP. The blackbox indicates the PTP domain. The shadedbox indicates the region of approximately 90 amino acid long, which shows high similarity. The hatchedbox shows the signal peptide or transmembrane domain as indicated. The numbers indicate the amino acid positions.
In addition, we found a sequence of 104 amino acids in the EMBL data (Hendriks, W., Brugman, C., Zeeuwen, J., Schepens, J., and Wieringa, B., accession No. S40280), which has 100% identity to a part of the PTPBR7 sequence (amino acids 482-585). This sequence most likely represents a partial PTP domain sequence of PTPBR7.
PTPBR7 has only one PTP
domain, whereas most receptor PTPs have two. So far, only three
receptor PTPs, HPTP, DPTP10D, and
SAP-1(13, 14, 17, 40) , are known to
have one cytosolic PTP domain. These three PTPs have a large
extracellular domain composed of FN-III repeats, and these structural
features define the type III receptor PTP. We could not find apparent
amino acid sequence similarity in the extracellular domain of PTPBR7
with HPTP
, DPTP10D, SAP-1, or any other molecules in the NCBI data
base. In the PTP domain, the similarity of PTPBR7 to HPTP
,
DPTP10D, or SAP-1 was lower than that to HePTP/LC-PTP or STEP.
To test the catalytic activity of the putative
PTP domain, we constructed a chimeric gene encoding a fusion protein,
GST-PTPBR7, in which the putative PTP domain was fused C-terminally to
glutathione S-transferase. The chimeric gene was expressed in E. coli, and the phosphatase activity was measured using p-NPP as a substrate (Fig. 5, A and B). The phosphatase activity was measured at pH 7.4, because,
in the preliminary experiment, the activity detected with the pH 7.4
assay buffer was approximately 300-fold higher than that detected with
the pH 5.0 assay buffer. The catalytic activity was inhibited by 3
mM NaVO
, a tyrosine phosphatase
inhibitor (Fig. 5B, third column).
Figure 5:
Catalytic activity of the GST-PTPBR7
fusion protein. A, 2 µg of purified glutathione S-transferase or GST-PTPBR7 protein, which was used for the
phosphatase assay, was electrophoresed on a 10% polyacrylamide gel and
stained with Coomassie Blue. Molecular sizes (kDa) are shown on the right. B, catalytic activity of glutathione S-transferase or GST-PTPBR7 was measured at pH 7.4 as
described under ``Materials and Methods.'' In a reaction (thirdcolumn), 3 mM NaVO
was included.
The expression of PTPBR7 was undetectable in all the cell lines tested, which included T-lineage (EL4 and 2B4), B-lineage (70Z/3 and NS-1), myeloid (WEHI3), mastcytoma (P815), thymic stroma (MRL104.8a), and fibroblast (BALB3T3) cell lines (Fig. 6A).
Figure 7:
In situ hybridization analysis of the
expression of PTPBR7. Horizontal 10-µm sections of adult mouse
brain were hybridized with a S-labeled antisense RNA probe
of PTPBR7 (A-C). A probe for CD45, which is specifically
expressed on hematopoietic cells, was used as a negative control (D-F). A and D, cerebellum; B and E, regions of the habenula; C and F, regions of the hippocampal
formation.
We have cloned a cDNA of a novel PTP, PTPBR7, which is expressed exclusively in the brain. PTPBR7 possesses typical features of a type I membrane protein. There is a stretch of hydrophobic amino acid, a putative transmembrane domain. The N-terminal amino acid sequence deduced from the putative translation initiation site resembles a signal peptide. The capability of the extracellular domain of PTPBR7 to translocate across the cytoplasmic membrane was confirmed using a chimeric PTPBR7 tagged with immunoglobulin Fc (PTPBR7-Fc). Taken collectively, we conclude that PTPBR7 belongs to the receptor PTP family.
The extracellular domain of PTPBR7 shows no apparent structural similarity with known molecules, including reported receptor PTPs. Thus, PTPBR7 defines a new subfamily of receptor PTPs.
Data base searches revealed that the cytoplasmic portion of PTPBR7 has high similarity with previously reported PTPs, HePTP/LC-PTP (45) and STEP(46) , in the PTP domain and in a short stretch (approximately 90 amino acids) preceding the PTP domain. Both HePTP and LC-PTP are specifically expressed in lymphoid cells and show an extremely high sequence similarity. The minor differences in the reported sequence of HePTP and LC-PTP could be explained as allelic variation of the same gene. STEP is expressed predominantly in striatum. Based on the differences in the sequence and in the tissue distribution pattern, it is clear that PTPBR7 is not the murine homologue of human HePTP/LC-PTP or rat STEP.
HePTP/LC-PTP and STEP are, unlike PTPBR7, non-receptor PTPs. This is surprising, because usually PTPs with high homology in the PTP domain have a similar overall structure. The reported sequences for HePTP and STEP are clearly not the partial cDNA sequences of receptor PTPs, because there are in-frame stop codons preceding the initiation codon. Interestingly, LC-PTP and STEP have two kinds of transcript, which are different in size, and the reported cDNA sequences are in good accordance with shorter transcript (2.9 kb for LC-PTP and 3 kb for STEP) in size. One attractive hypothesis is that the longer transcripts (4.0 kb for LC-PTP and 4.4 kb for STEP), generated by alternative splicing or different usage of the transcription initiation sites, are encoding receptor forms. So far, there is no evidence for the presence of receptor forms of HePTP/LC-PTP and STEP.
Several PTPs, for example, DLAR, DPTP10D,
and DPTP99A in Drosophila(13, 14, 49) and RPTP/PTP
(9, 27) and STEP (46) in mammals, are
expressed primarily in the nervous system. Among these PTPs,
RPTP
/PTP
has been reported as the first cloned receptor PTP
of which expression is restricted to the nervous system. PTPBR7 is a
new example of a receptor PTP primarily expressed in the nervous
system.
There is an apparent overlap in the expression of PTPBR7 and
RPTP/PTP
(9, 27) . Both are expressed in
Purkinje cell layer of cerebellum and the hippocampal formation. The
differences between PTPBR7 and RPTP
/PTP
in the structure
might result in different functions of these PTPs in a neuron. For
example, the difference in the extracellular domains could imply a
difference in (putative) ligand-specificity. In addition, PTPBR7 has
one cytoplasmic PTP domain, whereas RPTP
/PTP
has two. The
catalytic activity of the second PTP domain of receptor PTPs is usually
low or absent, and its regulatory role or binding to
tyrosine-phosphorylated proteins has been
proposed.(6, 19, 50, 51) . Thus,
PTPBR7 lacking the second PTP domain might be involved in a signal
transducing pathway different from that of RPTP
/PTP
.
The physiological role of PTPs in the nervous system remains to be elucidated. Because tyrosine phosphorylation by PTKs plays a critical role in the proliferation, survival, and differentiation of neuronal cells, PTPs might have regulatory function through dephosphorylation of the target protein. We found that PTPBR7 is expressed predominantly in the cerebellum. Interestingly, one of the highest levels of PTK activity in the brain has been found in the cerebellum(3) . It was also reported that nerve growth factor, a ligand of a receptor-PTK, has effects on the survival or morphogenesis of Purkinje cells(52) .
It is also possible that PTPBR7 is primarily active in immature nervous systems rather than in adult brain. During embryonic development, receptor/ligand systems seem to be involved in the specific guidance of migrating neurons and outgrowing axons, neuron survival, and proliferation. For example, three transmembrane PTPs, DLAR, DPTP10D, and DPTP99A are expressed on axons of developing nervous systems in Drosophila, and their involvement in axon outgrowth and guidance has been suggested(13, 14) . Therefore, it would be important to investigate the expression of PTPBR7 more precisely in adult and developing nervous systems as well as to determine the distribution of PTPBR7 within a neuron, i.e. whether it is expressed in the cell body, axon, or both.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D31898[GenBank].