(Received for publication, June 5, 1995; and in revised form, August 25, 1995)
From the
A novel protein-tyrosine phosphatase, PTP, was cloned from
a murine macrophage cDNA library. As a result of alternative splicing,
macrophage PTP
mRNAs are predicted to encode two
membrane-spanning molecules and a cytosolic enzyme with identical
catalytic domains. The membrane-spanning forms differ in the
juxtamembrane region, while a start codon downstream of this region is
utilized in the translation of the putative cytosolic form. Expression
of PTP
mRNA is low and restricted to macrophage cell lines,
macrophage-rich tissues, and brain, kidney, and heart. The mRNA in
macrophages and heart is
2.8 kilobases (kb). However, a
5.5-kb transcript in brain and kidney indicates a fourth isoform
encoding a large extracellular domain. The
5.5-kb PTP
brain
mRNA encodes the mouse homolog of GLEPP1, a recently reported
glomerular epithelial protein. The level of expression of the mRNA
encoding the cytosolic form was very low, and only the
membrane-spanning proteins (43 and 47 kDa) could be detected in
macrophages. Following addition of colony stimulating factor-1 to
quiescent BAC1.2F5 macrophages, PTP
mRNA and protein were
down-regulated. The restricted expression of the shorter isoforms of
PTP
and their regulation by colony stimulating factor-1 in
macrophages suggest that PTP
may play a role in mononuclear
phagocyte survival, proliferation, and/or differentiation.
The importance of phosphorylation on tyrosine residues of
proteins involved in the signaling of cell proliferation,
differentiation, and transformation has been well established (reviewed
in (1) ). Despite the fact that the protein-tyrosine
phosphatases (PTPs) ()were first described only relatively
recently(2, 3) , they have been found in species
ranging from viruses to mammals (reviewed in (4) ). However,
unlike the protein-tyrosine kinases, which exhibit sequence similarity
with serine/threonine kinases, the PTPs do not show any sequence
similarity with the serine/threonine phosphatases.
PTPs can be divided into two main groups: the low molecular weight, cytoplasmic, single catalytic domain-containing molecules and the high molecular weight, membrane-spanning, receptor-like forms, almost all of which contain two tandem repeats of the intracellular catalytic domain (4) . The well conserved phosphatase domain consists of about 240 amino acids, within which is a particularly well conserved consensus sequence of 11 residues ((I/V)HCXAGXGR(S/T)G). The cysteine residue in this signature sequence is essential for the specific catalytic activity of the enzyme (5, 6) and has been shown to participate in the formation of a covalent phosphoenzyme intermediate as part of the catalytic process(7) .
The membrane-spanning PTPs frequently
have very large extracellular domains containing immunoglobulin-like
repeats and/or fibronectin III-like repeats, suggesting a receptor-like
function. However, no ligands have yet been identified. In view of the
similarity of the extracellular domains to neural cell adhesion
molecules, it has been suggested that homophilic or heterophilic
cell-cell interactions rather than soluble ligands may modulate the
activity of some PTPs leading to cell contact inhibition(5) ,
and this has been confirmed in the case of PTPµ (8) and
PTP(9) . The functional significance of the tandemly
repeated catalytic domains present in all but a few of the
receptor-like PTPs is uncertain. The amino-terminal domain alone is
active with evidence for a complete lack of phosphatase activity in the
carboxyl-terminal domain (5, 6) or the presence of low
activity(7, 10) . However, the carboxyl-terminal
domain appears to regulate the substrate specificity of the first
domain(5, 6, 7, 10) .
The cytosolic PTPs comprise a more disparate group. Subfamilies within this group are characterized by different amino- and carboxyl-terminal domains, many of which are important in the subcellular localization of these molecules(11, 12, 13) . Recent evidence indicates that both subcellular localization (12) and phosphorylation status (14) are important in the cellular regulation of the activity of the cytosolic tyrosine phosphatases. Other methods of regulating activity include transcriptional control, which is responsible for the pattern of expression of an immediate, early gene whose product, 3CH134, is a dual specificity phosphatase that dephosphorylates mitogen-activated protein kinase (15) and alternative splicing in the catalytic domain, which down-regulates the in vitro affinity of PTP1D for substrates (16) .
Alternative splicing is a relatively common phenomenon in PTPs resulting in extracellular variants (17, 18) and intracellular variants (19, 20) of the receptor-like PTPs. A membrane-spanning PTP isolated from rat pheochromocytoma cells has alternative splicing that results in two isoforms, one of which contains one catalytic domain while the other has the more usual tandemly repeated domains(21) . Extracellular splicing in CD45 produces isoforms that are differentially expressed in lymphocytes(22) , whereas the biological significance of the splice variants in the other molecules has not been clearly determined.
We have used the polymerase chain reaction (PCR) with degenerate
oligonucleotide primers to identify the PTPs that are expressed in the
colony stimulating factor-1 (CSF-1)-dependent murine macrophage cell
line, BAC1.2F5. In this report, we describe a novel PTP, designated as
PTP, whose primary transcript is alternatively spliced to yield
messages encoding two membrane-spanning forms and a putative cytosolic
form and whose expression is regulated by CSF-1. A recent study
reported the cloning of a novel rabbit PTP derived from the renal
glomerulus and designated glomerular epithelial protein 1,
GLEPP1(23) , for which one isoform of PTP
appears to be
the mouse homolog.
Organs were collected from 17- and 20- day-old fetal, 2-day-old neonate, and adult C57B/6 mice and directly processed for RNA isolation or immediately frozen in liquid nitrogen prior to storage at -80 °C.
Casein was
phosphorylated by incubating 100 units of the protein kinase
A-catalytic subunit (P-8289, Sigma) with 660 µg of casein (C-4032,
Sigma) in 40 mM Hepes, pH 7.0, 20 mM MgCl, 1 mM benzamidine HCl, 1 µg/ml
pepstatin A, 1 µg/ml leupeptin, 5 µg/ml aprotonin, 2 mM ATP with 600 µCi of [
-
P]ATP (3000
Ci/mmol) (total volume of 600 µl) for 1 h at 37 °C before
termination of the reaction by the addition of 400 µl of
trichloroacetic acid at 4 °C for 1 h. The trichloroacetic
acid-precipitated protein was pelleted by centrifugation at 12,000
g for 1 h at 4 °C, washed, and dialyzed against
water in a Centricon-30 concentrator as described above.
Serine
phosphorylation and conversion of phosphorylase b to
phosphorylase a by phosphorylase kinase was carried out
according to the protocol outlined in the Life Technologies, Inc.
phosphatase assay system (Life Technologies, Inc.). The substrate was
then precipitated in ice-cold 90% saturated ammonium sulfate at 4
°C for 30 min, pelleted, washed five times with ice-cold 45%
ammonium sulfate, and resuspended in 1 ml of buffer B (50 mM Tris HCl, pH 7.0, 0.1 mM EDTA, 15 mM caffeine,
0.1% mercaptoethanol (w/v)). The sample was then successively
diluted and concentrated against buffer B in a Centricon-30
concentrator as described above.
Serine phosphatase assays
were performed as for the tyrosine phosphatase assays with the
following changes: 1 mM MnCl and 30 µg/ml
protamine sulfate were included in buffer A (34, 35) and 100 pmol of
P-labeled casein
or 600 pmol of [
P]phosphorylase a,
prepared as stated above, replaced the
P-labeled
poly(Glu-Tyr) (4:1).
The pH activity profile for PTP was
determined using a previously described mixed buffer system (50 mM acetic acid, 50 mM Mes and 100 mM
triethanolamine) that maintained constant composition and ionic
strength within the pH range tested (5-8.5)(36) .
Tyrosine phosphatase assays were performed as described above with the
mixed buffer replacing Hepes in buffer A and a decrease in the
concentrations of NaCl and KCl to 35 mM. Assays to determine
the optimum ionic strength for PTP
activity were carried out in
buffer A without KCl but containing from 0 to 500 mM NaCl.
Screening of a BAC1.2F5 cDNA
library with the subcloned PCR product yielded two overlapping partial
clones. The library was rescreened with a 2.1-kb fragment of one of
these, 10-1, and five additional clones (10-1/1,
10-1/3, 10-1/5, 10-1/7, 10-1/8) were obtained,
all of which contained the 10-1 PTP sequence. However, three of
the five clones contained an insert of either 190 or 274 bp that was
spliced in upstream of the catalytic domain (Fig. 1). The 274-bp
insert in clones 10-1/1 and 10-1/7 had a unique 84-bp
stretch at the 3`-end of the 190-bp insert in clone 10-1/3 but
was otherwise identical (Fig. 2). The sequences of the two
groups of clones that have inserts differed from each other by a finite
number of codons so that the reading frame was maintained, but the
sequence of the original insert-free clone, 10-1 (Fig. 1),
differed by 63 codons + 1 bp and 91 codons + 1 bp from the
short (10-1/3) and long (10-1/7) insert clones,
respectively, resulting in the loss of reading frame. All clones had
the same 5`- and 3`-untranslated region (UTR) sequences, the 5`-UTR
containing an in-frame ochre stop codon at nucleotide 111. The
insert-containing clones had a start codon with strong consensus for
protein initiation (42) that was just upstream of the 5`-end of
each alternatively spliced insert. Both inserts encoded the same
25-amino acid putative transmembrane domain, followed by a variable
juxtamembrane domain upstream, and thus the insert-containing variants
putatively coded for two very similar membrane-spanning PTPs with short
extracellular domains. In contrast, the start codon for 10-1 lay
78 bp downstream of the insert site (Fig. 2), and the sequence
predicted a low M cytosolic PTP. The alternative
splice sites have been confirmed by mapping and sequencing of the
exon-intron boundaries (Fig. 2). (
)
Figure 1:
Schematic model of the
alternatively spliced cDNA variants of PTP. 10-1/7, long
insert variant; 10-1/3, short insert variant; 10-1,
no-insert variant. The open reading frame is represented by a box containing the putative transmembrane (filled) and
catalytic (cross-hatched) domains. The asterisk indicates the loss of reading frame in 10-1, resulting in
the use of the alternative downstream start codon (ATG
2).
Figure 2:
The
nucleotide sequence and deduced amino acid sequence of PTP in
BAC1.2F5 cells. The 5`- and 3`-UTRs are in lower case with the
5`-in-frame stop codon and the 3`-poly(A)
adenylation
signal underlined. The two start codons are boxed,
and the transmembrane domain is shaded. The catalytic domain
is outlined with the heavy border, and the invariant
cysteine is outlined with the double line. The short insert is
within the dashed box, and the additional sequence of the long
insert is within the dotted box.
Data base
searching (GenEmbl, 1994) for the catalytic domain alone revealed close
homology with the murine homolog of PTP (49.8% similarity by
Lipman-Pearson protein alignment), a single catalytic domain-containing
PTP(43) , with DPTP10d (50.2%), a Drosophila PTP
expressed in the central nervous system(44, 45) , and
with leukocyte antigen-related PTP (42.1%)(46) .
Figure 3:
Expression of PTP mRNA in cell
lines. A, Northern blot showing expression of PTP
mRNA
in cell lines. The blot was exposed to the phosphorimager screen for 7
days. B, ethidium bromide staining of total RNA in the
agarose/formaldehyde gel showing the 28 and 18 S ribosomal bands as a
control for loading.
Figure 4:
Tissue distribution of PTP mRNA. A, Northern blot showing expression of PTP
mRNA in
C57B/6 adult mouse tissues. Molecular size markers on the left indicate the size of PTP
transcripts. The blot was
autoradiographed for 14 days. B, ethidium bromide staining of
total RNA in the agarose/formaldehyde gel showing the 28 and 18 S
ribosomal bands as a control for loading.
Both an adult and a neonatal mouse brain cDNA library were screened to sequence the extra 2.7 kb of brain transcript revealed by Northern analysis. Of the six independent clones identified, only one contained sequence extending 5` of the short extracellular domain seen in BAC1.2F5 cells. Sequence analysis of this clone, which contained 116 bp of new 5`-sequence revealed a fibronectin-like repeat homology region (results not shown). All six brain clones spanned at least part of the alternatively spliced region, and all contained the insert characteristic of long insert (10-1/7) variant.
In view of the
failure to discern separate transcripts corresponding to the different
cDNA clones obtained from the library screens by Northern analysis,
RT-PCR was carried out on RNA extracted from BAC1.2F5 cells, mouse
brain, kidney, and bone marrow using oligonucleotide primers that
framed the insert region. BAC1.2F5 RNA showed three bands corresponding
to the long (10-1/7), short (10-1/3), and no-insert)
variants (Fig. 5). The short insert variant appeared to be more
highly expressed than the long insert variant, while the level of
expression of the no-insert variant was substantially lower. Since all
three amplicons used the same primer pair in the same PCR reaction
tube, it is likely that the results reflected the relative levels of
expression of the three variants. The major product seen on
amplification of whole brain RNA was the long insert variant with a
faint band corresponding to the short insert variant and no evidence of
expression of the no-insert variant. The major product in kidney was
the short insert variant with some expression of the long insert
variant but no evidence of the no-insert variant. The pattern of
expression of PTP mRNA in bone marrow was the same as the pattern
of BAC1.2F5 macrophage expression.
Figure 5: Tissue-specific expression of alternatively spliced transcripts. 2% agarose/TBE gel was stained with ethidium bromide and shows RT-PCR products with oligonucleotide primers framing the alternative splicing sites. The bands representing the long, short, and no-insert variants are indicated at 453, 369, and 179 bp, respectively.
To confirm that the sequences of the BAC1.2F5 mRNAs in the alternatively spliced regions corresponded to the sequences of the cDNA clones obtained by library screening, all three RT-PCR products were subcloned and sequenced. The mRNA sequences for the two insert-containing variants and the putative cytosolic variant were identical to those of their corresponding cDNA clones.
Figure 6:
Macrophage PTP has alternative start
codons. [
S]Methionine-labeled in vitro translation products of PTP
cDNA clones run in a
7.5-17.5% gradient SDS-PAGE minigel. The negative control
contained no DNA. Molecular size markers are shown on the left with expected mass of the three splice variant products indicated
on the right.
The long insert variant (10-1/7) was expressed as a
GST-PTP fusion protein in E. coli to characterize its
catalytic activity with respect to substrate specificity, pH, ionic
strength, and kinetic parameters. GST-PTP
possessed PTP activity
for the tyrosine-phosphorylated poly(Glu-Tyr) substrate that was
substantially decreased in the presence of 1 mM vanadate,
while GST alone had no measurable activity (Fig. 7A).
No GST-PTP
activity could be detected toward the serine/threonine
phosphatase-specific substrates, phosphorylated phosphorylase a, and casein (Fig. 7A). The pH optimum for
GST-PTP
of 6.5 was similar to that reported for leukocyte
antigen-related PTP toward protein substrates (7) (Fig. 7B). The optimum ionic strength for
GST-PTP
activity was achieved with 100 mM NaCl, with
activity declining at higher NaCl concentrations (Fig. 7C). Michaelis-Menton analysis revealed a K
of 2.6 µM toward phosphotyrosine on
the phosphorylated poly(Glu-Tyr) (4:1) with a k
of 0.2 s
(Fig. 7D).
Figure 7:
Kinetic analysis and substrate specificity
of recombinant PTP. A, activity of GST-PTP
(closed diamond), GST (open square), and
GST-PTP
+ 1 mM vanadate (open diamond)
toward
P-labeled poly(Glu-Tyr) (4:1) and of GST-PTP
toward phosphorylase a (open circle) and casein (X). B, pH profile of GST-PTP
phosphatase
activity. C, effect of added NaCl on the phosphatase activity
of GST-PTP
. D, kinetic analysis of GST-PTP
.
Phosphatase activity is expressed in picomoles of phosphate released
from substrate per 20 min at 25 °C.
To
confirm the presence of the various protein isoforms of PTP in
BAC1.2F5 cells, whole cell lysates, membrane, and cytosolic fractions
were Western blotted and probed with
PTP
antisera.
Consistent with our in vitro translation results and their
predicted transmembrane domain, the
PTP
fusion protein
antibody detected a doublet at 43-47 kDa corresponding to the
short and long insert forms in both whole cell lysate and the membrane
fraction that was not detected by preimmune serum (Fig. 8). The
PTP
antibody binding was inhibited by purified GST-PTP
protein but not by GST alone. The three anti-peptide PTP
antisera
also detected the 43- and 47-kDa isoforms. Neither the
PTP
antibody nor the two anti-peptide antibodies, which would be expected
to recognize the putative cytosolic form, were able to detect the
predicted 33-kDa band (see Fig. 6) in the whole cell lysate or
cytosolic fraction either by Western blotting or by immunoprecipitation
followed by Western blotting (data not shown). As PTP
is not an
abundant cellular protein and the relative expression of the 43- and
47-kDa isoforms reflects the relative expression of their predicted
mRNAs assessed by RT-PCR (Fig. 5), the much lower expression of
mRNA for the putative 33-kDa cytosolic isoform is consistent with the
failure to detect the protein. The
38-42-kDa doublet
detected by the fusion protein antibody in the whole cell lysate and
cytosolic fraction ( Fig. 8and 9D) was detected by all
the anti-peptide antibodies. This doublet may represent proteolytic
cleavage products of the putative membrane-spanning isoforms since the
extent of doublet band separation is unchanged and there are potential
cleavage sites (dibasic residues) immediately after the transmembrane
domain (Fig. 2).
Figure 8:
Expression of PTP in BAC1.2F5 cells.
PTP
Western blot of whole cell lysates (WCL),
membrane (memb), and cytosolic (cyt) fractions from
BAC1.2F5 cells is shown. The transfer membrane was blotted with
pre-immune serum (PI), immune serum in the presence of GST (I + GST), or immune serum in the presence of
GST-PTP
(I + GST-PTP
). Molecular size markers
(prestained) are indicated on the left.
Figure 9:
Regulation of PTP expression by
CSF-1. A, Northern blot of PTP
in BAC1.2F5 cells, which
were rendered quiescent by overnight incubation in the absence of CSF-1
and incubated with the growth factor for the times indicated. B, Northern blot of 28 S ribosomal RNA as a control for
loading. C, quantitative analysis of the level of PTP
mRNA expression, corrected for loading. D,
PTP
Western blot of fractions from quiescent (0 h), log-phase (cycling),
and restimulated (5 h) BAC1.2F5 cells. Molecular size markers are
indicated on the right. The sharp band above the 47-kDa band
is an artifact observed in many PTP
Western blots and, when
present, is also seen in preimmune lanes. WCL, whole cell
lysates; memb, membrane; cyt,
cytosolic.
We have cloned and characterized a novel PTP from a murine
macrophage cell line, BAC1.2F5, and have designated it PTP.
Uniquely among PTPs, PTP
encodes, as a result of alternative
splicing, both membrane-spanning, single catalytic domain-containing
PTPs and a putative low M
cytoplasmic PTP. The
alternative splicing seen in PTP
is unusual in that the
transcript encoding the cytoplasmic variant has a change of reading
frame compared with the membrane-spanning transcripts.
In the putative membrane-spanning isoforms an identical 5`-sequence is found within both the short and long inserts, while the long insert has an extra 28 codons spliced in at the 3`-end compared with the short insert providing evidence for two separate alternatively spliced regions. These putative membrane-spanning isoforms encode 43- and 47-kDa proteins that each have a very short extracellular domain of 8 residues.
In vitro transcription-translation demonstrates
that a second, downstream start codon is responsible for initiation of
protein synthesis in the putative cytosolic PTP, yielding a
catalytic domain that possesses only short amino-terminal (32 residues)
and carboxyl-terminal (22 residues) stretches (Fig. 1). RNA
processing to produce both a single and a double catalytic
domain-containing, receptor-like PTP has been reported
previously(21) , but there have been no reports of any PTPs
that, through alternative splicing of a transmembrane domain, may exist
as membrane-spanning and cytosolic forms.
Northern analysis reveals
the presence in brain and kidney of a further splice variant that, at
5.5 kb, is almost twice the length of the transcripts seen in
BAC1.2F5 cells and bone marrow. Preliminary studies in our laboratory
indicate that the 5.5-kb PTP
message expressed in brain encodes a
form of PTP
with a large extracellular domain containing
fibronectin type III repeats.
As this manuscript was being
completed, a report of a membrane-spanning rabbit PTP, GLEPP1,
containing a single catalytic domain and a large extracellular domain
consisting of 8 fibronectin type III repeats was
published(23) . PTP
appears to be the mouse homolog of
GLEPP1 (Fig. 10), which was detected in the renal cortex and
brain only. The failure to detect GLEPP1 mRNA in other tissues probably
resulted from the use of a GLEPP1 extracellular domain probe in the
ribonuclease protection assay, preventing detection of both the
cytosolic and short extracellular domain-containing forms. Although 2
of the 13 GLEPP1 cDNA clones showed evidence of a short stretch of
alternative splicing in the juxtamembrane region leading to the
exclusion of 12 amino acids immediately 3` to the inserts described for
PTP
, none of these clones indicated alternative splicing of the
extracellular domain or of the transmembrane domain (Fig. 10A). Thus, the
2.8-kb messages encoding the
membrane-spanning forms and the putative cytosolic form were not
described. Moreover, as the sequence of GLEPP1 in the insert region
corresponds to one of the short insert variants of PTP
, the
unique 3` 84-bp sequence of the long insert is not represented in the
published GLEPP1 sequence (Fig. 10B). The cDNA sequence
for the 5`-UTR of PTP
is homologous to that of the extracellular
domain of GLEPP1 in the juxtamembrane region for 270 bp before it
diverges completely at nucleotide 111 for PTP
(nucleotide 2340
for GLEPP1) (Fig. 10C). The point of divergence
coincides with the in-frame stop codon in the 5`-UTR of PTP
and
is therefore likely to represent a splice site. Consistent with its
homology to GLEPP1, the brain PTP
clone that extended 5` of the
BAC1.2F5 sequence diverged from the BAC1.2F5 sequence at this point
also. The 3`-UTR of GLEPP1 and PTP
exhibit a
67.5% sequence
similarity, and the ATTAAA polyadenylation site at nucleotide 2718 in
PTP
is not seen in GLEPP1, for which there is no reported
polyadenylation site.
Figure 10:
Comparison of PTP and
GLEPP1.(23) . A, schematic comparison of the two
membrane-spanning isoforms of PTP
expressed in macrophages and
the larger, receptor-like GLEPP1 expressed in kidney and brain. The
signal sequence and transmembrane domains are represented by the filled boxes, the lighter cross-hatching indicates
the fibronectin type III repeats, and the darker cross-hatching indicates the catalytic domains. B, alignment of the open
reading frame of the long insert variant of PTP
(10-1/7)
with the corresponding amino acid sequence of GLEPP1. Identical
residues are boxed, and the dashes represent the lack
of corresponding GLEPP1 sequence. C, alignment of the DNA
sequence of the 5`-UTR of PTP
with the juxtamembranous
extracellular domain of GLEPP1. The start codon for PTP
is boxed, and the in-frame stop codon is underlined.
The pattern of expression of PTP is
quite restricted and the level of expression very low with Northern
blots requiring a 2-week autoradiograph exposure time. Like several
other membrane-spanning PTPs (18, 44, 45) ,
it is most highly expressed in brain, both developing and adult, but
even there at low levels and at even lower levels in kidney. However,
expression of the
2.8-kb form is mostly limited to macrophages and
tissues that contain significant numbers of macrophages such as bone
marrow, lung, spleen, and thymus(28, 47) . The lack of
detectable
2.8-kb message in liver, which contains significant
numbers of macrophages, may be a result of the large amounts of mRNA
produced by hepatocytes and a consequent dilution of PTP
mRNA
below the levels of detection by Northern blotting. A similar problem
may exist in the brain where the proportion of macrophages (microglia)
is lower. A
2.8-kb message was also detected in heart but not
skeletal muscle. It is notable that regulation of expression of
PTP
mRNA at a tissue-specific level is very complex, with the
5.5-kb long insert variant almost exclusively expressed in the
brain, the
5.5-kb short insert variant expressed in renal
glomeruli, and the shorter
2.8-kb insert-containing variants
expressed in macrophages. Expression of PTP
mRNA in hematopoietic
cells appears to be limited to mature cells of the monocyte-macrophage
lineage. Primary macrophages express PTP
at a level at least
equivalent to the brain, and this, together with the regulation of
PTP
by CSF-1 in BAC1.2F5 macrophages, suggests that this enzyme
plays an important role in these cells.
An antibody raised to the
recombinant PTP fusion protein expressed in E. coli recognized 43- and 47-kDa proteins exclusively expressed in the
membrane fraction of BAC1.2F5 macrophages. The predicted 33-kDa
cytosolic PTP
protein could not be detected by Western blotting,
consistent with the very low level of mRNA expression.
Apart from
the evidence for both restricted tissue expression and tissue-specific
alternative splicing, we have shown that expression of macrophage
PTP is regulated by CSF-1 with a pronounced increase in the level
of expression of PTP
mRNA in quiescent BAC1.2F5 macrophages
compared with cells in log-phase growth. The level of expression
decreases rapidly 2 h after CSF-1 is reintroduced, to a level that is
lower than that seen in log-phase cells. Commensurate with the
increased expression of PTP
mRNA in quiescent BAC1.2F5
macrophages, the expression of PTP
protein is increased in cells
deprived of CSF-1.
Thus, PTP is a heteromorphic PTP that is
not only expressed in a restricted range of tissues but has
differential tissue expression of several alternatively spliced mRNA
variants encoding two membrane-spanning forms with very short
extracellular domains and a putative cytosolic form detected in
macrophages and bone marrow, a large membrane-spanning form that
contains fibronectin type III repeats in the extracellular domain
expressed in brain, and a slightly shorter fibronectin
repeat-containing membrane-spanning form found in kidney (23) .
The restricted expression of the shorter isoforms of PTP
and the
demonstration that CSF-1 down-regulates their expression suggests a
role for PTP
in the regulation of macrophage survival,
proliferation, or differentiation. It will be of interest to determine
whether expression of PTP
is induced in myeloid cells undergoing
differentiation toward the monocyte/macrophage lineage under the
influence of CSF-1. It would also be of interest to determine whether
the higher molecular weight forms of PTP
expressed in kidney and
brain are similarly regulated during proliferation and differentiation
of the cells expressing them.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37465[GenBank], U37466[GenBank], U37467[GenBank].