(Received for publication, July 11, 1996, and in revised form, December 3, 1996)
From the Departments of Molecular Oncology and
§ Neuroscience, Genentech, Inc.,
South San Francisco, California 94080
Here we describe a novel member of the
receptor-like protein-tyrosine phosphatases (PTPs) termed PTP ,
which is homologous to the homotypically adherent PTPs
and µ.
Murine PTP
contains MAM, IgG, fibronectin type III, and dual
phosphatase domains. As has been demonstrated for PTPs
and µ, PTP
mediates homotypic adhesion in vitro, and PTP
is
associated with
catenin in kidney epithelial cells. The
extracellular domain of PTP
is proteolytically processed in cell
culture as well as in vivo. Northern blot analysis reveals
that PTP
is expressed throughout embryonic development and is
predominately found in adult brain, lung, and kidney. In situ hybridization to 15.5-day old rat embryos reveals that PTP
is expressed in a variety of embryonic neuronal sites as well as in
the esophagus, lung bronchiolar epithelium, kidney glomerular epithelium, olfactory epithelium, and various cartilagenous sites. Analysis of neonatal brain demonstrates expression in cells of the
hippocampus, cortex, and the substantia nigra. Finally,
immunohistochemical analysis reveals expression of this PTP on specific
neurons of the spinal cord as well as on isolated cortical neurons.
Tyrosine phosphorylation is induced by a plethora of receptor-like molecules as well as by a wide range of intracellular enzymes. The effects of tyrosine phosphorylation are numerous, and they modulate a range of developmental as well as other cellular operations. Of course, the importance of tyrosine phosphorylation is underlined by the need for mechanisms that carefully regulate the levels of these events. Thus, protein-tyrosine kinases represent positive mediators of tyrosine phosphorylation, while protein-tyrosine phosphatases (PTPs)1 induce the removal of phosphate from tyrosine. The balance of the levels of tyrosine phosphate is thus mediated by the relative activities of these two types of enzymes. It is therefore clear that the mechanisms which regulate cellular function via tyrosine phosphorylation require specific proteins that mediate both the up-regulation as well as the down-regulation of the levels of this modified amino acid.
PTPs represent a growing family of enzymes that are found in both
receptor as well as non-receptor forms (1-3). The receptor PTPs are a
highly diverse group that are unified by the inclusion of a hydrophobic
domain that disposes them to the plasma membrane of the cell. Recently,
the receptor PTPs have been subdivided into eight types based upon
their domain content (4). These subtypes all contain one or two PTP
domains on their cytoplasmic sides, with a variety of extracellular
motifs including heavily O-glycosylated mucin-like domains
(for example, CD45), chondroitin sulfate domains (for example, PTP
), and short, highly glycosylated segments (for example, PTP
).
The largest family of PTPs is the family that contains various motifs
related to those found in adhesion molecules. These motifs include
immunoglobulin-like (IgG) domains and fibronectin type III (FNIII)
regions similar to those found in cell adhesion molecules such as ICAM,
N-CAM, and Ng-CAM (5). In addition, a subset of these adhesion-like
PTPs, including the PTPs
and µ, contain a third domain termed the
MAM, for
eprin/
5/PTP
, motif
(6). The MAM motif has been previously shown to be involved with
cell-cell recognition in neurons (7-9). Interestingly, recent data
suggest that three of these adhesion-like PTPs appear to be involved
with neuronal pathfinding during Drosophila development (10,
11).
PTPs and µ are the receptors that are most well characterized as
homotypic adhesion molecules (4, 12, 13). Thus, a diversity of assays,
including cell- as well as molecule-based, have demonstrated that the
extracellular domain of these enzymes can bind with high specificity in
a homophilic manner (14-16). Interestingly, mixing experiments have
revealed that these related PTPs will not bind to each other in a
heterophilic manner, suggesting that the extracellular region is meant
to recognize other cells specifically expressing identical receptors, a
situation highly reminiscent of the cadherin homotypic adhesion system
(17). While the extracellular domains required for this homotypic
binding remain controversial, it appears likely that both the MAM motif as well as the IgG region are involved with homophilic interactions (18, 19). While these data suggest that these homophilic adhesion enzymes are involved with the recognition of other cells expressing similar types of receptors, other data have suggested that this recognition event may play a role in the attachment of such cells to
each other. Thus, Tonks and coworkers (20) have recently demonstrated
that the receptor PTP µ specifically associates with the
catenin/cadherin complex of homotypic cell adhesion molecules. They
also demonstrated that treatment of cells with the PTP inhibitor pervanadate resulted in the up-regulation of tyrosine phosphorylation of cadherins and catenins, a result which suggested a role for a PTP,
potentially PTP µ, in the maintenance of the cadherin/catenin complex
in an underphosphorylated state. However, another recent report casts
somewhat of a shadow on these findings (21). Interestingly, two other
recent reports suggest that PTPs
and LAR also appear to interact
with the catenin/cadherin complex in a specific manner (22, 23).
Importantly, previous work suggested that the level of tyrosine
phosphorylation of this complex was correlated with the adhesive
capacity of the cadherins (24), a result which is consistent with the
hypothesis that the adhesion between cells mediated by the cadherins
might be regulated by their tyrosine phosphorylation levels as
determined by homotypic interactions between receptor PTPs such as
and µ.
The finding that PTPs and µ mediated homotypic adhesion, together
with the somewhat restricted tissue distribution of these PTPs (12,
13), has suggested that additional members of this family of adhesive
enzymes might exist. Here we report the cloning and characterization of
the third member of this receptor PTP family, termed murine PTP
.
The receptor reported here contains structural motifs that are very
similar to those found in PTP
and µ and is capable of mediating
homotypic adherence and associating with
catenin. In addition, this
receptor PTP reveals a tissue distribution, particularly with respect
to its expression on neurons in the central nervous system, which is
divergent from that previously described for the other members of this
family. These data are consistent with a burgeoning family of
MAM-containing receptor PTPs potentially involved with cell adhesion in
various tissues.
Messenger RNA
was isolated from the non-adherent LinloCD34hi
fraction of fetal yolk sac hematopoietic cells (25) (Micro-FastTrack, InVitrogen). Poly(A)+ RNA was reverse transcribed with
random hexamers (Promega) and Molony murine leukemia virus reverse
transcriptase (SuperScript II, Life Technologies, Inc.). One quarter of
this cDNA was amplified by PCR using degenerate mixed
oligonucleotides primers. Sense and antisense primers corresponding to
the amino acid sequences (H/D)FWRM(I/V)W
(5-A(C/T)T(C/T)TGG(A/C)GIATG(A/G)TITGG-3
) and WPD(F/H)GVP
(5
-GGIAC(G/A)(T/A)(G/A)(G/A)TCIGGCCA-3
), respectively, were used.
PCRs were carried out in 1× Taq DNA polymerase buffer (Life
Technologies, Inc.) plus 0.2 mM each dNTP, 10% dimethyl sulfoxide, and 5 units Taq polymerase (Life Technologies,
Inc.) for 25 cycles at 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1 min. The PCR products were treated with Klenow enzyme
(New England Biolabs Inc.) at 30 °C for 30 min, cloned into the
SmaI site of pRK-5 plasmid (Genentech, Inc.), and
subsequently sequenced (Sequenase, U. S. Biochemical Corp.).
Adapter-linked double-stranded
cDNA was prepared from A+ RNA of day 10 mouse embryos
(Marathon ready cDNA synthesis kit, Clontech) using either random
hexamer or oligo(dT) primer. Full-length cDNA was isolated by 5 or
3
rapid amplification of cDNA ends (RACE) of the marathon ready
cDNAs. A
cDNA library derived from adult mouse lung was
screened following standard protocols using cDNA fragments isolated
by RACE as probes. The DNA sequences of the RACE products as well as
the
clones were determined using standard sequencing protocols.
cDNA sequences encoding amino acids 43-741 containing the extracellular region of the PTP were obtained by PCR using primers containing SalI or NotI. The PCR fragment was treated with SalI and NotI restriction enzymes and cloned into the pGEX-4T-1 plasmid (Pharmacia Biotech Inc.). Fusion protein was affinity purified using glutathione-Sepharose columns (Pharmacia). Polyclonal anti-serum against the extracellular region was generated by repeatedly immunizing rabbits with the purified GST-fusion protein. Affinity-purified extracellular domain antibody was obtained by binding serum IgG to the receptor GST-fusion protein immobilized on glutathione-Sepharose, and eluting the bound antibody with low pH and high salt.
Expression and Purification of the MAM-IgG-FnIII Immunoglobulin Fusion ProteinTo express the extracellular portion of PTP ,
including MAM, IgG-like, and FnIII domains fused with human IgG hinge,
CH2, and CH3 domains, fragments were subcloned into the pRK5 vector (26). The fusion proteins were expressed under the control of a
cytomegalovirus promoter with a herpes simplex virus glycoprotein D
signal sequence and monoclonal antibody 5B6 binding epitope. Transfection was done in 293 cells using the calcium phosphate precipitation method with 40 150-mm plates. One day after transfection, the cells were transferred to serum-free conditional media for an
additional 5-6 days. Enzyme-linked immunosorbent assay in 96-well plates to test the expression levels was performed on the 3rd or 4th
day. To purify protein, the supernatant (about 1 liter) was spun to
remove cell debris, filtered, and concentrated ~10-fold. The sample
was then added to a protein G column three times, and the column was
washed with at least 10 bed volumes of PBS. Washing was monitored by
A280. Finally, the fusion protein was eluted with 0.1 M glycine, pH 2.8, and neutralized with 0.1 volume of 1 M Tris, pH 8.0. The eluted protein was dialyzed against PBS overnight.
Fluorescently-labeled
green and red spheres (~2 µm, Duke Scientific) were incubated in
300 µg/ml solutions of either the PTP -IgG or FLT 4-IgG (27)
fusion proteins in PBS for 30 min at room temperature. The beads were
blocked with 10 mg/ml bovine serum albumin in PBS for an additional 30 min at room temperature and subsequently washed three times with PBS,
after which they were suspended in their original volume of PBS and
used immediately. Different combinations of protein-coated beads were
incubated in microtitre wells at room temperature for 30 min on a
rotary shaker. 10-µl aliquots were removed and examined under the
fluorescence microscope at a wavelength of 359 nM. At this
wavelength, the green and red beads appear to be blue and orange,
respectively, and both can therefore be visualized simultaneously.
Various truncation mutants
of PTP , which contained the herpes simplex virus glycoprotein D
signal sequence and the monoclonal antibody 5B6 binding epitope, were
produced in the PRK 5 vector using the polymerase chain reaction.
Constructs were transfected into 293 cells and incubated for up to 4 days. The supernatant was collected and centrifuged to remove cell
debris. Then it was incubated with 5B6 at 1 µg/ml for 2 h at
4 °C and protein G-Sepharose (Pharmacia) was added for another hour.
The immune complexes were washed 5 times with 0.2% deoxycholic acid,
0.2% Tween 20 in PBS, boiled for 5 min with SDS sample buffer, and
analyzed on 4-20% SDS-PAGE. The gel was transferred to a ProBlott
membrane (Applied Biosystems) with electroblotting buffer (10 mM CAPS, pH 11, and 10% MeOH). Immunoblots were probed
with the 5B6 antibody and developed using chemiluminescence (ECL,
Amersham Corp.). Alternatively, if the cell lysates were used, the
transfected cells were washed once with PBS and lysed in E1A lysis
buffer (0.25 M NaCl, 5 mM EDTA, 0.1% Nonidet
P-40, 50 mM HEPES, pH 7.6, and 1 µl/ml aprotinin) at
4 °C. After centrifugation, the incubation of the lysate with antibodies and protein G-Sepharose, SDS-PAGE separation of the immune
complex, and immunoblot detection were as described above. Neonatal
brains were dounce-homogenized in the same lysis buffer and
immunoprecipitated with affinity purified anti-extracellular domain
antibody, transferred to membranes, and probed with the same
antibody.
Lysates of human embryonic
kidney (293) cells, which had been previously transfected with either
the full-length PTP or a truncated version missing the dual
phosphatase domains but containing the juxtamembrane region, were
immunoprecipitated with an anti-
catenin monoclonal antibody
directed against the C terminus of the protein (Transduction
Laboratories) using the immunoprecipitation conditions described above.
The anti-
catenin immunoprecipitates were run on SDS gels,
immunoblotted, and probed with the monoclonal antibody directed against
the glycoprotein D 5B6 epitope on the N terminus of PTP
. The blots
were exposed, stripped, and reprobed with the anti-
catenin
monoclonal antibody used for immunoprecipitation. Neither the 5B6 nor
the anti-
catenin monoclonal antibodies revealed cross reactivity
with
catenin or PTP
, respectively, in Western blot
experiments.
A 2.5-kilobase cDNA fragment encoding
the cytoplasmic region of PTP was used to probe the murine
multi-tissue Northern blot (Clontech)
Rat E15.5 embryos and postnatal day 1 brains were immersion fixed overnight at 4 °C in 4%
paraformaldehyde, then cryoprotected overnight in 15% sucrose. Adult
rat brains were fresh frozen with powdered dry ice. All tissues were
sectioned at 16 µm and processed for in situ hybridization
for PTP using [33P]UTP labeled RNA probes. Sense and
antisense probes were synthesized from a 2.5-kilobase DNA fragment of
PTP
using SP6 or T7 polymerase, respectively.
The cerebral cortex from postnatal day
1 (P1) rat pups was dissected out and minced into small pieces in
Hanks' balanced salt solution. The tissue was dissociated in papain
(Worthington) for 30 min. Triturated cells were resuspended in
Neurobasal medium with B27 supplement (Life Technologies, Inc.) and
plated onto chambered microscope slides (Nunc) coated with
poly-D-lysine. The cells were maintained in an incubator
for 1 day to allow the neurons to extend neurites and then fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for at
least 15 min. Brains from embryonic day 14 (E14) rat pups were
immersion fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose,
frozen in OCT mounting medium, and sectioned at 20 µm in the coronal
plane through the spinal cord. For immunostaining, cortical cells or
spinal cord sections were rinsed for 30 min in PBS, blocked with goat serum, and then incubated with affinity purified antibody against the
external portion of PTP (15 ng/ml) overnight at 4 °C followed by
a Texas Red-conjugated goat anti-rabbit secondary antibody (1:200,
Vector). Immunostaining reagents were diluted in 1 mg/ml bovine serum
albumin in PBS. All stained tissues were imaged on a Zeiss Optiphot
microscope with an Optronics 3 CCD color video or a Xillix digital
10-bit camera. Staining with pre-immune serum under identical
conditions did not reveal any detectable signal (data not shown).
In order to isolate novel PTPs expressed in murine primitive
hematopoietic cells, we undertook the cloning of PCR fragments produced
by priming with sequences directed against conserved protein motifs
found in PTPs from a number of different genes and species (28).
Analysis of 70 different PCR-derived subclones revealed an array of
previously described PTPs as well as two novel PTPs. One of these novel
PTPs, termed PTP HSCF, is a member of the PTP PEST family of enzymes,
and it has been previously described (29). The second novel PCR
fragment was homologous to PTPs and µ, both related receptor-type
PTPs that mediate homophilic adhesion (4). In order to further
characterize the cDNA encoding this novel PTP, a combined cloning
approach that utilized RACE as well as cloning from phage cDNA
libraries was performed. The composite cDNA and derived protein
sequences determined from these various clones is shown in Fig.
1. The ATG start codon utilized for translation of this
large open reading frame was embedded within a consensus Kozak
sequence, and there are several translational stop codons upstream of
this initiator codon. As can be seen from this figure, the protein
derived from this cDNA is a large receptor-like molecule of 1,436 amino acids and a molecular mass of approximately 161,176 Da. Closer
perusal of the sequence reveals 13 consensus N-linked
glycosylation sites in the extracellular region of the receptor,
consistent with a potentially significant degree of glycosylation. Fig.
2 illustrates that the novel, hematopoietically derived
PTP-related protein reported here shows a high degree of homology to
both PTP
(~60%) and PTP µ (~53%) throughout their entire
lengths (12-13). Thus, the novel PTP contains MAM, IgG, four
fibronectin type III, and two cytoplasmically localized phosphatase domains (Fig. 2) (4, 12, 13). These homologies with the novel PTP are
somewhat less than the homology between PTP
and µ (~62%),
suggesting that the novel PTP reported here is more distantly related
to these two PTPs rather than they are to each other. These data
suggest that this novel PTP is the third member of the homotypically
interacting PTP family containing PTPs
and µ, and we have
therefore named the novel receptor PTP
.
As can be seen from Fig. 3, the relative sequence
homologies in each of the domains of these three receptors suggests
that they are indeed closely related. Interestingly, previous data suggested that both the MAM and IgG domains mediated specific homotypic
adhesion between PTPs and µ (18, 19), and it is clear from the
sequence comparisons between these three related proteins that these
two domains are homologous. However, the fact that there are a large
number of sequence changes between these two motifs is also consistent
with the supposition that they can mediate specific homotypic
interactions. The overall sequence homologies between the three
proteins is also relatively high in the FnIII domains although the
homology in the first of these domains is significantly higher than in
the others. Previous work has also demonstrated that a juxtamembrane
site between the transmembrane domain and the first phosphatase domain
is distantly homologous to a similar region in the cadherins (20), and
this site shows a high degree of homology between these three
receptors, a point that will be discussed further. A significant degree
of sequence homology is also found between the first PTPase domains of
these three receptors, with a somewhat lower level of homology between the second PTPase domains of these proteins. This latter result may be
important since it has been reported that the first phosphatase domain
is the critical enzymatic motif of the dual phosphatase regions in the
receptor PTPs (30, 31). The homology between these PTPase domains
includes many of the residues previously found to be important for
substrate recognition and tyrosine dephosphorylation in the PTP 1B (32)
although not all of these residues are identically conserved, and
immunoprecipitation studies have demonstrated that PTP
has weak,
but detectable, phosphatase activity against two different
tyrosine-phosphorylated peptide substrates as well as against the
aritificial substrate p-nitrophenyl phosphate (data not
shown).
Homotypic Adhesion Mediated by PTP
Previous data have
demonstrated that the PTP -related receptor PTPs
and µ induce
homotypic adhesion and that these adhesive events appear to be mediated
by the MAM and IgG domains. In order to examine the ability of PTP
to mediate such homotypic adhesion, a soluble construct containing the
MAM, IgG, and first FnIII domain attached to the hinge, CH2, and CH3
regions of human IgG was produced (
-IgG chimeric protein). This
chimeric protein was purified to ~90% by passage over protein
A-Sepharose (data not shown) and was used in in vitro
binding experiments. As a control, the extracellular domain of the
tyrosine kinase receptor FLT 4, in the form of an IgG chimera, was used
(FLT 4-IgG) (27). As can be seen in Fig. 4, fluorescent
beads containing the FLT 4-IgG were unable to aggregate, while beads
containing the
-IgG protein formed large mixed aggregates. As a
further control, the aggregation of a mixture of red
-IgG and green
FLT 4-IgG beads was examined. Fig. 4 shows that only the red beads
aggregated in this experiment, with the green beads remaining largely
unaggregated. This in vitro binding assay thus demonstrates
that a form of PTP
can mediate homotypic aggregation and is
consistent with previous data suggesting that the MAM and IgG domains
are critical for this adhesive event (18, 19).
Proteolytic Cleavage of PTP
A number
of previous studies have suggested that various receptor PTPs,
including and µ, appear to have a regulated, specific proteolytic
cleavage that gives rise to a secreted region derived from the
extracellular domain (12, 33). We examined this possibility for PTP
in two different ways. In vivo proteolytic cleavage was
examined by probing extracts of neonatal mouse brain with an affinity
purified polyclonal antibody directed against a GST fusion of the
extracellular domain of PTP
. This source of tissue was chosen
because of the high transcript levels in Northern blots of adult brain
as well as the in situ hybridization results (both described
below). As can be seen in Fig. 5, immunoprecipitation of
brain extracts with this antibody and subsequent Western blotting revealed three specific bands in neonatal brain. The highest molecular mass band (~190 kDa) appears to correspond to a glycosylated
full-length form of this PTP. A second band at ~115 kDa and a smaller
band at ~70 kDa are observed and are likely to be proteolytic
fragments of the full-length receptor since they specifically react
with the affinity purified antibody directed against the extracellular domain. These data thus demonstrate that PTP
appears to be
proteolytically processed in brain extracts to two lower molecular
weight forms, and additionally demonstrates the specificity of the
affinity purified antibody directed against this phosphatase.
In order to examine this processing event in greater detail, we
produced a number of different truncated constructs to examine the
cleavage of the protein in vitro. All of the constructs
contained a herpes simplex virus glycoprotein D signal sequence and
N-terminal monoclonal antibody (5B6) epitope to aid in the analysis. As
can be seen in Fig. 6, expression of a full-length form
of the receptor in 293 cells results in a ~190-kDa protein as well as
a ~115-kDa proteolytically clipped fragment, very similar to those
seen in neonatal brain extracts. Examination of cell supernatants
reveals that the smaller ~115-kDa fragment appears to be shed from
the cells. This cleavage occurs in the absence of the PTP domains since
the truncation mutant lacking the dual phosphatase domains also shows a
~115-kDa fragment in both the cell lysates as well as in the
supernatants. Interestingly, this cleavage appears to occur when the
MAM domain is truncated from the receptor (appearing as a smaller band
in the cell lysates transfected with construct number 3 in Fig. 6)
although this form of the protein does not appear to be secreted,
possibly due to incorrect folding of this form of the protein and
intracellular degradation. Finally, removal of both the MAM and IgG
domains results in ~64- and ~50-kDa fragments in the cell lysates,
with the ~50-kDa fragment being very efficiently secreted into the
media. The molecular mass of the secreted ~50-kDa fragment is
consistent with the proteolytic cleavage event occurring within the
fourth FnIII repeat (12; also see "Discussion" below). Finally, the
construct containing only the FnIII repeats also shows a smaller
~25-kDa fragment in the medium, suggesting that the ~50-kDa
fragment may be further cleaved after secretion to give rise to this
smaller fragment although such a smaller fragment was not readily
apparent in the cells transfected with either construct 1 or 2 (Fig.
6). These data strongly suggest that the extracellular domain of PTP
is efficiently and specifically processed in 293 cells as it is in
neonatal brain, and further demonstrates that the processing site
appears to be contained within the fourth FnIII domain, a result
similar to that found previously for PTP
(12).
PTP
Previous data have suggested that PTPs µ and interact
specifically with the cadherin/catenin complex (20, 22). In addition, recent data have also suggested that PTP LAR interacts with this homotypic adhesion complex in neuronal PC12 cells (23). However, other
investigators have cast some doubts about the interactions between the
catenin/cadherin complex and PTP µ, predominantly due to antibody
cross-reactivity (21). In order to examine if PTP
interacts with
this adhesion complex, a coprecipitation experiment was performed.
Because transcript analysis suggests that PTP
is expressed in
vivo in embryonic kidney epithelial cells (see below), a human 293 embryonic kidney cell line was transfected with either the full-length
construct or a construct lacking the phosphatase domains but containing
the juxtamembrane domain homologous to cadherin cytoplasmic domains
(constructs 1 and 2 in Fig. 6). Lysates from these transfected cells
were then immunoprecipitated with a monoclonal antibody directed
against
catenin, and the immunocomplex was electroblotted and
probed with antibodies against the glycoprotein D tag contained within the N terminus of PTP
. As can be seen in Fig. 7,
immunoprecipitation with the
catenin monoclonal antibody resulted
in the specific coprecipitation of the full-length PTP
but not of
the cleaved extracellular domain. In addition, this figure illustrates
that only the intracellular juxtamembrane domain is required for an association with
catenin, consistent with previous data suggesting that this domain of PTP
is required for catenin association (22).
The relatively low percentage (~10%) of PTP
in the
immunoprecipitated
catenin complex can be explained in a number of
ways. For example, the system analyzed the coprecipitation of a murine
form of the PTP complexed with human
catenin, and the wash
conditions utilized may have been overly stringent. While a recent
report suggests that some degree of antibody cross-reactivity may have
been responsible for the apparent interactions between PTP µ and
catenin or E-cadherin (21), the use of highly specific monoclonal
antibodies for both the
catenin immunoprecipitation and PTP
blotting steps of this experiment eliminates any such potential
artifacts. Thus, we have not observed any indication that the anti-
catenin antibody recognizes PTP
on Western blots, nor have we found
that the anti-gD antibody used to detect PTP
reacts with
immunoprecipitated
catenin (data not shown). These data thus
support previous studies and suggest that PTP
, like PTPs µ,
,
and LAR, interacts with the cadherin/catenin adhesion complex.
Tissue Expression of the PTP
As can be seen in
Fig. 8, Northern blot analysis of fetal as well as adult
tissues demonstrates that PTP is expressed in a diversity of organs
outside of the hematopoietic progenitor cells from which it was
originally cloned. Thus, the expression of this PTP is detected
throughout embryonic development beginning in the very early embryo at
day 7. Interestingly, analysis of adult organs reveals that this
receptor is expressed specifically in only a subset of tissues. Thus,
there appears to be a very high level of expression of the enzyme in
brain, lung, and kidney, a much decreased level in heart, skeletal
muscle, and testis, and a lack of obvious expression at this exposure
in spleen and liver. The high level expression in lung and brain
together with the lack of expression in liver are in contrast to PTP
, a PTP which is expressed at high levels in liver but is almost
undetectable in lung and brain (12). Thus, in spite of the fact that
PTP
was originally isolated from hematopoietic stem cells, there is
no obvious expression in two sites which contain hematopoietic cells,
the spleen, and the liver. The lack of signal in the spleen, an organ
which contains mostly mature hematopoietic cells, suggests, therefore,
that this receptor may be expressed specifically in earlier
hematopoietic progenitor cells and not in the predominately mature
cells found in this organ as was previously found for PTP HSCF (34).
Interestingly, there appears to also be an alternatively spliced
transcript in the lung, which is not detected in the other two organs
that express this receptor at high levels or in the embryos although
the nature of this alternatively spliced transcript has not been
determined. In summary, these data suggest that PTP
is specifically
expressed in a subset of adult tissues, some of which are divergent
from PTP
.
In Situ Hybridization Analysis
We performed in
situ mRNA analysis of the rat E15.5 embryo, and P1 and adult
rat brain to determine potential sites of PTP production. Extensive
PTP
expression was observed in developing skeletal, epithelial, and
neuronal structures throughout the E15.5 embryo (Fig.
9). Systemic expression was observed in various
developing skeletal elements such as vertebral perichondrium,
intervertebral discs, teeth, mandible, and maxilla (Fig. 9,
A and B). Expression within urogenital structures
included the genital tubercle (panels A and B),
urethra, and urogenital sinus (not shown). Other positive areas of PTP
expression included the anal canal (not shown), skin, olfactory and
oral epithelium, esophagus (panels A and B), pituitary (panels A-C), dura mater (panels
A, B, and D), kidney (panels A
and B), and lung (panels A and B).
Higher magnification reveals expression restricted to developing
glomeruli in the cortical region of the kidney (panels F and
G), and bronchiolar epithelium of the lung (panels
H and I). Within the E15.5 embryonic nervous system,
high levels of expression were observed in the developing cerebral
cortex (panels A and B), floor of the midbrain,
choroid plexus primordium, gigantocellular reticular nucleus of the
brain stem (panels A-C), dura mater, and spinal cord
(panels A, B, and D). High
magnification of the spinal cord reveals highest expression of PTP
in the ventrolateral motor column (panel D).
In P1 and adult brain, expression of PTP was localized to regions
derived from embryonic anlage that also contained high levels of
expression. For instance, expression in the embryonic midbrain preceded
the high levels of PTP
expression in the P1 and adult substantia
nigra (Fig. 10, C and E,
respectively). Expression in the embryonic forebrain (Fig.
9A) preceded expression observed in the inner layers
(cortical layers 5 and 6) of the P1 and adult cortex (Fig. 10,
A, B, D, and E
respectively). Expression in the choroid plexus primordia of the embryo
begets high levels of expression in the P1 brain (Fig. 10A)
and low levels of expression in the adult brain (Fig. 10D).
In general, PTP expression in the adult brain appears to be
down-regulated relative to the P1 brain (Fig. 10). However, other areas
of prominent expression in both P1 and adult brain include piriform
cortex and endopiriform nucleus (Fig. 10, A and
D, respectively), amygdaloid nuclei, subiculum, and CA1,
CA2, and to a lesser extent, CA3 of the hippocampal formation (Fig. 10,
B and E, respectively). The P1 brain also
exhibits strong expression throughout the septal area, basal ganglia,
thalamus, and midbrain (panels A, B, and C). Weak expression is observed in the adult superior
colliculus as well as scattered expression throughout the thalamus
(panel E).
The Western blot, Northern blot, and in situ
analyses suggested that PTP is expressed in the nervous system
although the resolution of these techniques was insufficient to
determine if the receptor was expressed on neurons. In order to examine
this possibility, immunostaining of the spinal cord and isolated
cortical neurons of the E14 rat was done. As Fig.
11A shows, a subset of spinal neurons,
including ventral and dorsal root neurons extending from the spinal
cord as well as those in ventral and ventrolateral funiculi within the
spinal cord, were specifically stained with the antibody. Examination
of isolated cortical neurons for reactivity with the antibody revealed
that both the soma as well as the axons and dendrites extending from
the neuron stained with the antibody. In addition, growth cones were
also positive for this receptor (Fig. 11, B and
C). Together, these data suggest, in agreement with the
in situ hybridization data, that spinal cord and cortical neurons express PTP
, consistent with a function for this receptor in the formation of the nervous system.
The relative levels of tyrosine phosphorylation of a diversity of
proteins are critical for the regulation of a number of activities
during embryonic differentiation and throughout the life of the
organism. The absolute levels of this modification are mediated through
the balance of the enzymatic activities of tyrosine kinases with those
of tyrosine phosphatases. In both cases, these large families of
proteins perform their roles through conserved enzymatic domains that
are coupled to a plethora of specificity-determining motifs. These
various motifs are found in the context of both membrane traversing,
receptor-like molecules as well as intracellular forms of the enzymes.
These similarities in overall structure of the kinases and phosphatases
suggest that they mediate their relative specific activities through
the use of these various domains. A subset of receptor phosphatases
also contain a diversity of domains, including immunoglobulin- and fibronectin-like, which are associated with cell adhesion and ligand
binding activities in other protein families. Among the most
interesting of these types of adhesion-associated PTPs are the and µ receptors which are involved with homotypic types of interactions.
Earlier predictions, based upon the likely function of these receptors
in mediating cell adhesion as well as their limited tissue
distribution, suggested that there might be other
and µ-like
receptor PTPs with different tissue dispositions (4). We report here
the isolation of the third member of this family of homophilically
interacting receptor PTPs which may be associated with the construction
of epithelial and neural structures during development and in the
adult.
The strongest data suggesting that the novel PTP described here, termed
PTP , is homologous to the
and µ receptors lies in the high
degree of sequence conservation among these three proteins. Analysis of
these three receptors clearly revealed that the novel PTP had a
significant degree of sequence homology throughout its entire length.
This homology included the four major types of domains contained in
this family including the MAM, the IgG, the FnIII, and the dual
phosphatase (PTP) domains (12, 13). Because previous data have
suggested that both the MAM as well as the IgG domain appear to be
involved with homotypic adhesion (18, 19), it is likely that these
motifs are used for a similar function in PTP
, a hypothesis that is
consistent with a role for this receptor in cell adhesion (see below).
However, the degree of sequence homology of these domains between the
newly reported receptor and the
and µ receptors is quite
divergent, suggesting that the novel receptor may also specifically
mediate a homophilic interaction only to itself and not to these
domains in the other family members (19). As will be discussed below,
these results, together with the tissue localization of this receptor,
suggest that it may be involved with the formation of very specific
edifices during development. While it is difficult to currently
interpret the significance of the conservation of the FnIII domains,
which may act as spacer domains to extend the functionally critical MAM
and IgG domains from the cell surface, the conservation of the dual PTP
domains lends itself to some comment. Thus, the higher degree of
conservation of the first domain as compared with the second
substantiates previous work suggesting that the N-terminal PTP motif is
the enzymatically active one, while the C-terminal domain may be
involved with the regulation of enzyme activity (30). Indeed, recent
structural data of the phosphatase domains of PTP
reveal that the
second domain may regulate the enzymatic activity of the first domain
(31). In summary, the data reported here are consistent with PTP
being the third member of the homotypically interacting receptor PTP
family.
As with other members of this family of PTPs, PTP can mediate
homotypic adhesion. The in vitro binding studies using the IgG chimeric protein containing the MAM, IgG, and first FnIII domains
of PTP
are consistent with previous data suggesting that the first
two of these domains appear to be critical for homotypic adhesion (14,
19). While this paper was under review, Ullrich and colleagues also
reported a novel receptor PTP, termed PCP-2, which appears to be the
human homologue of PTP
(35). As predicted by the homotypic adhesion
studies reported here, PCP-2 is concentrated at the intracellular
contact points of cells expressing this receptor. Together, these data
support the hypothesis that PTP
is the third member of this family
of proteins to mediate homotypic adhesion and further suggest that this
adhesion is induced by, at most, the first three N-terminal domains of
the protein. The proteolytic cleavage studies reported here also are
consistent with previous data on this family of homotypically adherent
PTPs (12, 33), and have implications as far as the adhesion modulating functions of this receptor. The proteolytic mapping studies suggested that a specific cleavage site within the fourth fibronectin domain was
the point where proteolysis occurred, and examination of this domain in
PTP
revealed a furin-like cleavage sequence
(636RLRR639) which is highly conserved in all
three members of this receptor PTP family (12, 22). Interestingly, this
site appears to be missing in the PCP-2 phosphatase in spite of its
otherwise high degree of homology with PTP
, and PCP-2 is apparently
not cleaved in transient transfection assays (35), which is in sharp
contrast to the results reported here. In addition, analysis of PTP
expression in neonatal brain extracts revealed that this proteolytic
cleavage event also appears to occur at a significant level in
vivo at a potentially similar site to that seen in
vitro although a further processed form of the extracellular
domain was also observed in brain extracts. Because the cleaved form of
the extracellular region of PTP
contains all of the motifs
necessary to mediate homotypic adhesion, it is likely that this soluble
protein can bind to, and potentially inhibit, the homotypic adhesion
mediated by the cell surface form of PTP
. Thus, it is possible that
this specifically cleaved form of the protein acts to regulate the cell-associated form of the enzyme.
Previous data have suggested a role for this category of receptor PTPs
in cadherin/catenin regulation, and other investigators have pointed to
an intracellular juxtamembrane site with significant homology to a
similarly localized region in the cadherins (4, 20). Analysis of catenin immunocomplexes clearly revealed the association of PTP
with this adhesion system. We have also found a very high degree of
sequence conservation in the juxtamembrane region with the other
members of this phosphatase family, consistent with a potential role
for this domain in catenin/cadherin interactions. In agreement with
this supposition, we have demonstrated that a PTP
construct
containing only this juxtamembrane region intracellularly can associate
with
catenin, consistent with previous data demonstrating that a
homologous region of PTP
alone can mediate catenin association in vitro (22). These data thus support the proposal from
Tonks and coworkers (4, 20), suggesting a role for this family of PTPs
in the regulation of cadherin-mediated adhesion. An interesting possibility along these lines, therefore, is that the lack of contact
inhibition observed in cells treated with the phosphatase inhibitor
vanadate might have been due to the inhibition of the dephosphorylation
of the catenin/cadherin complex by one or more members of this receptor
PTP family (36). Together with the in situ data
demonstrating expression of PTP
on epithelial sheets in lung and
kidney particularly, this possibility suggests that the
cadherin-mediated adhesion of these cells may in part be regulated by
the activity of PTP
during development and in the adult. In
addition, the possibility that such phosphatase-catenin interactions in
neuronal cells exist has recently been suggested by work from Reichardt
and coworkers (23). The demonstration here that PTP
is expressed in
neurons of the central nervous system, together with the suggestion
that it mediates homotypic adhesion and associates with the
catenin-cadherin complex, is consistent with the possibility that this
novel PTP may be involved with specific adhesion during neural
development as well (see below).
The in situ hybridization analysis of the expression of PTP
in the developing embryo and adult suggest some potentially important hypotheses. The expression of this receptor in a diversity of
developing skeletal areas, as well as in epithelial sites which line
various organ systems with a layer of these cells, coupled with the
proposed role for PTP µ (20) (4) and potentially PTP
(22) in the
control of cadherin-mediated adhesion, suggests that the novel PTP
might be involved in a similar type of adhesion control in the
developing embryo. For example, the development of epithelial layers in
the lung bronchioles and kidney glomeruli requires that a sheet of
epithelial cells that is 1-cell thick be constructed. Thus, as the
cells grow and migrate during embryogenesis, they would require a
mechanism where they sensed the location of other epithelial cells that
were in contact with them so that this cellular contiguity initiated an
adhesive response that inhibited further epithelial movement via the
enhancement of cell adhesion. The formation of 1-cell thick epithelial
structures in these embryonic organs could be mediated by such a
sensing mechanism using the homotypic adherence of PTP
in
association with the catenin-cadherin complex. The expression of this
receptor PTP in bone forming chondrocytes would also be expected to
perform a similar type of sensing and adhesion function to assemble
these structures although this type of anatomy, which is more complex
than the thin walled epithelial-like morphology described above, would
be expected to involve more elaborate types of sensing and adhesive
mechanisms. Finally, because many common types of tumors of the lung
and other organs involve epithelial cells, it is possible that
disruptions in the proposed function of this type of adhesion sensing
mechanism might be involved with the disorganized morphology and often
high rate of metastasis of these tumors (17, 24). Together, these
hypotheses suggest a critical role for PTP
in the formation of
various epithelial-like structures in the embryo.
Recent data from the Drosophila system also suggest
interesting possibilities for the function of PTP in the developing nervous system (10, 11). In these reports, three different Drosophila receptor PTPs, termed DPTP69D, DPTP99A, and DLAR,
which all contain IgG and fibronectin type III adhesion domains similar to those found in PTP
, were shown to be critically involved with
neuronal pathfinding in the developing nervous systems. Thus, mutations
in either of these receptors resulted in a loss of the ability of
certain neural subsets to become reoriented during their formation in
the embryo. These data, together with the recent observations on the
association of mammalian LAR with the catenin-cadherin complex, are
consistent with a role for receptor PTPs in specific neural pathfinding
and adhesion. Because PTP
, a homotypically adhering PTP associated
with the catenin complex, is expressed in a number of developing neural
sites, it is possible that it plays a similar role in the pathfinding
of nerves in mammals. Thus, the expression of this PTP in the
developing midbrain, forebrain, and other neural sites would dispose it
to function as a mediator of pathfinding in these maturing systems.
Interestingly, the expression of this receptor in these embryonic
anlage was confirmed by expression in the adult sites which arise from
these embryonic structures. However, the expression in the adult
appeared to be somewhat reduced as compared with that observed in the
embryo, and it was far more organized. These data suggest that this
enzyme might be utilized during adult neuronal formation although the
apparent decrease in adult expression suggests a potentially more
critical role during embryogenesis. The observation that PTP
is
expressed on neurites of isolated cortical neurons as well as on the
growth-cone like structures at the tips of these processes is also
consistent with a potential role for this receptor in neuronal
pathfinding in the mammalian nervous system. Interestingly, we have
observed that PTP
is specifically expressed on layer 5 and 6 cortical neurons only during their differentiation and pathfinding
phases, again consistent with a role for this phosphatase in aspects of neural architecture.2 Finally, while the
clear observation of the loss of pathfinding in Drosophila
will be difficult to recapitulate in the mouse due to the relatively
high complexity of the mammalian nervous system, it will nevertheless
be potentially of great interest to examine the formation of the
nervous system in animals which have been made null for the expression
of this receptor.
In summary, the data reported in this paper demonstrate the existence of a third member of the family of receptor PTPs that appear to be involved with homotypic adhesion and, potentially, cadherin/catenin-mediated organ formation. The role that this novel receptor might play in the formation of epithelial sheets and neuronal structures remains to be determined. However, the existence of three of these types of receptors further suggests that this growing family may be involved with the specific formation of various types of complex structures during development, as well as in the adult.
We thank Louis Tamayo and David Wood for production of the figures.