From the
The receptor-like protein tyrosine phosphatases (RPTP) µ and
RPTP
Receptor-like protein tyrosine phosphatases (RPTPs)
We and others recently showed that
two closely related members of the type II RPTPs, RPTPµ and
RPTP
To date, little is
known about the structural features of RPTPµ and RPTP
In the present study we addressed two major
questions. First, given their structural similarity, can RPTPµ and
RPTP
To investigate the importance of the MAM domain in
mediating homophilic interactions, we deleted the entire MAM domain
(157 amino acids including three potential N-glycosylation
sites) from the C-terminally truncated RPTPµ construct, termed ExJ;
the ExJ protein, which lacks both catalytic domains, has adhesive
properties indistinguishable from those of full-length
RPTPµ(4) . MAM-deficient ExJ was termed ExJ
Having
established that the chimeric ExJ
Our results with the MAM
deletion mutants imply that the MAM domain can be considered as an
independent module that can be exchanged between related receptors
without loss of primary function. Based on our results obtained with
the chimeric receptor, we propose that the MAM domain may have a
``fine tuning'' function by contributing to the specificity
of homophilic binding. Deletion of the MAM domain abolishes homophilic
interaction indicating that the Ig-like and/or FN III-like domains are
not sufficient for mediating homophilic binding. This is substantiated
by the lack of interaction between the MAM deletion mutant and
full-length RPTPµ (Fig. 3A).
While this paper was
in preparation, Brady-Kalnay and Tonks (18) suggested that the
Ig domain of RPTPµ is both necessary and sufficient for homophilic
binding, with no apparent role for the MAM domain, as assessed under
non-physiological conditions using ``Covaspheres'' coated
with various soluble fragments of the RPTPµ ectodomain. However, as
the authors themselves point out(18) , their in vitro results do not exclude the possibility that the MAM domain is
critical for homophilic binding in vivo. The apparent
discrepancy with our findings is likely due to the very different assay
conditions used in both studies (coated beads versus intact
cells). Furthermore, the protein fragments used by Brady-Kalnay and
Tonks (18) are expressed in either Escherichia coli or
insect cells using cDNA constructs that lack appropriate signal peptide
sequences; this implies that the encoded proteins may well be misfolded
or otherwise inappropriately processed.
The present results strongly
suggest that the MAM domain has a general role in mediating
protein-protein interaction. Thus, in meprins the MAM domain may
mediate the observed dimerization or oligomerization of meprin
subunits(15, 19) . The MAM-containing A5 protein, when
transfected into L-cells, can promote neurite outgrowth of
A5-expressing neurons but not of A5-deficient neurons(17) ,
again consistent with the MAM domain being involved in homophilic
binding. Monoclonal antibodies against the MAM domain should help to
further elucidate its biological function and importance. Such studies
are currently under way.
We thank Lauran Oomen for assistance with confocal
microscopy and image processing.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
have a modular ectodomain consisting of four fibronectin type
III-like repeats, a single Ig-like domain, and a newly identified
N-terminal MAM domain. The function of the latter module, which
comprises about 160 amino acids and is found in diverse transmembrane
proteins, is not known. We previously reported that both RPTPµ and
RPTP
can mediate homophilic cell interactions when expressed in
insect cells. Here we show that despite their striking structural
similarity, RPTPµ and RPTP
fail to interact in a heterophilic
manner. To examine the role of the MAM domain in homophilic binding, we
expressed a mutant RPTPµ lacking the MAM domain in insect Sf9
cells. Truncated RPTPµ is properly expressed at the cell surface
but fails to promote cell-cell adhesion. Homophilic cell adhesion is
fully restored in a chimeric RPTPµ molecule containing the MAM
domain of RPTP
. However, this chimeric RPTPµ does not interact
with either RPTPµ or RPTP
. These results indicate that the MAM
domain of RPTPµ and RPTP
is essential for homophilic cell-cell
interaction and helps determine the specificity of these interactions.
(
)are transmembrane proteins that are thought to
transduce external signals by dephosphorylating phosphotyrosine
residues on cytosolic substrates. As such, they can be considered as
the counterparts of the receptor tyrosine kinases. Intracellularly,
RPTPs contain one or two conserved catalytic domains, but the
extracellular domains show great structural diversity. Based on this
diversity, the RPTPs were originally classified into four
subtypes(1, 2) . Later, a fifth subtype was identified
that contains an N-terminal domain homologous to carbonic
anhydrase(3) . The ``type II'' RPTPs are characterized
by an ectodomain containing one to three Ig domains and several FN
III-like repeats, which resembles the structure of cell adhesion
molecules of the Ig superfamily.
, can mediate homophilic cell-cell interaction when expressed
in non-adherent insect cells(4, 5, 6) ,
suggesting that these receptors serve a normal physiological function
in cell-to-cell signaling. We also showed that the intrinsic catalytic
activity is not required for homophilic binding.
that are
important for cell-cell interaction. In addition to an Ig domain and
four FN III-like repeats, the ectodomain of both receptors contains a
recently identified N-terminal MAM domain of unknown
function(7) . This MAM domain (meprin/A5/µ), which comprises
about 160 amino acids including four conserved cysteine residues, is
also present in the unrelated transmembrane proteins meprin and A5
glycoprotein(7) .
interact with each other? In fact, some molecules of the Ig
superfamily can mediate both homophilic and heterophilic
interactions(8) . Second, what is the role of the MAM domain in
mediating cell-cell interactions? Our results show that despite their
structural similarity, RPTPµ and RPTP
fail to interact with
each other, indicating a high degree of binding specificity for both
receptors. Furthermore, we show that the MAM domain is essential for
RPTPµ-mediated homophilic binding and confers binding specificity.
Plasmid Construction
Cloning of full-length
human RPTPµ and the C-terminally truncated ExJ mutant have been
described (4). Plasmid pVL-RPTP was constructed by cloning a
3.5-kb HpaI-EcoRI fragment from pK
(9) into the SmaI and EcoRI sites of
pVL1393 (PharMingen), followed by insertion of the 1.1-kb EcoRI fragment from pK
containing the remaining
C-terminal RPTP
sequences. Full-length cDNA coding for mouse
RPTPµ was cloned from pMT2 mFL1 (10) into pVL1392 using NotI-XbaI digestion to generate pVL-mFLµ. The MAM
domain of RPTPµ was deleted by a two-step PCR amplification, which
preserved the Kozak and signal sequences. With pBS-hFL (4) serving as template, primers M1 (5` GGTGGCCGCGTCCATCTG 3`)
and M2 (5` GTGAGGAGTCCTGCCACCTGAGAACGTCTC 3`) were used to amplify the
sequence upstream of the MAM domain and primers M3 (5`
TTCTCAGGTGGCAGGACTCCTCACTTCCTG 3`) and M4 (5` CTTTCCAGCATCTCGTTTGG 3`)
to amplify the downstream sequence up to the BstEII site at
position 705. Both PCR products were mixed and used as a template in a
second PCR using primers M1 and M4. The resulting product lacking the
MAM domain sequences was digested with SacI/BstEII
and cloned into pBS-hFL digested with the same enzymes. The 2.3-kb BstEII fragment, which was lost from the coding region in the
last step, was cloned back, resulting in pBS-FL
MAM. By replacing
the 2.5-kb BglII fragments of pVL-ExJ with the 2.0-kb BamHI-BglII fragment of pBS-hFL
MAM the
baculotransfer plasmid pVL-ExJ
MAM was generated. Exchange of the
RPTPµ MAM domain with that of RPTP
was also approached by a
two-step PCR procedure. The location of the fusion between RPTP
and RPTPµ sequences was chosen in the conserved region
Pro-His-Phe-Leu-Arg directly following the MAM domain at amino acid
positions 195-199 in RPTP
and 186-190 in RPTPµ.
Primers S1 (5` CGGAGATCTAACCGCCATGGATGTGG 3`) and S2 (5`
TTCTGAATTCGCAGGAAATGAGGAGATTTATCG 3`) were used on RPTP
cDNA to
amplify a 610-bp fragment ranging from the start codon up to and
including the RPTP
MAM domain. Using primers S3 (5`
CTCCTCATTTCCTGCGAATTCAGAATGTGG 3`) and S4 (5`
CCTTCTAGAATTCTTTAACTACCAACTC 3`) a 348-bp fragment was amplified from
the RPTPµ cDNA (bp 553-850) containing sequences downstream
of the MAM domain. Both fragments were mixed and amplified in a second
PCR using primers S1 and S4 to yield a hybrid product. This hybrid
fragment was cloned into a BglII-XbaI-digested
pVL1392 vector resulting in pVL-
MAM-µIg. An internal BstEII site in the pVL1392 vector was used to remove a 3.8-kb BstEII fragment from pVL-
MAM-µIg containing the
polyhedrin promoter region fused to the amplified product. This
fragment was subsequently cloned into pVL-ExJ digested with BstEII. The resulting chimeric construct was named
pVL-ExJ
MAM. The cDNA constructs were sequenced to verify the
desired mutations.
Cell Culture, Infections, and Recombinant Baculovirus
Generation
The Spodoptera frugiperda cell line Sf9 was
propagated in supplemented Grace's insect medium with 10% fetal
calf serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Life
Technologies, Inc.). Generation of baculovirus encoding full-length
human RPTPµ and C-terminally truncated RPTPµ (ExJ, lacking both
catalytic domains) has been described(4) . Recombinant
baculoviruses encoding the full-length mouse RPTPµ and RPTP,
the deletion mutant ExJ
MAM, and the chimeric ExJ
MAM protein
were generated with the corresponding pVL constructs using the
BaculoGold transfection kit (PharMingen) according to the
manufacturer's instructions. For infection, subconfluent
monolayers of Sf9 cells were inoculated in a small volume of
recombinant baculovirus with a multiplicity of infection of 10 or
higher. After a 1-h incubation at 26 °C, fresh medium was added,
and cells were further incubated for 2 days at 26 °C.
Antibodies
Monoclonal antibody 3G4 is directed to
the fibronectin type III-like repeats of human RPTPµ(4) .
Polyclonal serum 116 is directed to part of the MAM domain of
RPTP(6) . Monoclonal antibody 4B7 is part of a panel of
monoclonal antibodies, directed to the extracellular domain of human
RPTPµ.
(
)
Protein Analysis
Two days after
infection, Sf9 cells were collected and lysed on ice in Nonidet P-40
lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5
mM EDTA, 10% glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2.5
µg/ml aprotinin) for 20 min. After pelleting of nuclei and cell
debris, the supernatant was mixed with 4 sample buffer (250
mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20%
-mercaptoethanol) and denatured for 5 min at 95 °C. Total
lysate was separated on 7.5% SDS-polyacrylamide (11) and
transferred onto nitrocellulose(12) . After blocking in 5%
nonfat dry milk in TBST (10 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.05% Tween 20), blots were probed with first
antibody, washed in TBST, incubated with peroxidase-conjugated second
antibody, and washed again. Blots were developed using enhanced
chemiluminescence (ECL, Amersham Corp.).
Cell Aggregation Assays
Two days after infection
with the appropriate recombinant baculovirus, infected Sf9 cells were
harvested and resuspended in 30-mm plastic dishes at 10 cells/ml of Grace's medium with 10% FCS and antibiotics.
Dishes were rotated on an orbital shaker for 2 h at room temperature to
allow cell aggregation. For mixing experiments cells were incubated for
60 min at 26 °C with either 50 µM 5-(and
6-)-carboxy-2`,7`-dichlorofluorescein diacetate succinimidyl ester
(CFDA, Molecular Probes Inc.) or 20 µM DiI
(1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate,
Molecular Probes Inc.). After labeling cells were washed twice in 1 ml
of Grace's medium with 10% FCS, mixed, and resuspended in dishes
and allowed to aggregate with gentle rotation. Aggregate formation and
composition were examined by confocal microscopy on a Bio-Rad model
MRC-600 instrument.
Immunofluorescence Microscopy
Approximately 4
10
Sf9 cells were harvested 2 days after viral
infection, washed once, and resuspended in 0.5 ml of cold Grace's
medium supplemented with 10% FCS, antibiotics, and 0.02%
NaN
. Purified monoclonal antibody 4B7 was added to 1
µg/ml, and cells were incubated for 60 min at 4 °C after which
they were washed once in 1 ml of cold medium. Cells were then
resuspended in 200 µl of fresh medium and incubated with
fluorescein isothiocyanate (FITC)-conjugated goat F(ab`)
anti-mouse IgG (Zymed Laboratories Inc.) for 30 min at 4 °C.
After two subsequent washes in 1 ml of medium, fluorescence was
examined by confocal microscopy. Recombinant baculovirus encoding a
chimeric EGFR/RPTPµ receptor (4) in which the extracellular
domain of RPTPµ is replaced by that of the EGF receptor was used
for control staining.
Sequence Alignments
DNA and protein sequences were
retrieved from the SWISSPROT and GenBank/EMBL data bases using the
Genetic Computer Group software package(13) . The similarity
between RPTPµ and RPTP was calculated for each domain
separately, using the GCG program BESTFIT.
Cell-Cell Interaction Mediated by RPTPµ and
RPTP
The predicted structures of RPTPµ and RPTP
are remarkably similar, with individual domains being conserved in both
position and sequence (Fig. 1A). The extreme N terminus
of both RPTPµ and RPTP
contains the MAM domain, sharing 68.6%
similarity, followed by a single Ig-like domain and four fibronectin
type III-like repeats. The high structural similarity between RPTPµ
and RPTP
suggests that both receptors may interact in a
heterophilic manner. To test this possibility we used the
baculovirus-Sf9 insect cell system for assaying cell-cell interaction,
since Sf9 cells are normally non-adherent. Recombinant baculovirus was
generated containing the full-length mouse RPTP
cDNA. To exclude
the possibility that the difference in species plays a role in possible
heterophilic interactions between RPTPµ and RPTP
, we also
generated recombinant baculovirus encoding the mouse homolog of
RPTPµ. As expected, cells infected with either RPTPµ or
RPTP
aggregate into large clusters. To study heterophilic
interactions, Sf9 cells expressing mouse RPTPµ or RPTP
were
fluorescently stained with CFDA (green) or DiI (red),
respectively. Both cell populations were mixed, resuspended, and then
allowed to aggregate. After 2 h of gentle agitation, distinct cell
clusters of either green RPTPµ cells or red
RPTP
cells are visible (Fig. 1B, upperpanel). There is no sign of mixed cell aggregates
consisting of both green and red cells. On the other hand, when cells
expressing RPTPµ were stained with either CFDA or DiI and then
mixed, all cell aggregates consist of a mixture of green and red cells (Fig. 1B, lowerpanel). Hybrid
aggregates were also observed when human and mouse RPTPµ-expressing
cells were mixed. It thus appears that RPTPµ and RPTP
cannot
undergo heterophilic interactions, implying that the homophilic binding
properties of these receptors are highly specific.
Figure 1:
Cell-cell interaction mediated by
RPTPµ and RPTP. A, schematic diagram showing the
modular structure of RPTPµ and RPTP
. Amino acid similarities
between corresponding domains were calculated using the BESTFIT program
and are given in percentage. B, mixed cell aggregation assays
showing homophilic but no heterophilic interaction. Upper
panel, Sf9 cells expressing mouse RPTPµ (stained green with CFDA) and RPTP
-expressing cells (stained red with DiI) were 1:1 mixed and then resuspended and allowed to
aggregate. Note the lack of heterophilic interaction between green and red cells. Lower panel, control experiment
in which RPTPµ-expressing Sf9 cells were stained either green or red, 1:1 mixed, and then resuspended and allowed to
aggregate. After 2 h of gentle rotation, cell aggregation was analyzed
by confocal microscopy. Yellowcolor (lowerpanel) results from the superposition of red and green
fluorescence.
Role of the MAM Domain in Cell-Cell
Interaction
The MAM domain is present not only in RPTPµ and
RPTP (9, 10) but also in the unrelated
transmembrane proteins meprin and Xenopus A5
antigen(7) . The MAM domain is about 160 amino acids long and is
characterized by four conserved cysteine residues, possibly involved in
disulfide bridging, and two conserved regions with no similarity to
known proteins. Furthermore, the MAM domain sequence contains several
aromatic and hydrophobic residues at conserved positions and predicts
the presence of a
-sheet(7) . Besides an extracellular
modular structure, proteins bearing the MAM domain have seemingly
little in common. Meprins are zinc-dependent metalloproteases (14, 15) and the A5 protein is believed to be involved
in neurite outgrowth or axonal guidance(16, 17) ,
whereas RPTPµ and RPTP
are thought to signal cell-cell
interaction.
MAM (Fig. 2A). To ensure proper transport to the cell
surface, the signal peptide sequences were preserved. As predicted,
expression of the ExJ
MAM construct in Sf9 cells results in a
protein of about 115 kDa (Fig. 2B). Correct surface
expression of the ExJ
MAM protein was confirmed by
immunofluorescence using a monoclonal antibody against the RPTPµ
ectodomain (Fig. 2C). However, the deletion mutant
failed to mediate any detectable cell aggregation as shown in Fig. 3A. Moreover, cells expressing ExJ
MAM did not
interact with cells expressing full-length RPTPµ. These results
suggest a critical role for the MAM domain in mediating homophilic
binding and indicate that the Ig- and FN III-like domains are not
sufficient for homophilic binding.
Figure 2:
Expression of various forms of RPTPµ
and RPTP and cell aggregation in Sf9 cells. A, schematic
diagram showing the structure of the various constructs used. The
extent of Sf9 cell adhesion was measured microscopically as described
under ``Experimental Procedures'' and in Ref. 4 (+, full
aggregation; -, no aggregation). PTP, protein
tyrosine phosphatase domain; B, expression of the various
constructs in Sf9 cells. Cells infected with recombinant baculovirus
were lysed 48 h after infection, and total protein was analyzed by
Western blotting. The extracellular domain of both human (h)
and mouse (m) RPTPµ was detected using monoclonal antibody
3G4 against the FN III-like repeats; the MAM domain of mouse RPTP
was detected using polyclonal antibody 116. The predicted molecular
masses for full-length RPTPµ and RPTP
are 195 and 140 kDa for
ExJ and chimeric ExJ
MAM and 115 kDa for truncated ExJ
MAM.
Additional bands observed with ExJ, mouse RPTPµ and RPTP
probably represent breakdown products. The positions of molecular mass
markers are indicated. C, immunofluorescence analysis of cell
surface expression. Sf9 cells expressing full-length RPTPµ (upperpanel), ExJ
MAM (middlepanel), and the EGFR/RPTPµ chimera (lowerpanel) were incubated with monoclonal antibody 4B7 to the
RPTPµ ectodomain, stained with FITC-conjugated second antibody, and
then visualized by confocal microscopy. Sf9 cells infected with the
EGFR/RPTPµ chimera (lowerpanel) served as a
control for specificity of staining.
Figure 3:
Role of the MAM domain in aggregation. A, Sf9 cells expressing the ExJMAM protein (stained green with CFDA) were 1:1 mixed with cells expressing
full-length RPTPµ (stained red with DiI) and then
resuspended and allowed to aggregate for 2 h. Cell aggregation was
monitored by confocal microscopy. Note that ExJ
MAM does not
mediate any homophilic interactions nor does it interact
heterophilically with full-length RPTPµ. B, two
populations of ExJ
MAM-expressing Sf9 cells were stained either green or red, 1:1 mixed, and allowed to aggregate.
The yellowcolor results from the superposition of
green and red fluorescence. C, mixed aggregation assay using a
1:1 mixture of cells expressing chimeric ExJ
MAM (stained green) and cells expressing full-length RPTPµ (stained red). D, mixed aggregation assays using a 1:1 mixture
of cells expressing ExJ
MAM (stained green) and cells
expressing full-length RPTP
(stained red).
Characterization of a Chimeric RPTPµ/RPTP
We next sought to replace the MAM domain of RPTPµ
with that of RPTP
Molecule
to examine if aggregation could be restored and
to assign a functional role to the MAM domain. A chimeric cDNA was
constructed in which the signal peptide and MAM domain of the
C-terminally truncated ExJ construct were replaced by those of
RPTP
(ExJ
MAM, Fig. 2A). As expected, the size
of the chimeric ExJ
MAM protein is similar to that of the ExJ
protein (Fig. 2B). Immunofluorescence analysis revealed
that the ExJ
MAM protein was present on the cell surface of
infected cells (not shown). When cells expressing the chimeric protein
were stained fluorescently green or red and allowed to aggregate, large
clusters consisting of both green and red cells were readily observed (Fig. 3B). In other words, the MAM domain of RPTP
can functionally substitute for that of RPTPµ. Thus, the MAM
domains of RPTPµ and RPTP
can be considered as independent
modules that are essential for homophilic binding.
MAM receptor mediates homophilic
interaction, we next examined the binding specificity of the chimeric
molecule toward either RPTPµ or RPTP
. Cells expressing the
chimeric ExJ
MAM protein and those expressing expressing
full-length RPTPµ were stained, mixed, and allowed to interact.
After 2 h of mixing by gentle rotation, aggregates had formed, which
consisted exclusively of cells expressing either chimeric ExJ
MAM
or full-length RPTPµ, with no sign of heterophilic interaction (Fig. 3C). We also mixed cells expressing chimeric
ExJ
MAM with cells expressing full-length RPTP
. This also
resulted in sorting out of cell clusters expressing either the chimera
or RPTP
, with again no sign of cross-interaction (Fig. 3D). This suggests that the MAM domain plays a
critical role in determining the specificity of homophilic binding.
Furthermore, it follows that the MAM domain is necessary but not
sufficient to mediate homophilic binding. Additional interactions in
the Ig- and/or FN III-like domains must therefore also participate in
homophilic binding.
Concluding Remarks
In conclusion, we have shown
that (i) RPTPµ and its close relative RPTP interact in a
homophilic but not heterophilic manner and (ii) the MAM domain is
necessary but not sufficient for mediating homophilic interaction. That
both receptors fail to bind to each other suggests that cell-cell
interactions mediated by RPTPµ and RPTP
are highly specific
and strictly homophilic in nature. As the tissue distribution of both
receptors appears to be different (i.e. RPTPµ expression
is highest in lung while RPTP
is predominantly expressed in
kidney(9, 10) ), it seems likely that RPTPµ and
RPTP
mediate similar homophilic interactions (and subsequent
signaling events) in different cell types.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.