(Received for publication, October 31, 1994)
From the
Protein-tyrosine phosphatases (PTPases) play an essential role
in the regulation of cell activation, proliferation, and
differentiation. A major subfamily of these enzymes is the
transmembrane-type PTPases that contain extracellular regions comprised
of Ig-like and fibronectin type III (FN-III)-like domains.
Characterization of the human transmembrane PTPase (HPTP
)
revealed the existence of multiple HPTP
isoforms that vary in
their extracellular regions. The full-length HPTP
isoform has an
extracellular region containing three Ig-like and eight FN-III-like
domains connected via a transmembrane peptide to an intracellular
region with two PTPase domains, whereas another isoform lacks four of
the eight FN-III like domains. Furthermore, other HPTP
isoforms
exist that lack 9 amino acids within the second Ig-like domain and 4
amino acids at the junction of the second and third Ig-like domains or
9 amino acids within the fifth FN-III-like domain. Reverse
transcription polymerase chain reaction analysis demonstrated that
HPTP
isoforms lacking these short peptides are expressed in
kidney, whereas isoforms containing these peptides are expressed in the
brain. Analysis of HPTP
biosynthesis demonstrated that HPTP
is expressed as a complex of two noncovalently associated subunits
derived from a proprotein and that the HPTP
ectodomain is shed
from the cell surface. Mutational analysis of the HPTP
proprotein
cleavage site revealed the existence of two or three functional and
overlapping furin-like endoprotease cleavage sites.
Reversible tyrosine phosphorylation of signal transduction
proteins by protein-tyrosine kinases and protein-tyrosine phosphatases
(PTPases) ()regulates diverse cellular processes including
cell activation, differentiation, and proliferation (1, 2, 3, 4, 5) . A subset
of these enzymes are transmembrane proteins that operate by
transmitting signals across the plasma membrane. For example, several
growth factor receptors are protein-tyrosine kinases, including the
insulin and the epidermal growth factor
receptors(6, 7) . The CD45 transmembrane PTPase is
essential for thymocyte differentiation and activation of T and B
lymphocytes via the T cell or B cell antigen receptors(8) .
However, besides CD45, little is known about the function of the
transmembrane PTPases.
A majority of transmembrane PTPases contain
extracellular regions comprised of varying numbers of fibronectin
type-III (FN-III)-like domains arranged in tandem or of FN-III-like
domains in combination with Ig-like domains(9) . The
ectodomains of the remaining transmembrane PTPases are essentially
unrelated to other proteins with the exception of PTP and
PTP
, which contain at their amino terminus a carbonic
anhydrase-like domain followed by a single FN-III-like
domain(10, 11) . Considerable structural diversity
exists for some of the PTPase ectodomains as a result of alternative
splicing (12, 13, 14, 15, 16, 17, 18, 19, 20) .
Because the ectodomains of several known cell-cell adhesion molecules
are also composed of Ig-like and FN-III-like domains(21) , it
is possible that some of the transmembrane PTPases may function as
cell-cell adhesion or cell-extracellular matrix protein adhesion
molecules. This suggestion is supported by the finding that the
mammalian PTP
and PTPµ PTPases, which have ectodomains
containing Ig-like and FN-III-like domains as well as a MAM domain
(meprin, A5, µ; an approximately 170-amino acid-long domain found
in structurally diverse proteins(22) ) have homophilic binding
properties(23, 24, 25) . Furthermore, the
PTP
ectodomain binds the extracellular matrix protein
tenascin(19) .
Several of the transmembrane PTPases that
have ectodomains comprised of Ig-like and FN-III-like domains,
including LAR(26, 27) , PTP(28) , and
PTP
(29) , are expressed on the cell surface as two
noncovalently associated subunits derived by intracellular processing
of a proprotein. For the LAR PTPase, it was demonstrated that
proprotein processing occurs at a furin endoprotease consensus cleavage
site, RXXR, located 82 amino acids amino-terminal of the
transmembrane peptide(26, 30) . The LAR extracellular
region, which is comprised of three Ig-like and eight FN-III-like
domains, can be released from the cell surface as a result of a second
proteolytic cleavage event at a site located near the transmembrane
peptide(30) . We report here the molecular characterization of
the human transmembrane PTPase
(HPTP
). HPTP
, like LAR,
has an ectodomain comprised of three Ig domains and eight FN-III-like
domains and is expressed on the cell surface as a two-subunit structure
derived by proteolytic processing of the proprotein at a furin-like
cleavage site. The HPTP
ectodomain is shed from the cell surface.
Furthermore, evidence is presented for the existence of multiple
HPTP
isoforms that are expressed in a tissue-specific manner.
Figure 2:
Deduced primary structure of HPTP. A, the HPTP
amino acid sequence deduced from cDNA cloning
is shown using the standard single-letter code and is compared with the
MPTP
-fl (16) and human LAR (35) sequences. The
predicted signal peptide (sp), transmembrane peptide (TM), and proprotein-processing site (proc. site) are indicated by underlining. The extent and
boundaries of the Ig-like domains, FN-III-like domains, processing
site, transmembrane peptide, and PTPase domains are indicated by dashes and horizontallines, respectively.
The predicted domain junctions are based on the lar gene
organization (20) . The relative positions of the alternatively
used HPTP
meA, meB, and meC peptides are shown, and putative N-linked glycosylation sites are indicated by #. Dots in the MPTP
sequence and LAR sequence indicate an identical
amino acid with HPTP
. Spaces in the sequences indicate
gaps. B, nucleotide sequences and deduced amino acid sequences
of various HPTP
cDNA clones isolated. Shown is a comparison of the
DNA sequences containing (spliced-in) or lacking (spliced-out) the sequences encoding the meA, meB, and meC
peptides or the FN-III-like domains 4-7. The position of the
sequence insertion or deletion is marked by an asterisk.
Numbering refers to the amino acid numbers shown in panelA. indicates that the valine residue at position 999 in
the HPTP
-fl sequence is replaced by a methionine residue in the
clone encoding the HPTP
-
F4-7 isoform. This change is
probably caused by a polymorphism in the HPTP
sequence.
Figure 5:
Identification of the HPTP proprotein
processing site. A and B, SDS-PAGE analysis of
anti-HPTP
immunoprecipitates from
[
S]methionine-labeled COS-7 transiently
expressing HPTP
-fl (wt) or HPTP
-fl mutants
containing amino acid substitution mutants at potential furin cleavage
sites. Transiently transfected COS-7 cells were metabolically labeled
with [
S]methionine for 3 h. Following labeling,
cell lysates were prepared, HPTP
proteins were precipitated with
the anti-HPTP
mAb D1.1, and immunoprecipitates were analyzed by
SDS-PAGE (8% gels). Mutations are designated, using the one-letter
code, with the wild-type amino acid, its residue number, and the amino
acid substitution. control (in panelA),
immunoprecipitation products from COS-7 transiently expressing
HPTP
-fl using an isotyped matched mAb; control (panelB), immunoprecipitation products from COS-7 transfected
with the pSP65-SR
.2 expression vector using the anti-HPTP
mAb
D1.1. C, amino acid sequences of the distinct HPTP
mutants at the putative furin-like cleavage consensus region. Dots indicate identical amino acid residues with wild-type HPTP
sequence. Underlined residues 1155-1164 contain the
three potential furin cleavage sites. The relative amount of HPTP
proprotein processing was calculated as described under
``Experimental Procedures.'' Proprotein processing values
given are the average of the two experiments shown in panelsA and B. The protein migrating slightly slower
than the E-subunit of the R1158A mutant may be a degradation product
and was not included in determining the amount of proprotein
processing.
Figure 1:
Structure of cDNA
clones encoding HPTP. At the top of the figure is shown a
composite 6.3-kb cDNA encoding HPTP
including the relative
positions of the 5`-untranslated (5` UT) and
3`-untranslated (3` UT) regions, and the open reading
frame that encodes the three Ig-like domains, the eight FN-III-like
domains, the transmembrane peptide (TM), and the two PTPase
domains. Below the schematic, thicklines represent lengths and relative positions of the various HPTP
cDNAs isolated. Dashedlines and brackets represent deleted sequences as compared with other
cDNAs.
In addition to the cDNAs isolated that encode the HPTP sequence
shown in Fig. 2A, several other cDNAs were isolated
that encode shorter HPTP
isoforms. Two cDNAs (Fig. 1, clone
70 and clone 73) lacking peptide sequences within the extracellular
region of HPTP
were found by screening a fetal kidney cDNA
library. Clone 70 lacks sequences encoding two HPTP
internal
regions: a 9-amino acid-long sequence, provisionally designated the
mini-exon A (meA) peptide (Fig. 2, A and B,
residues 141-149) that is predicted to affect the length of a
loop region between the D and E
-strands of the second Ig-like
domain(37) . Clone 70 also lacks a 4-amino acid sequence
designated the meB peptide (Fig. 2, A and B,
residues 187-190) that is predicted to affect the length of the
sequence between the second and third Ig-like domains. Clone 73 (Fig. 1) lacks another 9-amino acid sequence, designated the meC
peptide (Fig. 2, A and B, residues
756-763), that is located within the fifth FN-III-like domain (Fig. 2B). Another clone (Fig. 1, clone 91) was
isolated from a fetal brain cDNA library that lacks the sequence
encoding the fourth to seventh FN-III-like domains (Fig. 2, A and B, residues 568-978). Comparison of the
sequences of the various HPTP
cDNA clones suggests that the
molecular basis for the observed variation is alternative splicing of
the HPTP
pre-mRNA. Supporting this possibility is the observation
that the meA, meB, and meC peptide coding sequences are exactly
inserted at exon-exon junctions of the HPTP
-related lar gene and that alternative splicing of LAR exon 13, which encodes
the LAR meC-like peptide, has been documented(20) . Isolation
of cDNA clones encoding isoforms containing or lacking FN-III-like
domains 4-7 has also been reported for the MPTP
(16) and PTP
PTPases(14, 17, 28) . Furthermore, generation
of structural diversity of ectodomains of other transmembrane PTPases
by alternative splicing has also been
reported(12, 13, 14, 15, 16, 17, 18, 19, 20) .
Taken together, these findings suggest the existence of several
HPTP
isoforms that are generated by alternative splicing of the
exons encoding the meA, meB, and meC peptides as well as the exons
encoding the fourth to seventh FN-III-like domains.
Figure 3:
HPTP mRNA expression. Northern blot
analysis of poly(A)
RNA isolated from different human
tissues using a radiolabeled HPTP
cDNA probe (A) or
-actin cDNA probe (B). Size markers in kilobases (kb) are shown at the left. The time of exposure for
the autoradiogram shown in panelA was 3 days and for panelB 2 h. C, RT-PCR analysis of
poly(A)
RNA isolated from human kidney and fetal brain
tissues or HPTP
plasmid cDNAs as standards, using HPTP
oligonucleotide pairs as PCR primers. control, RT-PCR with no
DNA or poly(A)
RNA template. Expected sizes of the
amplified fragments are as follows: oligo-pair 1, 232 or 271 bp;
oligo-pair 2, 290 or 317 bp; oligo-pair 3, 365 bp. M, DNA size
marker in bp.
To examine the expression of HPTP mRNAs encoding the putative
HPTP
mRNA isoforms with or without the meA, meB, and meC peptide
sequences or lacking the fourth to seventh FN-III-like domains, RT-PCR
analysis was performed using poly(A)
RNA isolated from
human kidney and fetal brain (Fig. 3C). HPTP
mRNA-derived cDNAs, or HPTP
plasmid cDNAs used as standards, were
amplified using oligonucleotide primers corresponding to sequences
flanking the alternatively used sequences, as described under
``Experimental Procedures.'' As seen in Fig. 3C, PCR analysis of HPTP
plasmid-derived cDNA
lacking or containing the meA and meB peptide-encoding sequences gave
the expected cDNA products of 232 bp (lane3) and 271
bp (lane4), respectively, using oligo-pair 1. For
the meC peptide sequence, the expected PCR products of 290 bp (lane8) and 317 bp (lane9), respectively,
were observed for the deletion or addition using oligo-pair 2. To
assess the deletion of the sequences encoding the fourth to seventh
FN-III-like domain, PCR primers corresponding to the second and eighth
FN-III-like domains (oligo-pair 3) were used and gave the expected PCR
product of 365 bp (lane13). The inclusion of the
fourth to seventh FN-III-like domains was assessed using oligo-pair 2,
which detects the presence of the fifth FN-III-like domain. RT-PCR
analysis revealed that the HPTP
mRNA isolated from human kidney
lacked the sequences encoding the meA and meB peptides (Fig. 3C, lane5) and that the
majority of mRNA also lacked the sequences encoding the meC peptide (Fig. 3C, lane10), whereas mRNA
isolated from fetal human brain contained the sequences encoding these
peptides (Fig. 3C, lanes6 and 11, respectively). In contrast, both the brain and kidney
samples contained the HPTP
isoform mRNA that included the fifth
FN-III-like domain (Fig. 3C, lanes10 and 11) and the HPTP
-
F4-7 isoform (Fig. 3C, lanes14 and 15,
respectively). These results suggest that a complex pattern of
HPTP
mRNA expression can be generated by tissue-specific
alternative splicing of the HPTP
pre-mRNA and that one tissue type
can express multiple HPTP
isoforms. Based on the cDNA cloning and
RT-PCR data, there is thus evidence for the existence of at least four
HPTP
isoforms: the full-length isoform (meA+, meB+,
meC+, and FN-III-4-7+), an isoform lacking the fourth
to seventh FN-III-like domains without the meA and meB peptides
(meA-, meB-, and FN-III
4-7), an isoform lacking
the fourth to seventh FN-III-like domains and containing the meA and
meB peptides (meA+, meB+, and FN-III
4-7), and one
isoform lacking the meA and meB peptides but containing the meC peptide
(meA-, meB-, meC+, and FN-III-4-7+). Given
the possibility of the combinatorial use of the alternatively used
exons, there may also be additional PTP
isoforms.
Alternative
splicing of pre-mRNA is a molecular mechanism to generate functional
diversity of a gene product. For a number of transmembrane PTPases,
including CD45, LAR, PTP, PTP
, PTP
, and PTP
,
alternative splicing appears to generate a number of various isoforms
with distinct functional
properties(12, 13, 14, 15, 16, 17, 18, 19, 20) .
For the CD45 PTPase, the developmental and tissue-specific alternative
splicing of three exons encoding amino-terminal sequences of the CD45
PTPase has been extensively documented, and there exists a strong
correlation between CD45 isoform expression and the functional
phenotype of the cell(38) . A regulatory role for
tissue-specific, alternatively used peptide sequences has been shown
for the neural cell adhesion molecule, N-CAM (39, 40) . Alternative splicing of the N-CAM VASE
exon, which encodes a 10-amino acid peptide located within the fourth
N-CAM Ig-like domain, regulates the neurite outgrowth promoting
activity of N-CAM(41) . Small peptide insertions between the
second and third Ig domains have also been reported for other cell
adhesion molecules, including the neural adhesion molecules, Nr-CAM and
neurofascin(42, 43) . Thus, it is possible that the
HPTP
meA, meB, and meC peptides play an active role in regulating
the interaction between HPTP
and its putative ligand(s).
Figure 4:
Biosynthesis of HPTP-fl and
HPTP
-
F4-7. SDS-PAGE analysis of anti-HPTP
immunoprecipitates from [
S]methionine-labeled
COS-7 transiently expressing HPTP
-fl and
HPTP
-
F4-7. A, for pulse-chase analysis of the
HPTP
-fl isoform biosynthesis, cells were metabolically labeled
with [
S]methionine for 15 min (Pulse)
or were labeled for 15 min and then incubated in medium containing an
excess of non-radioactive L-methionine for 1 h (Chase). Following incubation, cell lysates were prepared, and
HPTP
proteins were precipitated using the anti-HPTP
mAb D1.1.
Immunoprecipitates were analyzed by SDS-PAGE (8% gels). Molecular mass
standards in kilodaltons (kDa) are shown on the left,
and the positions of the HPTP
-fl proprotein (Pro),
E-subunit (E), and P-subunit (P) are shown on the right. control, immunoprecipitation products from
COS-7 transfected with the pSP65-SR
.2 expression vector using the
anti-HPTP
D1.1 mAb. B, prior to harvesting cell culture
supernatants or preparing cell lysates for immunoprecipitation analysis
using the anti-HPTP
mAb D1.1, cells expressing the HPTP
-fl or
-
F4-7 isoforms were metabolically labeled with
[
S]methionine for 3 h. The positions of the
HPTP
-fl proprotein (Pro), E-subunit (E), and
P-subunit (P) are shown on the left, and the
positions of the HPTP
-
F4-7 isoform proprotein (Pro/
F4-7) and E-subunit (E/
F4-7) are shown on the right.
The ectodomains of both the
HPTP-fl and -
F4-7 isoforms were present in the cell
culture supernatants, demonstrating that the ectodomain of HPTP
,
like LAR(26) , can be shed from the cell surface (Fig. 4B). Ectodomain shedding has been observed for a
number of transmembrane proteins and is thought to play a role in
decreasing the responsiveness of the cells to the cognate
ligands(44) . Alternatively, HPTP
ectodomain shedding may
be important for signal transduction, membrane localization, and/or
internalization of the remaining portion of HPTP
.
Because the
LAR PTPase proprotein is known to be processed at a furin-like cleavage
site (RXXR)(45, 46, 47) ,
located 82 amino acids amino-terminal of the putative transmembrane
peptide(26, 30) , we examined whether the HPTP
proprotein is also processed at a furin-like cleavage site (Fig. 5). To this end, amino acid substitution mutants were
generated that substituted one or two basic residues between
HPTP
-fl residues located about 85 amino acids from the putative
transmembrane peptide (Fig. 2A). Between residues 1155
and 1164 (RKRRSIRYGR) there are three potential furin cleavage sites:
RKRR, RSIR, and RYGR (see Fig. 5C). Each of these
potential sites was altered by site-directed mutagenesis, and resulting
pSP.SR
-HPTP
plasmid DNAs harboring the R1158A, R1161A, and
R1164A mutants were transfected into COS-7 cells. Transfected cells
were metabolically labeled with [
S]methionine,
and HPTP
-fl proteins were analyzed by SDS-PAGE following
immunoprecipitation with the anti-HPTP
mAb D1.1. As seen in Fig. 5A and summarized in Fig. 5C,
mutation of the potential cleavage site RKRR to RKRA (mutation R1158A)
decreased proprotein cleavage about 2.5-fold, whereas mutation of the
other two potential sites (RSIR to RSIA and RYGR to RYGA) did not
significantly affect HPTP
proprotein processing (R1161A and R1164A
mutations, respectively). However, a double mutation, R1158/R1161A,
that destroyed all three potential furin cleavage sites, completely
blocked HPTP
-fl proprotein cleavage (Fig. 5, B and C). Thus, HPTP
proprotein processing can occur at two or
three overlapping furin-like cleavage sites located 81-87 amino
acids NH
-terminal to the putative transmembrane peptide.
Because there are no cysteine residues between the proprotein cleavage
sites and the transmembrane peptide, the HPTP
E- and P-subunits,
like the LAR subunits(26) , are not disulfide-bonded.
The
functional significance of the HPTP two-subunit architecture is
not known. Proprotein processing of HPTP
and LAR is not essential
for surface expression, since cleavage-defective LAR and HPTP
mutants were expressed on the cell surface ( (26) and data not
shown). In the case of LAR, the two-subunit structure also does not
appear to play a role in ectodomain shedding(30) . However, the
fact that proprotein cleavage is a conserved feature of several
PTPases, including LAR, HPTP
, PTP
, and PTP
, as well as
several adhesion molecules including Ng-CAM (48) and
Nr-CAM(49) , suggests that this proteolytic processing plays an
important role in the function of these molecules. Possibly, proprotein
cleavage is required for correct conformation of the ectodomain, for
localization within the plasma membrane, and/or for signal
transduction. In the case of the insulin receptor, cleavage of the
insulin proreceptor is necessary for high affinity insulin binding as
well as efficient signal transduction following ligand
binding(50, 51) , and naturally occurring insulin
proreceptor processing mutations may cause insulin-resistant diabetes
mellitus(52, 53) .
Comparison of the amino acid
sequences and structural properties of the LAR, HPTP, and
PTP
PTPases suggests that these molecules comprise a subfamily of
transmembrane PTPases with highly conserved architecture and common
alternative splicing patterns (Fig. 6). However, whereas LAR is
expressed in most tissues (26) , both PTP
(16) and
PTP
(14, 17, 28) appear to have a more
restricted tissue distribution, most notably to neural tissue. It is
conceivable that this subfamily of PTPases shares a common mechanism to
regulate their catalytic activity but exerts its biological function by
dephosphorylating tissue-specific substrates. Further studies with
specific mAbs will be necessary to document in detail the differential
expression of these enzymes. In addition, the expression of multiple
alternative isoforms affecting the structure of the ectodomains may
determine their differential ligand specificity in the distinct
tissues. The identification of such ligands constitutes a key point in
understanding the physiological function and regulation of this
subfamily of transmembrane PTPases.
Figure 6:
Comparison of the structure and
alternative isoform patterns of LAR, PTP, and PTP
. A
schematic representation of LAR, PTP
and PTP
isoforms is
depicted with numbers corresponding to the various Ig-like and
FN-III-like domains. The locations of the HPTP
meA, meB, and meC
peptides and the meC-like LAR peptide (E13) are indicated by arrows. D1 and D2, PTPase domains 1 and 2,
respectively. The structure of the PTP
-fl isoform is derived from
Zhang et al.(54) . The existence of a MPTP
isoform cDNA that lacks the first and second Ig-like domains was also
reported(16) . A PTP
isoform cDNA that lacks D2 has also
been described(14) .