©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of the Human Transmembrane Protein-tyrosine Phosphatase
EVIDENCE FOR TISSUE-SPECIFIC EXPRESSION OF ALTERNATIVE HUMAN TRANSMEMBRANE PROTEIN-TYROSINE PHOSPHATASE ISOFORMS (*)

(Received for publication, October 31, 1994)

Rafael Pulido (1) (2) Neil X. Krueger (1) (3) Carles Serra-Pagès (1) (4) Haruo Saito (1) (3) Michel Streuli (1) (2)

From the  (1)Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 and the Departments of (2)Pathology, (3)Biological Chemistry and Molecular Pharmacology, and (4)Medicine, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Reversible tyrosine phosphorylation of signal transduction proteins by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases) (^1)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 PTPkappa 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 PTPkappa(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.


EXPERIMENTAL PROCEDURES

cDNA Cloning and Sequencing

HPTP cDNA clones 40 and 52 were isolated from a gt11 human placenta cDNA library(31, 32) ; clone 139 from a human fetal liver cDNA library (Clontech, Palo Alto, CA); clone 206 from a human fetal lung cDNA library (Clontech); clones 70 and 73 from a human fetal kidney cDNA library (Clontech); and clones 91, 242, and 243 from a human fetal brain cDNA library (Clontech), according to standard procedures(33) . HPTP cDNA was subcloned into plasmid vectors. DNA manipulations and DNA sequence determination using the chain termination method were done according to standard procedures (33) . The complete HPTP cDNA sequence appears in the EMBL/GenBank/DDBJ nucleotide sequence data bases (accession number L38929). The putative initiation methionine residue shown in Fig. 2A is encoded by nucleotides 154-156.


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-DeltaF4-7 isoform. This change is probably caused by a polymorphism in the HPTP sequence.



Plasmid Constructions

HPTP expression plasmids were generated by combining appropriate restriction fragments into the pSP65-SRalpha.2 expression vector(34) . pSP65-SRalpha.2.HPTP-fl encodes HPTP amino acids 1-1892 plus the signal peptide (see Fig. 2A), and pSP65-SRalpha.2.HPTP.DeltaF4-7 contains the same sequence except that the sequences encoding amino acids 588-998 are deleted. The amino acid substitution mutants shown in Fig. 5were generated by oligo-directed mutagenesis, and mutants were confirmed by nucleotide sequencing.


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-SRalpha.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.



Northern Blot Analysis

A tissue Northern blot containing about 2 µg of poly(A) RNA/lane was obtained from Clontech and hybridized to P-labeled DNA probes using the hybridization conditions recommended by the supplier. The HPTP cDNA probe spans nucleotides 1-2144, which encode amino acids -20 to 645 of the full-length HPTP isoform.

Detection of HPTP mRNA from Cells by Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Poly(A) RNA isolated from human kidney and fetal brain tissue was obtained from Clontech. cDNA was synthesized with SuperScript reverse transcriptase (Life Technologies, Inc.), using random hexamers as primers, and 10 ng of poly(A) RNA. Control PCR was done using appropriate HPTP cDNA. PCR reactions (35 cycles) consisted of sequential incubations for 1 min at 92 °C, 2 min at 55 °C, and 2 min at 75 °C with a final extension of 5 min at 75 °C. Products of RT-PCR or PCR were analyzed on 1.5% agarose gels. Synthetic oligonucleotide pairs used as PCR primers were as follows (HPTP nucleotide numbering is based on the complete HPTP nucleotide sequence): 1 (sense) (HPTP; residues 662-684), 5`-ACAACAATGGTCGTATTAAGCAG-3`; 1 (antisense) (HPTP; residues 913-933), 5`-CACGGCCACACAGGTGATATT-`; 2 (sense) (HPTP; residues 2295-2319), 5`-TCCTCGCAAAGTCGAGGTAGA-3`; 2 (antisense) (HPTP; residues 2593-2612), 5`-GTGGTGGACACCAGTTTGGG-3`; 3 (sense) (HPTP; residues 1681-1701), 5`-ATCACTCAGACAGGAGTACCA-3`; 3 (antisense) (HPTP; residues 3259-3279), 5`-CTCTGGAATCTCCCAAGACAG-3`.

Production of an Anti-HPTP Monoclonal Antibody

Anti-HPTP mAb D1.1 (IgG1, kappa) was generated by immunizing Balb/c mice with a murine 300-19 pre-B cell line expressing a HPTP-LAR chimeric protein consisting of HPTP amino acids -20 to 1076 and LAR amino acids 1061-1881(35) . Expression of this chimeric protein in 300-19 cells was monitored using the anti-LAR mAb 11.1A(26) . Splenocytes from immunized mice were fused to NS-1 myeloma cells, and culture supernatants from hypoxanthine/aminopterin/thymidine medium-resistant hybridomas were screened for reactivity to the 300-19(HPTP-LAR) cell line used as immunogen and lack of reactivity to a 300-19 cell line expressing human LAR (300-19(LAR)). Screening was performed by flow cytofluorography using fluorescein isothiocyanate-conjugated goat anti-mouse as secondary antibody on a Profile EPICS cell sorter (Coulter Immunology).

Cell Transfections, Cell Labeling and Immunoprecipitations

Simian COS-7 cells (ATCC CRL 1651) were maintained in RPMI medium containing 10% v/v fetal calf serum, 2 mML-glutamine, and 50 µg/ml gentamicin sulfate. COS-7 cells were transfected with the pSP65-SRalpha.2.HPTP expression plasmids or control pSP65-SRalpha.2 using the DEAE-dextran method as described(33) . Following transfection, cells were cultured for 48-56 h, washed with phosphate-buffered saline, and then metabolically labeled with [S]methionine in L-methionine-free RPMI medium (Life Technologies, Inc.) containing 2% (v/v) dialyzed fetal calf serum (0.1 mCi of [S]methionine/5 times 10^5 cells). After labeling, cell culture supernatants were harvested, and cells were washed with phosphate-buffered saline and then lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin). Resulting cell lysates or culture supernatants were precleared with protein G-Sepharose (25 µl of slurry) (Pharmacia Biotech Inc.) prior to use for immunoprecipitation studies with the anti-HPTP mAb D1.1 or a control isotype-matched mAb (1 µl of ascites fluid or 5 µg of mAb/0.5 ml of lysate) and 25 µl of protein G-Sepharose slurry. Immunoprecipitated proteins were washed 4 times with phosphate-buffered saline containing 0.1% Triton X-100 and then analyzed by SDS-PAGE (8% gels) using reducing conditions, followed by autoradiography. The relative amounts of HPTP 220-kDa proprotein (Pro) and 150-kDa E-subunit (E) were determined by densitometric scanning of autoradiographs using an Ultrascan XL laser densitometer (Pharmacia). For determining the relative amount of HPTP proprotein processing, the values obtained by densitometric scanning were corrected for the number of methionine residues present in the various HPTP proteins, the amount of proprotein processing was calculated as E/(Pro + E), and resulting values were standardized to give a value of 1.0 for wild-type HPTP proprotein processing.


RESULTS AND DISCUSSION

Isolation of cDNAs Encoding Human PTP Isoforms

Previously, we described the isolation of cDNA clones encoding the partial amino acid sequence of HPTP(32) . Now we report the complete HPTP sequence as well as the sequence of a number of HPTP isoforms deduced from the sequence analysis of cDNA clones (Fig. 1). The longest composite cDNA (clones 243, 242, 139, and 52; Fig. 1) spans about 6.3 kb and contains an open reading frame of 5736 bp encoding a protein of 1912 amino acids. The deduced amino acid sequence of this HPTP isoform is shown in Fig. 2A and is aligned with the sequence of the mouse homologue, MPTP (16) and the human LAR transmembrane PTPase (35) . Since the amino-terminal 20 amino acids are likely to serve as a signal peptide(36) , the 21st amino acid (glutamic acid) is predicted to be the amino terminus of the mature 1892-amino acid-long HPTP protein and is designated the +1 residue (Fig. 2A). A hydrophobic stretch of 25 amino acids located from residue 1246 to 1270 (underlined in Fig. 2A) is likely to be the transmembrane peptide. Sequence comparisons suggest that the 1245-amino acid extracellular region of HPTP is comprised of three Ig-like domains and eight FN-III-like domains and contains four putative N-linked glycosylation sites (indicated by # in Fig. 2A). The 622-amino acid-long HPTP intracellular region contains two PTPase domains that were previously demonstrated to be catalytically active in vitro(32) . Overall, the HPTP and MPTP amino acid sequences are 95% identical, and HPTP and LAR are 72% identical. Thus, PTP is evolutionarily well conserved, and its overall architecture and sequence are very similar to the LAR PTPase.


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 beta-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.

Expression of mRNAs Encoding Distinct HPTP Isoforms

The expression of HPTP mRNA was examined by Northern blot analysis using poly(A) RNA isolated from various human tissues and a cDNA probe corresponding to the HPTP ectodomain (Fig. 3A). HPTP mRNA was detected in the kidney and brain samples but not in the heart, placenta, liver, or skeletal muscle samples (Fig. 3A). In the kidney mRNA sample, there was a dominant HPTP mRNA of about 9.5 kb and a minor mRNA of about 8.5 kb, whereas in brain there was only an 8.5-kb mRNA. The expression of HPTP predominantly in brain and kidney is consistent with the finding that MPTP is also primarily seen in these tissues(16) . The difference in length of the 6.3-kb composite HPTP cDNA and the 8.5-9.5-kb mRNA suggests that additional 3`-untranslated sequences are not represented in the isolated HPTP cDNAs. Control hybridization of the Northern blot with a beta-actin cDNA probe is shown in Fig. 3B.


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 beta-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-DeltaF4-7 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-IIIDelta4-7), an isoform lacking the fourth to seventh FN-III-like domains and containing the meA and meB peptides (meA+, meB+, and FN-IIIDelta4-7), 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 PTPalpha, 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).

Characterization of PTP Biosynthesis and Proprotein Cleavage Site

To study the biosynthesis of HPTP, we generated an anti-HPTP mAb, termed D1.1, by immunizing mice with a murine 300-19 pre-B cell line expressing an HPTP-LAR chimeric protein as described under ``Experimental Procedures.'' The expression of the HPTP isoforms or HPTP mutants was then analyzed by immunoprecipitation studies using the anti-HPTP mAb D1.1. To this end, pSP.SRalpha-HPTP expression plasmids were transfected into COS-7 cells, cells were grown for 48-56 h, and then cells were metabolically labeled with [S]methionine. Following labeling, cell extracts were prepared, and HPTP protein was isolated by immunoprecipitation and visualized by autoradiography following SDS-PAGE (Fig. 4). Pulse-chase analysis demonstrated that the full-length HPTP (HPTP-fl) isoform was expressed as a proprotein (Pro) of about 220 kDa that, after a 1-h chase incubation, was processed into two products of 150 (E) and 85 kDa (P) (Fig. 4A). Because of the sequence similarity of HPTP with LAR (26) , it was likely that the HPTP-fl isoform was synthesized as a proprotein of 220 kDa (Pro) that is cleaved intracellularly into two subunits of 150 and 85 kDa. The 150-kDa E-subunit is predicted to contain the three Ig-like and eight FN-III-like domains, whereas the 85-kDa P-subunit contains a short ectodomain segment, the transmembrane peptide, and the intracellular PTPase domains. The HPTP-DeltaF4-7 isoform was synthesized as a smaller proprotein (Pro/DeltaF4-7) of about 175 kDa, the HPTP-DeltaF4-7 E-subunit (E/DeltaF4-7) resolved poorly with a molecular mass between 95,000 and 125,000, and the P-subunit was 85 kDa (Fig. 4B).


Figure 4: Biosynthesis of HPTP-fl and HPTP-DeltaF4-7. SDS-PAGE analysis of anti-HPTP immunoprecipitates from [S]methionine-labeled COS-7 transiently expressing HPTP-fl and HPTP-DeltaF4-7. 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-SRalpha.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 -DeltaF4-7 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-DeltaF4-7 isoform proprotein (Pro/DeltaF4-7) and E-subunit (E/DeltaF4-7) are shown on the right.



The ectodomains of both the HPTP-fl and -DeltaF4-7 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.SRalpha-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(2)-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 PTPkappa, 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) .




FOOTNOTES

*
This work was supported by Grant CA55547 from the National Institutes of Health (to M. S.), a post-doctoral fellowship award from the Fundación Ramón Areces (Spain) (to R. P.), a Fulbright fellowship award from the Ministerio de Educación y Ciencia (Spain) (to C. S. P.), and a Pew Scholar in the Biomedical Sciences Award (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: PTPase, protein-tyrosine phosphatase; LAR, leukocyte common antigen-related protein; HPTP, human protein-tyrosine phosphatase ; MPTP, murine protein-tyrosine phosphatase ; FN-III, fibronectin type III; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Drs. Paul Anderson, John Gribben, and Quintus Medley for critical review of the manuscript, Dr. Timothy Ernst for oligonucleotide synthesis, and Dr. Stuart F. Schlossman for encouragement and support.


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