Identification of the homophilic binding site of the receptor protein

THE Jouru.4~ OF BIOUX~ICAL CHEMTSTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 45, Issue of November 11, pp. 28472-26477,1994 Printed in U.S.A.

Identification of the Homophilic Binding Site of the Receptor Protein Tyrosine Phosphatase PTPp*

(Received for publication, July 8, 1994)

Susann M. Brady-KalnayS and Nicholas K. Tanks§

From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208

The receptor-type protein tyrosine phosphatase PTPp comprises an extracellular segment containing a MAM domain, an immunoglobulin domain and four fibronectin type 111 repeats, a transmembrane segment, and two intracellular PTP domains. We have previously shown that PTPp binds homophilically, i.e. PTPp on the surface of one cell binds to PTPp on an apposing cell, and that the extracellular segment alone is sufficient for homophilic binding. In this study we report that in MvLu cells PTPp is proteolytically processed into two noncovalently associated fragments, one comprising most of the extracellular segment (-100 kDa) and the other containing predominantly the transmembrane and intracellular portions (-100 ma). We have also identified the homophilic binding site within the extracellular segment. We have generated, expressed, and purified various fragments of the extracellular segment of PTPp and have used fluorescent beads (Covaspheres) coated with these fragments in three binding assays: (i) measure- ment of bead aggregation, (ii) binding of beads to surfaces of dishes coated with purified PTPp, or (iii) binding to MvLu cells. Only beads coated with recombinant fragments that contained the immunoglobulin domain underwent aggregation or bound to surfaces displaying PTPp, suggesting that neither the MAM domain nor the fibronectin type I11 repeats bound homophilically in these assays. The fragment containing the Ig domain alone bound as well as any other Ig domain-containing fragment, suggesting that the Ig domain is both necessary and sufficient for homophilic binding under these conditions.

Many cellular processes are regulated by reversible tyrosine phosphorylation, controlled by the balanced and opposing actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs).’ PTPs exist in both soluble and transmembrane, receptor-like forms (RPTPs) (1, 2). In the case of the receptor PTPs there is the potential for regulating activity by the binding of ligands to the extracellular segment of the protein. Several of the transmembrane PTPs are members of the

CA53840 and the Mellam Family Foundation. The costs of publication *This work is supported by National Institutes of Health Grant

of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a National Institutes of Health “raining Grant Fel- lowship 5T32CAO9311.

should be addressed: Cold Spring Harbor Laboratory, Demerec Bldg., 1 5 Pew Scholar in the Biomedical Sciences. To whom correspondence

Bungtown Rd., Cold Spring Harbor, NY 11724-2208. Tel.: 516-367-8846; Fax: 516-367-6812; E-mail: tonks @ cshl.org.

The abbreviations used are: P T P , protein tyrosine phosphatase; RPTP, receptor protein tyrosine phrosphatase; LAR, leukocyte common antigen-related protein; FN, fibronectin; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; N-CAM, neural cell adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule.

immunoglobulin superfamily and display structural motifs in their extracellular segments that are suggestive of a role in cell-cell adhesion (3, 4). For example, leukocyte common antigen- related protein (LAR), shows homology to N-CAM in that it bears an extracellular segment containing multiple immunoglobulin (Ig) and fibronectin type I11 (FN 111) repeats (5). This arrangement of Ig domains and FN I11 repeats has now been observed in the extracellular segments of several other transmembrane PTPs (reviewed in Ref. 6).

Our studies have focused on PTPp, the extracellular segment of which comprises one Ig and four FN 111 repeats as well as one copy of the recently described MAM domain (7,8) (see figure 1). Ig domains are disulfide-bonded structures that are found in a variety of proteins including cell surface receptors. These domains contain the homophilic binding site of some cell-cell adhesion molecules such as N-CAM (reviewed in Ref. 6). FN I11 motifs were originally observed in the extracellular matrix protein fibronectin but have now been detected in more than 50 eukaryotic proteins. They comprise 90-100 amino acids that are characterized by highly conserved hydrophobic residues. In most cases their function is unknown, however one FN 111 repeat in fibronectin has been shown to mediate cell attach- ment through binding of the tripeptide sequence RGD to inte- grins (reviewed in Ref. 6). The MAM domain is a sequence motif identified in five proteins to date: meprin A and B, the A5 glycoprotein, PTPp, and PTPK (8). The A5 antigen is a transmembrane protein which, upon optic nerve innervation, is expressed in pre- and post-synaptic neurons of the visual centers of the brain ofXenopus laevis, an area where cell-cell contact is important (9). Meprins are cell surface homo- or heterodimers that have metalloendopeptidase activity (10). The MAM domain (meprinlA5PTPp) that is common to these proteins comprises 170 amino acids containing 4 conserved cysteine residues and two conserved sequence motifs, tChtFahhXXtt and ttGhhXhD-hXh (where h = hydrophobic, a = aromatic, and t = turn or polar residues) (8). At present the function of this motif is not known.

PTPp is also characterized by a juxtamembrane segment that is -70 residues longer than the equivalent segment in most other RPTPs and displays homology to the intracellular segment of members of the cadherin family (6). This homology is of interest because it is unique among members of the immunoglobulin superfamily. The intracellular segment of the cadherins is essential for adhesion. This domain binds to proteins termed catenins which mediate the interaction between cadherins and the actin cytoskeleton. Thus it is possible that PTPp may associate with the cytoskeleton through an interaction with a catenin-like molecule. The structural features of PTPp therefore suggest that it may play a unique role in regu- lation of cytoskeletal function through changes in tyrosine phosphorylation in response to cell-cell contact.

We have previously shown that PTPp can mediate aggregation of cells via homophilic binding, ie. one PTPp molecule binds to another PTPp molecule on an adjacent cell (11). Ex-

28472

Identification of the Homophilic Binding Site of PTPp pression of PTPp, by infection of Sf9 cells with recombinant baculoviruses, induced their aggregation independent of the presence of the PTP domains. Another study has confirmed these Sf9 cell aggregation data (12). In addition, we reconsti- tuted the binding reaction in vitro demonstrating that PTPp- coated beads bound specifically to a bacterially expressed glutathione S-transferase fusion protein containing the extracellular segment of PTPp (PTPp-extra) adsorbed to Petri dishes (11). Furthermore, PTPp-extra-coated fluorescent beads (Covaspheres) aggregated in vitro. In addition we demonstrated binding between PTPp-coated Covaspheres and PTPp endogenously expressed on MvLu cells (11). These observations demonstrate that the ligand for this transmembrane PTP is another molecule of PTPp on the surface of an adjacent cell. A structurally similar molecule, PTPK, which has a MAM domain, an Ig domain and four FN I11 repeats in its extracellular segment, has also been shown to bind homophilically (13).

w 1 , PTPK, and PTPp contain potential proteolytic cleavage sites, which consist of a cluster of basic amino acids, in their extracellular segment. LAR was the first RPTP shown to be proteolytically processed, at the sequence RRRRR, to generate a noncovalently associated complex comprising an E subunit, containing most of the extracellular segment, and a P subunit which consists predominantly of the transmembrane and intracellular segments (14, 15). PTPK is cleaved in a similar region of the protein, at the sequence RTKR (16). We present data to illustrate that PTPp, which also possesses a cluster of basic amino acids in a similar region of the protein (RPRRTKK), was proteolytically processed in MvLu cells which express the protein endogenously. We have adopted the nomen- clature of E and P subunit for these fragments that was first proposed in the LAR study (14).

Since the E subunit contains most of the extracellular segment, it was important to demonstrate that the E subunit retains the capacity to bind homophilically and to identify the site responsible for the binding interactions. To address this issue, we generated various fragments of the extracellular segment of PTPp and expressed them either as histidine tagged proteins in recombinant baculovirus-infected Sf9 cells or in Escherichia coli as glutathione S-transferase fusion proteins. We present the results of various binding assays that establish the importance of the immunoglobulin domain in promoting homophilic interactions.

MATERIALS AND METHODS Plasmid and Baculovirus Construction-Polymerase chain reaction

was used to amplify cDNA corresponding to three fragments; the MAM domain (amino acids 20-184), the Ig domain (amino acids 185-289), and IgFN containing the Ig domain and two FN I11 repeats (amino acids 185-434), which were ligated into pAcSG-His-NT (Pharmingen Corp., San Diego, CA) to generate histidine-tagged fusion proteins. The recombinant viruses were made using the BaculoGold Transfection System (Pharmingen). PTPp-ACD has been previously described (11). The various fragments of human PTPp were expressed in Sf9 cells from recombinant baculoviruses as described previously (11). Other PTPp recombinant fragments were made as GST fusion proteins in E. coli. PTPp- extra has been described (ll), and MIF which expresses the MAM, Ig domains, and two FN I11 repeats (amino acids 20-434) was generated by polymerase chain reaction and ligated into pGEX-KG.

Antibodies-A monoclonal antibody to the intracellular segment of PTPp (SBK15) was used as described previously (11). A peptide, corresponding to amino acids 42-60, was coupled to keyhole limpet hemocy- anin and used to generate monoclonal (BK series) or polyclonal (CSH 494) antibodies toward the extracellular segment. Peptides corresponding to part of the immunoglobulin domain (residues 231-256) and the C terminus (1439-1452) of PTPp were coupled to keyhole limpet hemo- cyanin and used to generate rabbit polyclonal antibodies (CSH 338 and CSH 471, respectively).

Protein Purification, Electrophoresis, and Immunoblotting-MvLu cells were lysed in bufferA(20 mM Tris, pH 7.6,1% Triton X-100,5 pg/ml

A B

28473

C 0

d

m,, FIG. 1. F"Pp is proteolytically processed. Lysates from MvLu

cells were immunoprecipitated and resolved on 6% SDS-PAGE gel. PTPp was immunoprecipitated with either a polyclonal (CSH 494, lane 1 ) or monoclonal (BK2, lane 2) antibody to the extracellular domain or a polyclonal (CSH 471, lane 3) or monoclonal (SK 15, lane 4 ) antibody to the intracellular domain. Panel A is an immunoblot of these immunoprecipitates probed with antibodies to the intracellular segment (SBK15) which detects the full-length form (200 kDa) and the intracellular, P subunit (100 kDa). Panel B is an immunoblot of these immunoprecipitates probed with antibodies to the extracellular segment (BK2) which recognizes the full-length form and the extracellular, E subunit (100 kDa). Panel C is a schematic diagram that illustrates the cleavage of PTPp. The molecule is cleaved into two noncovalently associated fragments, the extracellular portion (100 kDa), and a predominantly intracellular portion (100 kDa). The lines on the side ofpanelsA and B indicate the migration of molecular mass standards 180 and 116 kDa, respectively.

leupeptin, 5 pg/ml aprotinin, 1 mM benzamidine), incubated on ice for 30 min, centrifuged a t 5,000 x g for 5 min, and immunoprecipitated with antibodies to PTPp that had been coupled to Protein A- Sepharose or CNBr-Sepharose 4B (Pharmacia Biotech Inc.). Sf9 cells were infected with recombinant baculoviruses, harvested 4 days post-infection, lysed in buffer A, and loaded onto a nickel-Sepharose column (Probond resin, Invitrogen, San Diego, CA) for 30 min at room temperature. The beads were washed with buffer B (50 mM sodium phosphate, 300 mM NaCl, 1% glycerol, 1% Triton X-100, pH 8.0) and eluted in buffer B plus 0.6 M imidazole. The PTPp-extra and MIF fusion proteins were expressed in E. coli and purified according to Guan and Dixon (17). Immunoblotting was performed as described elsewhere (11).

Couasphere Preparation and Binding Assays-Covaspheres (0.9-pm; Duke Scientific, Palo Alto, CA) were prepared as described previously (11). Covaspheres were visualized with a Zeiss Axiophot microscope equipped for epifluorescence using a 20x lens. In aggregation assays, Covaspheres (20 pl) were briefly sonicated and added to 1 ml of phosphate-buffered saline and incubated a t 25 "C a t 90-100 rpm in a gyra- tory shaker for l h (11). The in vitro binding assays utilized surfaces coated with purified preparations (5 &well) of fragments of the extracellular segment of PTPp which were adsorbed to 8-well chambers (Nunc, Naperville, IL) for 30 min, then remaining unbound sites were blocked with 1 mglml bovine serum albumin in phosphate-buffered saline. PTPp-linked Covaspheres (20 pl) were added to the dishes in a final volume of 300 pl of phosphate-buffered saline. The various protein- coated Covaspheres (20 pl) were tested for binding to the surface of subconfluent MvLu cells in multiwell chambers as described previously (11). For inhibition studies, the MvLu cells were incubated for 30 min with the appropriate antibodies to either the MAM domain (50 p1 of negative control or BK2 ascites) or the Ig domain (100 pl of preimmune or immune serum CSH 338) prior to the addition of 20 p1 of PTPp-linked Covaspheres.

RESULTS

PTPp Is Proteolytically Processed in Vivo-The juxtamembrane portion of the extracellular segment of PTPp, like other RPTPs, is characterized by a stretch of basic residues that would be consistent with a site of proteolytic cleavage. To in- vestigate whether PTPp is proteolytically processed, we im- muoprecipitated PTPp from MvLu cell lysates (Fig. l ,A and B). The immunoprecipitates were probed with antibodies either to the intracellular (Fig. LA) or extracelluar segment of PTPp (Fig. 1 B ) . Antibodies to the extracellular segment react with two bands, one corresponding to the full-length form (200 kDa)

Identification of the Homophilic Binding Site of PTPp 28474

A

0 0

B

116 -

87 - 67 -

m

1 2 3

C 55 - 36 -

V

1 2 3 FIG. 2. Expression of the various forms of PTF'p. Panel A is a

schematic diagram of the various mutants of FTPp in the order that they appear in Panels B and C. The stippled oval represents the MAM domain. The Ig domain is the half circle with the S-S bond. The hatched rectangles represent the FN 111 repeats. The stippled box on PTPpACD is the transmembrane region and 54 amino acids of the intracellular segment. Panel B is an immunoblot of lysates run on an 8 6 SDS-PAGE gel and probed with an antibody to the extracellular segment (BK2): lane 1 is FTPpACD, lane 2 is PTPp-extra, and lane 3 is MIF. Panel C is an immunoblot of lysates run on a 15% SDS-PAGE gel; lane 1 is MAM probed with antibody BK2, lane 2 is IgFN, and lane 3 is Ig which are probed with antibody CSH338.

and one corresponding to the E subunit (100 kDa). Antibodies to the intracellular segment of PTPp also react with two bands, one corresponding to the full-length form (200 kDa) and one corresponding to the P subunit (100 kDa). Upon immunopre- cipitation with antibodies to the intracellular segment of PTPp, the truncated extracellular segment (E subunit) is observed to coprecipitate. Similarly, antibodies to the extracellular fragment coprecipitate the P subunit of PTPp indicating that, fol- lowing proteolysis, the E and P subunits remain associated. The results presented above have been confirmed with both monoclonal and polyclonal antibodies to the extracellular segment (lanes 1 and 2) and the intracellular segment of PTPp (lanes 3 and 4 ) . The E and P subunit of PTPp have been observed in immunoblots of lysates from Wi38, A549, CHO, and 293 cells in addition to MvLu cells (data not shown). The cleavage of PTPp is represented schematically in Fig. 1C. The presence of the full-length and cleaved forms in multiple PTPp- expressing cell lines indicates that PTPp is proteolytically processed in cells in which it is normally expressed. This raises the issue of whether the E subunit alone can function in homophilic interactions and which sequence motif(s) within this subunit is responsible for binding.

Expression of the PTPp Fragments-Fragments were generated that encompass the different domains within the extracellular segment of the protein. A schematic diagram of these fragments is shown in Fig. 2A. Four fragments were expressed in recombinant baculovirus-infected Sf9 cells; PTPpACD, MAM, Ig, and IgFN. Two other forms were expressed in E. coli as GST fusion proteins, PTPp-extra and MIF. All forms were expressed and migrated at the expected apparent molecular weight upon SDS-PAGE, as confirmed by immunoblotting (Fig. 2, B and C). PTPpACD, which lacks the catalytic domain of

C

o n MAM domain

does not inhibit

lg domain inhibits binding

n

lgFN fragment

inhibits bindmg

.. . . -

inhibited by Ig domain-containing fragments of PTPp. Covas- FIG. 3. Aggregation of €"p-extra-linked Covaspheres is

pheres coated with the F'TPp-extra fragment were allowed to aggregate under low shear conditions in the presence of isolated MAM domain (Panel A ) or Ig domain-containing fragments (Panels B and C). Only the Ig domain-containing fragments Ig (Panel B ) and IgFN (Panel C) inhibited aggregation of the PTPp-extra coated Covaspheres.

PTPp, migrates at 110 kDa (Panel B, lane 1). PTPp-extra, which contains the entire extracellular segment of PTPp, migrates at 110 kDa (Panel B , lane Z) , while MIF, which contains the MAM domain, the Ig domain, and two FN I11 repeats migrates a t 72 kDa (Panel B, lane 3). MAM, which contains only the MAM domain of PTPp, migrates a t 19 kDa (Panel C, lane 1 ). IgFN, which contains the immunoglobulin domain and two FN I11 repeats of PTPp, migrates a t 27 kDa (Panel C, lane 21, while Ig, which contains only the immunoglobulin domain of PTPp, migrates a t 12 kDa (Panel C, lane 3 ) .

Immunoglobulin Domain-containing Fragments of PTPp Linked to Covaspheres Mediate Aggregation-In our previous study we demonstrated that PTPp-extra-coated fluorescent beads (Covaspheres) aggregated in suspension under low shear conditions (11). We have developed this line of study by adding the various fragments of PTPp to the PTPp-extra-coated Cova- spheres and testing their ability to inhibit the Covasphere aggregation. As shown in Fig. 3, the Ig domain-containing fragments, Ig and IgFN, prevented aggregation of PTPp-extra- coated Covaspheres whereas the MAM domain fragment did not. To confirm that the site responsible for aggregation was located in the immunoglobulin domain, the different fragments of PTPp were linked to Covaspheres indirectly via appropriate antibodies. The resulting protein-coated Covaspheres were used in several assays. First, the beads were tested for their ability to self-aggregate. As shown in Fig. 4A the Covaspheres coated with the MAM domain alone did not aggregate. In contrast, both types of Covaspheres coated with the Ig domain- containing fragments of PTPp aggregated (Fig. 4, B and C).

Identification of the Hornophilic Binding Site of PTPp 28475

, ' i

C m

'4 - .

MAM domain covaspheres

lg domain covaspheres

lgFN fragment covaspheres

FIG. 4. Aggregation of PTPp fragment-linked Covaspheres. Covaspheres coated with various fragments of PTPp were allowed to aggregate in suspension. The fluorescence micrographs illustrate that MAM domain-linked Covaspheres do not aggregate (Panel A ) whereas both Ig domain-linked (Panel B ) and IgFN-linked Covaspheres (Panel C ) form aggregates.

This indicates that in this assay the site responsible for aggregation resides in the Ig domain.

Only Zg Domain-containing Covaspheres Bind to PTPp- coated Surfaces-Since the fragments were linked to the beads via antibody, it is possible that binding to the antibody could affect the ability of FTPp fragments to function in homophilic interactions. Therefore, we utilized a different assay in which the fragment was not linked via antibody but was coupled to a surface directly. The various fragments of PTPp were purified, adsorbed to the surface of Petri dishes, and the ability of various Covas- pheres to bind to the purified protein-coated surface was then determined. Table I summarizes the results of these experiments in which full-length FTPp-coated Covaspheres were tested for their ability to bind to surfaces coated with different fragments of FTPp. The full-length PTPp-coated Covaspheres bound only to surfaces coated with the Ig domain-containing fragments, Ig and IgFN. This interaction was inhibited by preincubating the protein-coated surface with appropriate antibodies to PTPp. In a second set of experiments, summarized in Table 11, Covaspheres coated with various fragments of FTPp were tested for their ability to bind to surfaces coated with the extracellular segment of PTPp, i.e. PTPpACD, "extra, or MIF. Similarly, only Covas- pheres displaying fragments containing the Ig domain (Ig and IgFN) bound to PTPpACD, PPp-extra, and MIF (Table 11). These data show that the Ig domain Covaspheres bound equally well to proteins expressed in baculovirus (PTPpACD) or E. coli (FTPp- extra or MIF).

Covaspheres Coated with Ig Domain-containing Fragments Bind to the Surface of MvLu Cells-To verify that these results accurately reflect homophilic binding in vivo, Covaspheres

TABLE I Quantitation of the binding of PTPp Covaspheres to surfaces coated

with fragments of PTPp Binding assays in vitro utilized purified preparations of fragments of

the extracellular segment of PTPp including MAM, Ig, and IgFN (5 pg)

pheres (20 pl) were added to the PTPp fragment-coated surfaces. adsorbed to multiwell chambers. The full-length PTPp-linked Covas-

Protein coated to surface Addition No. bounda

MAM domain Control Ab 16 (24) MAM domain PTPp Ab 8 (fl) Ig domain Control Ab 379 ( d 9 ) Ig domain PTPp Ab 8 (el) IgFN fragment Control Ab 461 (279) IgFN fragment PTPp Ab 29 (23)

" This is the number of Covaspheres bound per field. The field is equal to 0.9 mm2. The number bound per field is the mean of at least four fields and the number in parentheses is the standard error of the mean.

TAHLE I1 Quantitation of the binding of Couaspheres coated with fragments of

PTPp to PTPp-coated surfaces

PTPp-extra, and MIF (5 pg) adsorbed to multiwell chambers. The var- Binding assays in vitro utilized purified preparations of PTPpACD,

ious PTPp fragment-linked Covaspheres (20 pl) were added to the protein-coated surfaces.

Covaspheres Protein coated to surface No. bound"

Full-length PTPpACD 426 (216) MAM domain PTPpACD 17 (?I) Ig domain PTPpACD 520 (229) IgFN fragment PTPpACD 417 ( ~ 2 7 ) Full-length PTPp-extra 522 (249) MAM domain PTPp-extra 19 (22) Ig domain PTPp-extra 552 (f7) IgFN fragment PTPp-extra 634 (213) Full-length MIF 421 (246) MAM domain MIF 18 (23) Ig domain MIF 578 (255) IgFN fragment MIF 433 (t21)

This is the number of Covaspheres bound per field. The field is equal to 0.9 mm2. The number bound per field is the mean of a t least four fields and the number in parentheses is the standard error of the mean.

coated with fragments of PTPp were tested for their ability to bind to PTPp expressed enodogenously on the surface of MvLu cells. These data are represented pictorially in Fig. 5 and quan- titated in Table 111. Confirming the results of our previous study (111, the full-length PTPp-coated Covaspheres bound to MvLu cells and this binding was inhibited by preincubating the cells with appropriate antibodies to PTPp (Table 111). The MAM domain-coated Covaspheres did not bind to MvLu cells and addition of anti-PTPp antibody did not affect their inability to bind (Fig. 5, A X ) . Both of the Ig domain-containing fragments, Ig (Fig. 5, D and E ) and IgFN (Fig. 5, G and H ) , bound equally well to the surface of MvLu cells. As shown in Panels F and I, preincubation of the cells with antibodies to PTPp inhibited the binding of both Ig and IgFN-coated Covaspheres. There did not appear to be any enhancement of homophilic binding by the presence of the FN I11 repeats (Table 111). Together, these results suggest that the Ig domain is both necessary and sufficient for homophilic binding in these assays.

DISCUSSION

Several of the transmembrane PTPs are members of the immunoglobulin superfamily and display structural motifs in their extracellular segments that are suggestive of a role in cell-cell adhesion (3, 4). The important finding that the ligand for a t least two receptor FTPs, PTPp and PTPK, is an identical molecule on an apposing cell, suggests that these molecules may transduce signals in response to cell contact. Interestingly, phosphacan, a chondroitin sulfate proteoglycan, which is proposed to be a splice variant of FTP< comprising the extracellu-

28476 Identification of the Homophilic Binding Site of PTPp

FIG. 5. Ig domain containing fragments of FTPp bind to the surface of MvLu cells. Panels A, D, and G represent phase contrast micrographs of monolayers of MvLu cells. Panels B , C , E , F , H , and I are fluorescence micrographs to indicate binding of coated Covaspheres on the surface of MvLu cells. The MAM domain-Covaspheres do not bind (Panel B ) , and preincubating the cells with antibodies to PTPp was without effect (Panel C). However, both PTPp constructs containing the Ig domain, Ig (Panel E) and IgFN (Panel H ) , bind to the surface of MvLu cells, and this interaction is inhibited by preincubating the cells with antibodies to PTPp (Panels F and I).

TABLE 111 Quantitation of the binding of Covaspheres coated with fragments of

PTPp to MvLu cells Various protein-coated Covaspheres were tested for their ability to

bind to the surface of MvLu cells. Covaspheres (20 111) were added in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum to subconfluent cultures of MvLu cells in multiwell chambers. Bound covaspheres were visualized with a Zeiss Microscope equipped for epifluorescence.

Covasphere Additions No. bound“

Full-length Control Ab 405 ( ~ 1 0 ) Full-length PTPp Ab MAM domain Control Ab

8 (?2)

MAM domain 7 (*1)

PTPp Ab 10 ( A 2 ) Ig domain Control Ab 641 (+25) Ig domain PTPp Ab 21 (23) IgFN fragment Control Ab 646 (213) IgFN fragment PTPp Ab 28 (*8)

This is the number of Covaspheres bound per field. The field is equal to 0.9 mm’. The number bound per field is the mean of a t least four fields and the number in parentheses is the standard error of the mean.

lar segment, has been shown to interact heterophilically with N-CAM, Ng-CAM, and tenascin (18, 19). These binding interactions raise the exciting possibility that triggering of a receptor PTP by cell contact may underlie the mechanisms of contact inhibition of cell growth. The advantage of the involvement of receptor PTPs in such a phenomenon is that their extracellular segments may “sense” directly cell-cell contact and transduce this into a signal through triggering the dephosphorylation of a defined subset of PTyr-containing proteins.

A common post-translational modification that has been observed in both cell-cell adhesion molecules such as neurofascin, Ng-CAMIL1 and BravoiNr-CAM (20-24) and RPTPs such as LAR and PTPK is cleavage a t basic sequences close to the ex- ternal face of the plasma membrane. Interestingly, mutation of the extracellular cleavage site in PTPK, so that i t could no longer be processed, had no effect on aggregation induced by

the expression of this PTP (16). We now demonstrate that a similar site found in PTPp also appears to be the proteolytically cleaved in MvLu and other cells which naturally express the protein (Fig. 1). Release of the extracellular segment of some adhesion molecules from the cell surface has been observed. For example, the 120-kDa form of N-CAM, which is linked to the membrane via a phosphatidylinositol anchor (GPI-linked), can be released by phosphatidylinositol-specific phospholipase C (25). In addition, the E subunit of LAR has been shown to be shed into the cell culture medium at high cell density (14). Although its structure is suggestive of involvement in homophilic binding interactions, the ligand for LAR is not known, and thus it is not clear how shedding of the E subunit may effect the function or activity of the phosphatase. In light of the structural similarity to LAR, one might predict that the extracellular segment (E subunit) of PTPp may be shed from cells a t high density, although this has yet to be verified. Since the E subunit contains the homophilic binding site, the potential shedding of this segment could generate a fragment of PTPp that would retain the capacity for homophilic binding but function in a context other than cell-cell contact. I t is also possible that proteolytic cleavage andor shedding of the E subunit may relieve a regulatory constraint on phosphatase activity medi- ated by the extracellular segment either directly or indirectly through control of localization. In this regard, a direct compar- ison of the PTP activity of the full length form of PTPp and a truncated soluble form, containing only the intracellular segment of PTPp, demonstrated that the latter had a higher intrinsic PTP activity (26).

It is important to resolve the issue of where the binding site is located in PTPp. Based on the fact that the MAM domain was an extracellular module, it was proposed that this domain has an “adhesive role” (8). There is no direct evidence that A5 or meprins function in cell-cell aggregation or homophilic binding and the function of the MAM domain in these proteins is not known. In our study, we did not detect a role for the MAM

Identification of the Hornophilic Binding Site of PTPp 28477

domain in homophilic binding of PTPp. However, we cannot rule out the possibility that the MAM or FN I11 repeats are involved in heterophilic binding or perform other roles that assist homophilic binding in uiuo, such as lateral association in the plane of the membrane.

This study highlights the importance of the Ig domain as both necessary and sufficient for homophilic binding interactions involving PTPp. Similarly, the homophilic binding site of N-CAM, the prototypical member of the Ig-superfamily of cell adhesion molecules, resides in an immunoglobulin domain. The extracellular segment of N-CAM contains five Ig domains and two FN I11 repeats (27) of which the first, second, and third Ig domains have been implicated in this process (28-30). Rao et al. (31) have reported that the peptide KYSFNYDGSE in the third Ig domain mediates homophilic binding. Based on these find- ings we would predict that the Ig domains of other type I1 RPTPs may be involved in binding interactions. Ig domain containing constructs such as reported here, which retain the binding capacity of the entire extracellular segment, may represent powerful probes with which to analyze the physiological significance of homophilic interactions between PTP molecules in developmental processes.

Acknowledgments-We would like to thank Peter Guida for technical assistance, Carmelita Bautista and Margaret Falkowski for help in generating monoclonal antibodies to PTPp, and Phil Renna and Jim Duffy for photography.

REFERENCES

2. I b n k s , N. K. (ed.) (1993) Semin. Cell Biol. 4, 373-453 1. Charbonneau, H., and Tonks, N. K. (1992) Annu. Reu. Cell Biol. 8,463-493

3. Streuli, M., Krueger, N., Tsai, A,, and Saito, H. (1989) Proc. Natl. Acad. Sci.

4. Fischer, E. H., Charbonneau, H., and Tonks, N. K. (1991) Science 263,401406 5. Streuli, M., Krueger, N., Hall, L., Schlossman, S., and Saito, H. (1988) J. Exp.

6. Brady-Kalnay, S. M., and Tonks, N. K. (1994) Adu. Protein Phosphatases 8,

U. S. A. 86,8698-8702

Med. 168, 1523-1530

241-274

7.

8. 9.

10.

11.

12.

13.

14.

15. 16.

17. 18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Gebbink, M., van Etten, I., Hateboer, G., Suijkerbuijk, R., Beijersbergen, R.,

Beckman, G., and Bark, P. (1993) 7bends Biochem. Sci. 18,4041 Takagi, S., Hirata, T., Agata, K., Mochii, M., Eguchi, G., and Fujisawa, H.

Jiang, W., Gorbea, C., Flannery, A., Beynon, R., Grant, G., and Bond, J. S.

Brady-Kalnay, S. M., Flint, A. J., and Tonks, N. K. (1993) J. Cell Biol. 122,

Gebbink, M., Zondag, G., Wubbolts, R., Beijersbergen, R., van Etten, I., and

Sap, J., Jiang, Y. P., Friedlander, D., Grumet, M., and Schlessinger, J. (1994)

Streuli, M., Krueger, N., Ariniello, P., Tang, M., Munro, J., Blattler, W., Adler,

Yu, Q., Lenardo, T., and Weinberg, R. A. (1992) Oncogene 7, 1051-1057 Jiang, Y. P., Wang, H., DEustachio, P. D., Musacchio, J. M., Schlessinger,

Guan, K., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267 Maurel, P., Rauch, U., Flad, M., Margolis, R. K., and Margolis, R. U. (1994)

Grumet, M., Milev, l?, Sakurai, T., Karthikeyan, L., Bourdon, M., Margolis, R.

Volkmer, H., Hassel, B., Wolff, J. M., Frank, R., and Rathjen, F. G. (1992) J.

Burgcan, M. P., Grumet, M., Mauro, V., Edelman, G. M., and Cunningham, B.

Faissner, A., Teplow, D. B., Kubler, D., Keilhauer, G., Kinzel., V., and

Kayyem, J. F., Roman, J. M., de la Rosa, E. J., Schwarz, U., and Dreyer, W. J.

Grumet, M., Mauro, V., Burgoan, M. P., Edelman, G. M., and Cunningham, B.

He, H. T., Barbet, J., Chaix, J. C., and Goridis, C. (1986) EMBO J. 5, 248%

Brady-Kalnay, S. M., and Tonks, N. K. (1993) Mol. Cell. Biochem. 1271128,

Brackenhury, R., Thiery, J., Rutishauser, U., and Edelman, G. (1977) J. Biol.

van Kessel, A,, and Moolenaar, W. (1991) FEES Lett. 290,123-130

(1991) Neuron 7,295307

(1992) J. Biol. Chem. 267,9185-9193

961-972

Moolenaar, W. H. (1993) J. Bwl. Chem. 268, 16101-16104

Mol. Cell. Biol. 14, 1-9

D., Disteche, C., and Saito, H. (1992) EMBO J. 11, 897-907

J., and Sap, J. (1993) Mol. Cell. Biol. 13, 2942-2951

Proc. Natl. Acad. Sei. U. S. A. 91, 2512-2516

K., and Margolis, R. U. (1994) J. Biol. Chem. 269, 12142-12146

Cell Biol. 118, 149-161

A. (1991) J. Cell Biol. 112, 1017-1029

Schachner, M. (1985) EMBO J. 4,3105-3113

(1992) J. Cell Biol. 118, 1259-1270

A. (1991) J. Cell Eiol. 113, 1399-1412

2494

131-141

Cole, G. J. Loewy, A., Cross, N. V., Akeson, R., and Glaser, L. (1986) J. Cell Biol.

Frei, T., von Bohlen und Halbach, F., and Wille, W., and Schachner, M. (1992)

Chem. 252,6835-6840

103, 1739-1744

J. Cell Biol. 118. 177-194 Zhou, H., Fuks, A,, kcaraz, G., Bolling, T. J., and Stanners, C. P. (1993) J. Cell

Rao, Y., Wu, X., Gariepy, J., Rutishauser, U., and Siu, C. (1992) J. Cell Biol. Biol. 122, 951-960

118,937-949

Documents

Identification of the homophilic binding site of the receptor protein