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• W2105470905 abstract "The receptor protein-tyrosine phosphatase μ (PTPμ) is a homophilic adhesion protein thought to regulate cell-cell adhesion in the vascular endothelium through dephosphorylation of cell junction proteins. In subconfluent cell cultures, PTPμ resides in an intracellular membrane pool; however, as culture density increases and cell contacts form, the phosphatase localizes to sites of cell-cell contact, and its expression level increases. These characteristics of PTPμ, which are consistent with a role in cell-cell adhesion, suggest that control of subcellular localization is an important mechanism to regulate the function of this phosphatase. To gain a better understanding of how PTPμ is regulated, we examined the importance of the conserved immunoglobulin domain, containing the homophilic binding site, in control of the localization of the enzyme. Deletion of the immunoglobulin domain impaired localization of PTPμ to the cell-cell contacts in endothelial and epithelial cells. In addition, deletion of the immunoglobulin domain affected the distribution of PTPμ in subconfluent endothelial cells when homophilic binding to another PTPμ molecule on an apposing cell was not possible, resulting in an accumulation of the mutant phosphatase at the cell surface with a concentration at the cell periphery in the region occupied by focal adhesions. This aberrant localization correlated with reduced survival and alterations in normal focal adhesion and cytoskeleton morphology. This study therefore illustrates the critical role of the immunoglobulin domain in regulation of the localization of PTPμ and the importance of such control for the maintenance of normal cell physiology. The receptor protein-tyrosine phosphatase μ (PTPμ) is a homophilic adhesion protein thought to regulate cell-cell adhesion in the vascular endothelium through dephosphorylation of cell junction proteins. In subconfluent cell cultures, PTPμ resides in an intracellular membrane pool; however, as culture density increases and cell contacts form, the phosphatase localizes to sites of cell-cell contact, and its expression level increases. These characteristics of PTPμ, which are consistent with a role in cell-cell adhesion, suggest that control of subcellular localization is an important mechanism to regulate the function of this phosphatase. To gain a better understanding of how PTPμ is regulated, we examined the importance of the conserved immunoglobulin domain, containing the homophilic binding site, in control of the localization of the enzyme. Deletion of the immunoglobulin domain impaired localization of PTPμ to the cell-cell contacts in endothelial and epithelial cells. In addition, deletion of the immunoglobulin domain affected the distribution of PTPμ in subconfluent endothelial cells when homophilic binding to another PTPμ molecule on an apposing cell was not possible, resulting in an accumulation of the mutant phosphatase at the cell surface with a concentration at the cell periphery in the region occupied by focal adhesions. This aberrant localization correlated with reduced survival and alterations in normal focal adhesion and cytoskeleton morphology. This study therefore illustrates the critical role of the immunoglobulin domain in regulation of the localization of PTPμ and the importance of such control for the maintenance of normal cell physiology. The physical interactions of individual cells with the local environment is critical to the control of their growth, differentiation, and fate within a multicellular organism. These responses are initiated by cell adhesion receptors, which bind either homophilic or heterophilic ligands on apposing cells or the extracellular matrix. A critical aspect in the cellular response to these interactions is reversible protein tyrosine phosphorylation of cell junction-associated proteins (1Zamir E. Geiger B. J. Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar, 2Daniel J.M. Reynolds A.B. BioEssays. 1997; 19: 883-891Crossref PubMed Scopus (285) Google Scholar). In fact, the bulk of the tyrosine-phosphorylated cellular proteins are localized to sites of cell-cell and cell-extracellular matrix adhesion. Several protein-tyrosine kinases and phosphatases have been implicated in the regulation of cell adhesion (3Garton A.J. Tonks N.K. J. Biol. Chem. 1999; 274: 3811-3818Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 4Harder K.W. Moller N.P. Peacock J.W. Jirik F.R. J. Biol. Chem. 1998; 273: 31890-31900Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 5Su J. Muranjan M. Sap J. Curr. Biol. 1999; 9: 505-511Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2166) Google Scholar). In particular, receptor protein-tyrosine phosphatases (RPTPs), 1The abbreviations used are: RPTPs, receptor protein-tyrosine phosphatases; PTP, protein-tyrosine phosphatase; BAEC, bovine aortic endothelial cells; trBAEC, transformed bovine aortic endothelial cells; MDCK, Madin-Darby canine kidney; BrdUrd, 5-bromo-2-deoxyuridine; WT, wild-type; Ig, immunoglobulin; MEM, minimum essential media; VE-cadherin, vascular endothelial cadherin. several of which possess extracellular domains with features characteristic of cell adhesion receptors, linked to a cytoplasmic PTP activity, are uniquely designed to mediate cellular responses to adhesive signals through protein tyrosine dephosphorylation (7Brady-Kalnay S.M. Tonks N.K. Curr. Opin. Cell Biol. 1995; 7: 650-657Crossref PubMed Scopus (188) Google Scholar, 8Beltran P.J. Bixby J.L. Front. Biosci. 2003; 8: D87-D99Crossref PubMed Google Scholar). Of particular interest is the potential for the extracellular domains of individual RPTPs to mediate interactions with, and control of, either cell-cell or cell-extracellular matrix adhesions. In this way, the targeting of RPTPs to specific junctions may be a key element in controlling the changes in cell junction-actin cytoskeleton interactions that occur as cells transition from a growing, migrating state, in which actin stress fibers are linked to cell-matrix junctions, to contact-inhibited cells, in which actin fibers are linked primarily to cell-cell junctions (9Larsen M. Tremblay M.L. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 700-711Crossref PubMed Scopus (109) Google Scholar). In this study, we focus on the receptor protein-tyrosine phosphatase PTPμ. PTPμ is synthesized as a single polypeptide chain, which is then glycosylated and proteolytically cleaved at a site N-terminal to the transmembrane segment to give two subunits that remain noncovalently associated (10Brady-Kalnay S.M. Tonks N.K. J. Biol. Chem. 1994; 269: 28472-28477Abstract Full Text PDF PubMed Google Scholar, 11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar). The E-subunit (see Fig. 1A), which constitutes the majority of the extracellular segment, consists of one immunoglobulin (Ig) domain; four fibronectin type III repeats; and a MAM (Mu, A5, and meprin homology) domain, which is a conserved domain unique to PTPμ, PTPκ, PTPρ, and PTPλ and some cell adhesion molecules (12Beckmann G. Bork P. Trends Biochem. Sci. 1993; 18: 40-41Abstract Full Text PDF PubMed Scopus (135) Google Scholar). The P-subunit (see Fig. 1A) contains a short extracellular segment; the transmembrane segment; and the entire cytoplasmic portion, including two PTP domains. PTPμ can function as a homophilic adhesion receptor whereby a PTPμ molecule on one cell can bind to an identical PTPμ molecule on an apposing cell (13Brady-Kalnay S.M. Flint A.J. Tonks N.K. J. Cell Biol. 1993; 122: 961-972Crossref PubMed Scopus (242) Google Scholar, 14Gebbink M.F. Zondag G.C. Wubbolts R.W. Beijersbergen R.L. van Etten I. Moolenaar W.H. J. Biol. Chem. 1993; 268: 16101-16104Abstract Full Text PDF PubMed Google Scholar). It is this binding that is proposed to direct the localization of PTPμ to sites of cell-cell contact, where it is thought to interact with the cadherin-catenin cell adhesion complex (11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar, 15Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar). Consistent with a role in cell-cell adhesion, the level of PTPμ protein increases as intercellular contacts are made and cell-cell junctions form (11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar). This phenomenon is seen with other RPTPs and may provide a means to control protein phosphotyrosine-based signaling in contact-inhibited cells (15Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar, 16Ostman A. Yang Q. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9680-9684Crossref PubMed Scopus (203) Google Scholar, 17Symons J.R. LeVea C.M. Mooney R.A. Biochem. J. 2002; 365: 513-519Crossref PubMed Scopus (23) Google Scholar, 18Gaits F. Li R.Y. Ragab A. Ragab-Thomas J.M. Chap H. Biochem. J. 1995; 311: 97-103Crossref PubMed Scopus (60) Google Scholar). PTPμ is expressed predominantly in vascular endothelial cells and to a lesser extent in bronchial epithelium and may play a role in endothelium-dependent processes such as the control of vascular permeability and the angiogenic responses to tumor formation and wound healing (19Bianchi C. Sellke F.W. Del Vecchio R.L. Tonks N.K. Neel B.G. Exp. Cell Res. 1999; 248: 329-338Crossref PubMed Scopus (37) Google Scholar). Considering the importance of PTPs in the control of signal transduction, it is critical that they be tightly regulated to facilitate protein tyrosine phosphorylation (20Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (463) Google Scholar). Based on studies with RPTPα, dimerization of RPTPs, which leads to occlusion of the active site, has been proposed as one mechanism for inhibition of phosphatase activity (21den Bilwes A.M. Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar, 22Majeti R. Bilwes A.M. Noel J.P. Hunter T. Weiss A. Science. 1998; 279: 88-91Crossref PubMed Scopus (219) Google Scholar, 23den Jiang G. Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (160) Google Scholar). However, it is not clear whether dimer-induced inhibition is widely applicable, as dimers formed in crystals of RPTP-LAR and RPTPμ are oriented such that the active sites are unobstructed (24Hoffmann K.M. Tonks N.K. Barford D. J. Biol. Chem. 1997; 272: 27505-27508Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 25Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). A number of studies indicate that the control of localization and substrate accessibility by intrinsic targeting domains is another important way to modulate cellular dephosphorylation by PTPs (20Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (463) Google Scholar) such as PTPμ. In contrast to the structurally related LAR-subtype RPTPs (LAR, PTPσ, and PTPδ), which can be found in both cell-matrix and cell-cell contacts (26Aicher B. Lerch M.M. Muller T. Schilling J. Ullrich A. J. Cell Biol. 1997; 138: 681-696Crossref PubMed Scopus (137) Google Scholar, 27Serra-Pages C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (293) Google Scholar, 28Wang J. Bixby J.L. Mol. Cell. Neurosci. 1999; 14: 370-384Crossref PubMed Scopus (91) Google Scholar), PTPμ is thought to function exclusively at sites of cell-cell adhesion, where it has the potential to regulate signaling events specific to contact-inhibited cells. In subconfluent cells, PTPμ is thought to cycle between the plasma membrane and membrane vesicles in the perinuclear region of the cell (11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar, 15Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar). Although it has been proposed that stable surface expression is dependent upon homophilic binding at cell-cell contacts (11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar), the mechanism whereby PTPμ localization is controlled has not been defined. Previous studies using purified fusion proteins indicate that the conserved Ig domain is necessary and sufficient for homophilic binding of purified PTPμ fusion proteins as well as binding of the fusion proteins to the surface of cells expressing endogenous levels of PTPμ (10Brady-Kalnay S.M. Tonks N.K. J. Biol. Chem. 1994; 269: 28472-28477Abstract Full Text PDF PubMed Google Scholar). Here, we have deleted the Ig domain of PTPμ and expressed the mutant protein in endothelial and epithelial cell lines to assess the role of this domain in regulating localization of the phosphatase. The results demonstrate the importance of the Ig domain for the localization of PTPμ to sites of cell-cell contact and also identify a role for this domain in localization of PTPμ in subconfluent cells in the absence of cell-cell contact. Cell Culture—Bovine aortic endothelial cells (BAEC) and the derived cell lines GM7372 and GM7373 (referred to here as transformed BAEC (trBAEC)) (29Grinspan J.B. Mueller S.N. Levine E.M. J. Cell. Physiol. 1983; 114: 328-338Crossref PubMed Scopus (84) Google Scholar) were obtained from the Coriell Institute for Medical Research (Camden, NJ). The cells were grown in basal medium (Sigma) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 1× MEM/essential and nonessential amino acid mixtures, 1× MEM/vitamin mixture, 2 mm l-glutamine, and 10 μg/ml gentamycin (Invitrogen). Madin-Darby canine kidney (MDCK) cells (catalog no. CCL-34) were purchased from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (Invitrogen) plus 10% fetal bovine serum, 2 mm l-glutamine, and 10 μg/ml gentamycin. Construction of Retroviral Expression Plasmids—Human PTPμ (GenBank™/EBI accession number NM_002845) DNA constructs were cloned with a C-terminal T7 epitope tag (30Lutz-Freyermuth C. Query C.C. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6393-6397Crossref PubMed Scopus (135) Google Scholar) into the retroviral vector pWZL-hygro (provided by S. Lowe, Cold Spring Harbor Laboratory) by PCR with pBSK/hFL (31Gebbink M.F. van Etten I. Hateboer G. Suijkerbuijk R. Geurts Beijersbergen R.L. van Kessel A.G. Moolenaar W.H. FEBS Lett. 1991; 290: 123-130Crossref PubMed Scopus (110) Google Scholar) as the template. pWZL/PTPμWT contains nucleotides 1–4359 of human PTPμ; pWZL/PTPμΔIg and pWZL/PTPμΔE have deletions of nucleotides 568–930 and 67–1914, respectively, in human PTPμ. The resulting constructs encoded proteins with a C-terminal extension of AGMASMTGGQQMG, where the underlined amino acids define the T7 epitope tag. All constructs derived from PCR were confirmed by DNA sequencing. Expression of PTPμ by Retroviral Infection—LiNX-A retroviral packaging cells (32Wang J. Xie L.Y. Allan S. Beach D. Hannon G.J. Genes Dev. 1998; 12: 1769-1774Crossref PubMed Scopus (574) Google Scholar) were transfected with pWZL/PTPμ plasmid DNA using calcium phosphate. The resulting supernatants, containing recombinant retrovirus encoding PTPμ, were used to infect monolayer cultures of GM7372 cells, trBAEC, or MDCK cells. Two days following infection, cells were placed in the appropriate medium supplemented with 100 μg/ml (GM7372 cells) or 400 μg/ml (trBAEC and MDCK cells) hygromycin B (Invitrogen). The medium was changed every 2–3 days, and cultures were passaged when they reached ∼70–90% confluence. Antibodies—The following primary antibodies were used in this study: anti-T7 epitope antibody (mouse IgG2b; Novagen); anti-VE-cadherin antibody 45C6 (mouse IgG1; ICOS, Bothell, WA); anti-vinculin antibody VIN-11-5 (mouse IgG1; Sigma); anti-phosphotyrosine antibody G104 (mouse IgG1) (33Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar); anti-paxillin antibody (mouse IgG1; Transduction Laboratories); and the PTPμ-specific antibodies SK7 (mouse IgG1), BK2 (mouse IgG2a), and BK3 (mouse IgG1) (15Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar). Indirect Immunofluorescence—Cells were plated on glass coverslips, grown to the desired density, and fixed with 2% paraformaldehyde in phosphate-buffered saline for 12.5 min. Cells were either permeabilized in phosphate-buffered saline, 3% normal goat serum, and 0.2% Triton X-100 for 20 min or left intact as indicated. Fixed samples were incubated either with single primary antibodies or, for co-staining, with two antibodies of different isotypes. The primary antibodies were detected with isotype-specific secondary antibodies (Molecular Probes, Inc., Eugene, OR) to mouse IgG1 (labeled with Alexa 594) or to mouse IgG2b or IgG2a (labeled with Alexa 488). Co-staining with antibodies SK7 and 45C6, both of which are IgG1 isotype, was performed as follows. Fixed cells were first incubated with antibody SK7, followed by a 20-fold excess of Fab fragments of rabbit anti-mouse IgG and then Texas Red-labeled anti-rabbit IgG. Then the cells were incubated with antibody 45C6, followed by fluorescein isothiocyanate-labeled rabbit anti-mouse IgG. A control sample in which antibody 45C6 was omitted did not show staining with the fluorescein isothiocyanate-labeled anti-mouse IgG, indicating that the excess, Fab fragment rabbit anti-mouse IgG, effectively blocked antibody SK7 from reaction with this secondary antibody. Coverslips were mounted on glass slides using ProLong anti-fade reagent (Molecular Probes, Inc.) and examined by epifluorescence using an Axiophot 2 microscope and digital imaging system with a ×63 objective lens (Carl Zeiss Microimaging, Thornwood, NY) unless indicated otherwise. Images were processed using OpenLab software (Improvision Software, Lexington, MA). Confocal images were obtained with a Zeiss LSM510 confocal microscopy system using a ×100 objective lens and processed with built-in software and ImageJ software (National Institute of Mental Health, available at rsb.info.nih.gov/ij/). Immunoblotting and Immunoprecipitation—GM7372 cells were grown to the desired density and solubilized in lysis buffer (30 mm HEPES (pH 7.5), 150 mm NaCl, 1% Triton X-100, 8% glycerol, 1 mm EDTA, 1 mm benzamidine, and 5 μg/ml each aprotinin and leupeptin) for 30 min. Lysates were centrifuged at 10,000 × g for 15 min, and the resulting supernatants were assayed for protein concentration with Bradford reagent using bovine serum albumin as the standard. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with anti-PTPμ monoclonal antibodies SK7 and BK2 as described (15Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar). For immunoprecipitations, equal amounts of protein from GM7372 cell lysates, prepared as described above, were incubated with anti-T7 epitope antibody-conjugated agarose (Novagen) for2hat4°C. Samples were washed four times in lysis buffer, and bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting. Assay of Cell Proliferation and Apoptosis—Proliferation rates of cell lines were assayed by measuring incorporation of 5-bromo-2-deoxyuridine (BrdUrd). Cells were plated on glass coverslips at a density of 4 × 103 cells/cm2, and 36 h later, 20 μm BrdUrd was added for 40 min. Cells were stained for BrdUrd incorporation using fluorescein isothiocyanate-conjugated anti-BrdUrd antibody (Roche Applied Science) according to the manufacturer's protocol. Samples were co-stained with Hoechst 33342 (Molecular Probes, Inc.) to visualize nuclei of all cells and imaged by epifluorescence using a ×10 objective. Total and BrdUrd-positive cells were counted in images using ImageJ software, and the percentage of BrdUrd-positive cells was calculated. To assay for apoptosis, cells were plated on glass coverslips at 4 × 103 cells/cm2 and incubated at 37 °C for 36 h. Cells were fixed as described above for immunofluorescence and stained with Hoechst 33342 according to the supplier's protocol. Cells displaying condensed chromatin and the formation of nuclear blebs containing DNA were counted as apoptotic. The percentage of apoptotic cells in six representative fields of each sample was calculated as described above for the assay of BrdUrd incorporation. Generation of Stable Lines of GM7372 Cells Expressing PTPμ Deletion Constructs—As an initial step in understanding the mechanisms involved in regulating PTPμ function in endothelial cells, we expressed various mutant constructs in GM7372 cells, a cell line derived from BAEC. GM7372 cells have similar morphology and growth properties compared with primary BAEC and express endothelium-specific cell junction proteins (Fig. 1) (data not shown) (29Grinspan J.B. Mueller S.N. Levine E.M. J. Cell. Physiol. 1983; 114: 328-338Crossref PubMed Scopus (84) Google Scholar). GM7372 cells expressed a similar level of endogenous PTPμ compared with the parental BAEC (Fig. 1B). In agreement with previous findings (15Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar, 19Bianchi C. Sellke F.W. Del Vecchio R.L. Tonks N.K. Neel B.G. Exp. Cell Res. 1999; 248: 329-338Crossref PubMed Scopus (37) Google Scholar), PTPμ was observed as a full-length ∼200 kDa protein (Fig. 1B, EP) and as processed proteolytic fragments of ∼110 kDa for the E-subunit (Fig. 1B, E) and P-subunit (data not shown). As described previously (19Bianchi C. Sellke F.W. Del Vecchio R.L. Tonks N.K. Neel B.G. Exp. Cell Res. 1999; 248: 329-338Crossref PubMed Scopus (37) Google Scholar), at high cell density, PTPμ co-localized at cell-cell contacts with the endothelial-specific VE-cadherin, and the level of PTPμ protein was increased (Fig. 1, B and C). In subconfluent cells, PTPμ was found in a perinuclear vesicular compartment (Fig. 1C). Ectopic expression of PTPμWT, PTPμΔIg (deletion of amino acids 190–310), and PTPμΔE (deletion of amino acids 23–638) was achieved by infection of GM7372 monolayers with recombinant retrovirus encoding the T7 epitope-tagged PTPμ constructs. We utilized a vector, pWZL, containing an internal ribosomal entry site, from which PTPμ and the selection marker are separately translated from the same mRNA. Stable cells were selected in hygromycin as pooled populations from an ∼90% confluent monolayer of infected GM7372 cells. Attempts to isolate clonal cell lines of GM7372 expressing PTPμ from dilute cultures were unsuccessful. We reasoned that any inhibitory effects on growth or survival caused by ectopic expression of PTPμ might be more pronounced at low cell density, when the normal level of PTPμ is low, than at high cell density, when expression of the phosphatase is naturally up-regulated. The use of the pWZL vector resulted in a tight correlation between hygromycin resistance and ectopic expression of PTPμ, producing cell populations in which 70–80% of the cells expressed detectable amounts of the specific PTPμ mutant, as determined by detection with anti-PTPμ antibodies (data not shown). Immunoblotting of cell lysates with antibodies specific to the P-subunit (SK7) and the E-subunit (BK2) indicated similar levels of expression from each construct (Fig. 2). Furthermore, the wild-type EP protein migrated as a doublet on SDS-polyacrylamide gel, with the slower migrating band, previously shown to represent the glycosylated form (11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar), predominating. In contrast, the hypoglycosylated form of PTPμΔIg predominated, which would contribute to the faster than expected migration of the E-subunit of this mutant on SDS-polyacrylamide gel. In addition, immunoprecipitation of the P-subunit of PTPμΔIg with the antibody to the C-terminal T7 epitope followed by immunoblotting of the E-subunit with antibody BK3 demonstrated that the two intact segments of the processed protein were associated in the cell, as seen with the WT protein (Fig. 2). The Ig Domain Is the Major Determinant of Localization of PTPμ to Sites of Cell-Cell Contact—It has been proposed that homophilic binding may be responsible for localization of PTPμ to cell-cell contacts (10Brady-Kalnay S.M. Tonks N.K. J. Biol. Chem. 1994; 269: 28472-28477Abstract Full Text PDF PubMed Google Scholar, 11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar). To test this idea in a cellular context, GM7372 cells expressing T7 epitope-tagged PTPμ mutants were analyzed by indirect immunofluorescence with the anti-T7 epitope antibody to determine the subcellular localization of the phosphatase. Full-length PTPμ-WTT7 localized to cell-cell contacts as indicated by co-localization with VE-cadherin (Fig. 3, WT panels). Deletion of the Ig domain, containing the homophilic binding site, greatly reduced the localization of PTPμ to cell-cell contacts (Fig. 3, ΔIg panels), resulting in a dispersed pattern consistent with expression at the cell surface. Therefore, the Ig domain, which contains the homophilic binding site (10Brady-Kalnay S.M. Tonks N.K. J. Biol. Chem. 1994; 269: 28472-28477Abstract Full Text PDF PubMed Google Scholar), is a major determinant for localization to cell-cell contacts, suggesting that homophilic binding is important for this localization. Deletion of the entire E-subunit, containing most of the extracellular domain, completely eliminated localization of PTPμ to cell-cell contacts (Fig. 3, ΔE panels), suggesting that this region may contain sequences in addition to the Ig domain that play a part in this localization. A role has been proposed for the MAM domain in homophilic binding (11Gebbink M.F. Zondag G.C. Koningstein G.M. Feiken E. Wubbolts R.W. Moolenaar W.H. J. Cell Biol. 1995; 131: 251-260Crossref PubMed Scopus (119) Google Scholar); however, we were unable to determine whether the MAM domain may contribute to the localization of PTPμ because constructs with a deletion of this domain could not be stably expressed at levels comparable with the other mutants (data not shown). This is consistent with studies of the metalloprotease meprin A, which have suggested that the MAM domain may be critical for protein stability (34Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To verify that the localization of PTPμΔIg in GM7372 cells is a property of the phosphatase and not a phenomenon unique to these cells, we examined the localization the PTPμ deletion constructs in two additional cell types, a transformed endothelial cell line trBAEC and MDCK epithelial cells, the latter being a well established model for studying cell-cell adhesion. In trBAEC and MDCK cells, as seen in GM7372 cells, deletion of the Ig domain resulted in reduced localization to cell-cell contacts and a more dispersed distribution over the cell compared with the WT protein (Fig. 4). These results confirm that the Ig domain is a major determinant for the localization of PTPμ to cell-cell contacts in multiple cell types. The PTPμΔIg Protein Is Present at the Cell Surface—As described above, deletion of the Ig domain led to a widespread distribution of PTPμ over what appeared to be the cell surface. To confirm that the PTPμΔIg protein was present at the cell surface, we used the extracellular domain-specific antibody BK2 to stain GM7372 cells that had not been permeabilized prior to antibody staining. PTPμWT was detected primarily at cell-cell contacts in intact non-permeabilized cells, whereas PTPμΔIg was dispersed over the surface of the cell (Fig. 5). Staining of permeabilized cells revealed additional localization of both PTPμWT and PTPμΔIg in a perinuclear region (Fig. 5) as described for the endogenous protein (Fig. 1). We were unable to determine whether PTPμΔE can reach the cell surface, as it did not react with the available antibodies directed against the extracellular region of PTPμ. To analyze the localization of the mutant PTPμ proteins in further detail, we examined MDCK cells, which, in contrast to GM7372 endothelial cells, form a monolayer in culture with sufficient depth to visualize cell morphology in three dimensions using confocal microscopy. Cross-section imaging of MDCK cells expressing full-length PTPμWT and PTPμWTΔD2, a mutant lacking the membrane distal catalytic domain, stained with an antibody directed against the P-subunit of PTPμ, indicated that these proteins, which contain an intact E-subunit, were found concentrated along most of the lateral cell-cell contacts (Fig. 6). Co-staining with antibodies to the tight junction protein ZO-1, a marker for the apical junction, showed that PTPμWT was absent from the apical terminal portion of the lateral surface (data not shown). In contrast, the PTPμΔIg protein was seen in the lateral junction and on the apical and basal surfaces" @default.
• W2105470905 created "2016-06-24" @default.
• W2105470905 creator A5005973308 @default.
• W2105470905 creator A5018714844 @default.
• W2105470905 date "2005-01-01" @default.
• W2105470905 modified "2023-10-17" @default.
• W2105470905 title "The Conserved Immunoglobulin Domain Controls the Subcellular Localization of the Homophilic Adhesion Receptor Protein-tyrosine Phosphatase μ" @default.
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