PROTEINS INVOLVED IN THE REGULATION OF CELL-CELL ADHESION AND USES THEREOF

Abstract: A p100 protein, which has a molecular weight of about 100 kDa and appears to be related to p120, is involved in the regulation of cell-cell adhesion.

Description: PROTEINSINVOLVEDINTHEREGULATIONOFCELL-CELLADHESIONANDUSESTHEREOF The present invention relates to certain proteins which may be involved in regulating physiological changes (e.g. changes in cell-cell adhesion) and to uses thereof. Cell aαhesion is important for a wide variety of regulatory and developmental processes. The cadheπns comprise a family of transmembrane. ceil surface giycoproteins that meαiate Ca :+ -αependent ceil-cell adhesion in a homorypic manner tTakeicru. 1991). In ceiis with weil developed intercellular junctions, the caαhenns are localized to tne aαherens juncnon (Boiler et al., 1985) but appear to influence other mterceiiuiar lunctions such as gap junctions ( atsuzaki et ai.. 1990; lusii et al.. 1990) and tight junctions (Gumbiner and Simons. 1986; GαmDiner et ai.. 1988). The adherens junction aiso plays a cruαal role m tne development ana maintenance of ceil poiaπty (see Nelson, 1992) and its dysfuncπon has been strongiy implicated m the invasiveness and carαnogenesis of tumour ceils i see e g. Behrens et al.. 1989; Fnxen et al., 1991; Vleminckx et al., 1991; Shimoyama et al.. 1992. znά Kedncx et ai.. 1993; Tsukita et al., 1993; BircT neier and Behrens. 1994) The conserveα vtoπasmic comain or cadheπns is known to associate with trree rrotems. termeα a-. JJ- ana γ-caterun (Ozawa et al., 1989), which serve to link caαhenns ro tne ac::n--?aseα cortical cvtosκeleton i Hirano et al., 1987 ) . The association or cααnenns witn ca.eruns is essential for intercellular Ca :+ -dependent adhesiveness ( Xaearucru and Takeichi. 1988; Ozawa et al., 1990; Kintner, 1992 ) . α- catenin is homologous to vmcu n i Herre knecht et al.. 1991; Nagafuchi et al., 1991), maiαns it a cooα canαidate ror interaction with the achn-baseα cvtoskeleton (see Ozawa et al.. 1990. Hirano et ai., 1992). β-catenin is homologous to the Drosoprula segment poianty gene armadillo, suggesting a role in developmental signalling in vertebrates (McCrea et ai.. 1991). γ-catenin is probably identical to plakogiobin (Knudsen and Wheeiock. 1992; but see Piepenhagen and Nelson, 1993), which again is homologous to armadillo (see Franke et ai., 1989; Peifer and Wieschaus, 1990 ) . Indeed, 3-catenin and plakogiobin appear to form a multigene family (Peifer et al.. 1992). A repeating 42 amino acid motif that was originally identified in armadillo (Riggieman et al., I9S9) has also been found in several other proteins, including β- caterun and plakogiobin. with a variety oi functions (Peifer et al., 1994). These include the APC gene product, a rumour suppressor protein (Kinzler et al., 1991), pl20, a ppόO sπ: substrate (Reynolds et ai.. 1992), smgGDS, an exchange factor for ras-reiated G proteins ( ' Kikuchi et ai., 1992), a suppressor of RNA poiymerase 1 mutations in yeast (Yano et al., 1992; 1994) and band 6 protein, a major desmosomai constituent (Hatzfeid et ai., 1994). The function of the repeats in these arm proteins is unknown. Interestingly, the APC gene product associates with β- catenin iRubinfeld et ai., 1993; Su et al., 1993), supporting an impoπant role for cateruns in intracellular processes that reguiate cell growth. Furthermore, this illustrates that cacherins are not exclusive ceilular partners of catenins, raising the possibility of other interactions among catenin≤, cadherins and arm proteins, important in a variety of bioiogicai processes. pl20 was initially identified as one of several substrates of the tyrosine kinase r όO 5 ^ (Reynolds et ai.. 19S9; Kanner et ai., 1990). It is membrane- associated and can be myπsroviated, but does not appear to be glycosyiated ( Kanner et al., 1991 ). λlutaπonai anaivsis suggested that tyrosine phosphorylation oi pl20 is necessarv ror ox rrsu- s"rc -mediated celluiar transformation i Under and Burr, 1988; Reynolds et al . , 1989) . Tyrosine phosphorylation of pl20 was also observed in response to epidermal growth factor, platelet-derived growth factor, colony-stimulating factor 1 and in polyoma virus middle T antigen-transformed cells (Dowing and Reynolds, 1991; Kanner et al . , 1991), but the exact role of pl20 in cellular physiology and pathology was not clear. According to the present invention, there is provided a protein having a molecular weight of about 100 kDa which is associated in vivo with the cadherin/catenin complex of epithelial or endothelial cells (e.g. by binding) . This protein is referred to herein as "plOO". plOO is immunologically related to pl20 by virtue of cross-reactivity of antibody to pl20 with plOO. Partial amino acid sequence data of human plOO is given in Fig. 9 where it is compared with sequence data already available for mouse pl20. The present invention covers the specific protein of molecular weight of about 100 kDa described in the present examples and other proteins having substantial amino-acid homology therewith (excluding pl20, and optionally also excluding proteins having greater amino acid homology with pl20 than with plOO) . Human plOO is particularly preferred. The term "substantial amino acid homology" is used herein to cover proteins having at least 50%, preferably more than 90% or more than 95% amino acid homology with another protein. The present invention also covers fragments of these proteins or of plOO itself. Preferably these fragments are at least twenty amino acids long, more preferably they are at least fifty or at least one hundred amino acids long. These proteins or fragments may be partially or totally tyrosine phosphorylated. They may be provided in glycosylated or non-glycosylated form. Desirably they are provided in substantially pure form. One definition of "substantially pure form" is a form which is substantially free of other proteins. The present invention also includes nucleic acid sequences (preferably DNA sequences) coding for the above mentioned proteins or fragments. These sequences may be in isolated form. They may be incorporated as part of a recombinant nucleic acid molecule e.g. as part of a vector. The vector may be incorporated into a host cell and used for expression of plOO. Nucleic acid sequences complementary to the aforesaid sequences may be useful in antisense studies to alter the expression of gene products. Such sequences are therefore also within the scope of the present invention. In view of the data provided herein, it is believed that the protein plOO may be involved in the regulation of cell tight junction permeability. Phosphorylation of one or more tyrosine residues of plOO may be involved in increasing tight junction permeability and dephosphorylation of one or more tyrosine residues may be involved in decreasing tight junction permeability. The above comments also apply in respect of pl20, which the present inventors have shown to be immunologically closely related to plOO. plOO and pl20 are therefore useful for studying cell-cell adhesion generally and tight junction permeability in particular. They may therefore be used, for example, to investigate the effect of tyrosine kinase and/or of tyrosine phosphatase on tight junction permeability. Variants of these proteins may be prepared in order to investigate which regions of plOO or pl20 are important in regulating tight junction permeability. Our data indicate that pl20 and plOO associate with the cadherin/catenin complex. Furthermore, since pl20 and plOO are substrates for tyrosine kinases, it follows that pl20 and plOO proteins per se and the phosphorylation of these proteins may influence cellular functions directly, and indirectly, regulated by cadherins and catenins. Such an influence could be mediated by the physical association between pl20, plOO and the cadherin/catenin complex, in which case modulation could be achieved by enhancing or blocking the expression of pl20 and plOO. Disruption of the association could also be achieved, for example, by small molecule mimetics of the site of binding. As indicated above, the function of pl20 and pi00 may also be regulated by their tyrosine phosphorylation, in which case appropriate modulation of kinases and phosphatases could modulate the function of cadherins and other cadherin-dependent processes. plOO and pl20 may be of utility in studying tumour cell invasion and metastasis, breast cancer, colorectal cancer, gastric carcinoma, gynaecological cancer, lung cancer, oesophageal cancer, prostate cancer, scirrhous cancer, tissue regeneration, for example. Such studies could lead to diagnostic methods or treatments of these disorders. The Drosophila gene product armadillo is homologous to β-catenin and has been strongly implicated in developmental processes. It is therefore possible that pl20 and plOO, as 3-catenin-related proteins that interact with cadherins, may also be involved in developmental defects and abnormalities. The present invention also provides an agent which alters or blocks association between plOO or pl20 with the catenin/cadherin complex of epithelial or endothelial cells for use in the treatment or diagnosis of a disease. The disease may be tumour cell invasion and metastasis, breast cancer, colorectal cancer, gastric carcinoma, gynaecological cancer, lung cancer, oesophageal cancer, prostate cancer, scirrhous cancer, tissue regeneration. The agent may be used for the preparation of a medicament and may be used with a pharmaceutically acceptable carrier. The present invention will now be described by way of Example only with reference to the accompanying drawings , which are briefly described below: - Figure 1. Characterization or " antι-pi20 and 2B12 immunoreacrivity. Various ceil lines and a primary culture of bovine brain entiotheϋal ceils (Brain EC) were ivseα in 5DS sampie buffer and analyzed by SDS-PAGΞ followed by lrnmunoblortine with anπ-pl20 antibody iPanei A) or the 2B12 antibody (Panel B). The exposure times were 1 minute for Panel A (apart from that for the MDBK ceils which was 10 seconds ) and 13 mmures for Panei 3. The migration of pl20 (o) and plOO (•) has been indicated. Figure 2. pl20 protein is recognized by the anri-pl20 antibody and cross-reacts with the 2B12 anπbotiv, whereas plOO is recognized oniy by anti-pl20. MDBK cells were iyseti in TDS burfεr. Immunoprecipitations were performed using the anti-pl20 anubocv cr 2512 foiloweα by cross-blottmg (Panel A). MDBK ceils were chosen because p:2 and lOO are well separated bv SDS-PAGE and react with both annboαies. Ir. Fzr.ei ' 3. MDBK ceils were iabeiled with 3 S]methionme. iyseα in TDS buffer ana then immunopreciritated usins antι-pl20 or 2B12. Proteins were separated bv ITS-PAGE ana detected bv auroradiography. Cleariy, the broad bands ( Panei A and see rig. 1 A) corresponαing to pl20 and plOO as detected by lmm r.ociotnn.: are resoiveά as multiple bands. The same bands as seen in the 2312 im unoprecipitate ι o) are seen m the anti-pl20 immuno- precipitates. In me antι-pi22 immunoprecipitates. additional bands ( • ) corresponαinε to riϋO are aiso observed. Figure 3. Comparison of anπ-pl20 and anti-E-cadherin immunoprecipitates from MDCK and Caco-2 ceils. Panel A: MDCK ceils were labelled with [ 35 S]methionine and then iysed in either TX buffer or TDS buffer. Immunoprecipitations were performed using anti-pl20 or anti-E-cadherin (rrl). Proteins were separated by SDS-PAGE followed by fluorography. Bands corresponding to E-cadherin (E), α- (α) and 3-catenin (β) which are seen strongiy in the rrl immunoprecipitates have been indicated. The major anti-pl20 reactive band migrates at approximately the position of that of plOO (see Fig. 1 A). 2B12 does not appear to react with canine protein (see Fig. 1 B) making positive identification of pl20 difficult. Panel B: Caco-2 cells were labelled with 3 S]methionine and then lysed in TX buffer. Immunoprecipitations were performed using anti-E-cadherin (HECD-1), anti-β- catenin or anti-pl20 antibodies. Proteins were separated by SDS-PAGE followed by fluorography. In ail cases, four major comigrating bands were immunoprecipitated. corresponding in order or " increasing mobility to E-cadherin, a-, β- and γ-caterun. Figure 4. Detection of anti-pi20 reactive material in anti-E-cadheπn immuno¬ precipitates and E-cadherin in the antι-pl20 immunoprecipitates. MDCK cells were iysed in TX buffer. Immunoprecipitations were performed with either antibody to E-cadherin (rrl), the anti-pl20 antibody or anti-focal adhesion kinase (anti-FAK). A sham lmmunoprecipitation was performed in the absence of primary antibody (-). Following separation by SDS-PAGE, parallel blots were probed using: rrl (arrowhead, E-cadheπn) ; anti- β-catenin (arrowhead, β-catenin); anti-pi20 (arrowhead, anπ-pl20 reactivity); anti-FAK (arrowhead, FAK). Cleariy, rrl and the anti-pl20 antibody immunoprecipitated E-cadherin and β-catenin. rrl could also immunoprecipitate anti-pl20 immunoreactive material, but to a lesser extent than that immunoprecipitated by the anti-pl20 antibody. Anti-FAK could only immunoprecipitate FAK, and the sham immunoprecipitation did not result in any detectable E-cadheπn. β-catenin or anti-pl20 reactive material. Figure 5. Comparison oi antι-3-catenιn. antι-pl20 and 2B12 immunoprecipitates from primary cultures of bovine brain endothelial cells. Cells were labelled with [ 35 S]methiorune and then iysed in TX buffer or TDS buffer. Immunoprecipitations were performed using anri-3-caterun, anti-pl20 and 2B12. Proteins were separated by SDS-PAGE followed by fluorography. In the β-catenin immunoprecipitation from TX Iysed ceils, bands corresponding to a cadherin (C), α-catenin (a) and β- catenin (β) have been indicated. Under TDS conditions, a band (o) corresponding to pl20 is seen in the antι-pl20 and 2B12 immunoprecipitates and a further band ( •) is only seen in the anti- l20 immunoprecipitate, corresponding to plOO. Figure 6. Detection of pl20/pl00 in β -catenin immunoprecipitates and β-catenin in anti-pl20 immunoprecipitates from human umbilical vein endothelial cells (Panel A) and ECV304 ceils (Panel B). Cells were iysed in either TX or TDS buffer. ysates were immunoprecipitated using anti-β-catenin or the anti-pl20 antibody, followed by analysis by SDS-PAGE and immunoblotting. The migration of β- catenin (arrowheads), plOO (•) and pl20 (o) have been indicated. Note the absence of anti-pl20 reactive material in the β-catenin immunoprecipitates obtained using TDS buffer. Even though β-catenin and plOO, as defined by its reactivity with anti- pl20 antibody, migrate very closely, they are clearly distinct proteins. Figure 7. Localization of anti-pl20 reactivity and β-catenin in MDCK cells, and brain endotheiial ceils. MDCK ceils (Panel a,b) and porcine brain endothelial cells (Panel c.d) were co-iabelled with anti-pl20 antibody (Panel a,c) and anti-β-catenin antibody (Panel b.d). Secondary antibodies were fluorescein-conjugated anti- mouse and rhodamme-conjugated anti-rabbit. In this instance, it was verified that each secondarv antibody was absolutelv specific for its designated species of primarv antibodv. Bar: 20 urn. Fig. 8. Distribution of anti-pl20 immunoreactivity in brain and skeletal muscle tissue of the rat. Brain tissue was co-labeiled with the anti-pl20 antibody (a,c) and anti-α-catenin (b,d). Anti-pl20 immunoreactivity and α-catenin co-localise at intercellular junctions of choroid plexus epithelium (arrows, a and b) and ventricular ependymai ceils (arrowheads, a and b). Both antigens also co-locaiise at interendothelial junctions of blood vessels of macrovascular origin (arrows in c and d). Microvascuiar profiles in brain sections were identified by labelling with anti-coilagen TV antibody (f); co-labeiling with the anti-pl20 antibody (e), revealed the presence of antigen at interendothelial junctions (e, arrows). In these microvessels. α-catenin co-locaiised with anti-pl20 immunoreactivity (not shown). In muscle tissue which had been cut perpendicular to the orientation of the muscle fibres. anti-pl20 immunoreactivity is limited to areas between muscle fibres where blood vessels are located (g, arrows). Higher magnification reveals a punctate staining pattern (h, arrows) which is likely to reflect anti-pl20 immunoreactivity at interendothelial junctions. In Panels a,c and e, bp depicts the brain parenchyma; in Panei a. ' the ventricular lumen and cp the choroid plexus; in Panel g, m refers to muscie tissue. Bars: (.., g), 100 μm; (c, e, h), IS μm. H .9. Sequence analysis of peptides derived by LysC proteolysis of plOO from Caco-2 cells. Caco-2 cells (20 x confluent 9 cm dishes) were Iysed in TX buffer to minimize nuclear lysis. Insoluble protein was removed by centrifugation. Deoxycholate and SDS were added to the supernatant to give (w/v) 0.5 % and 0.2 % final concentration, respectively. Addition of these detergents results in dissociation of plOO and pl20 from the cadherin/catenin complex. pl20 and plOO were immunoprecipitated from the lysate using the anti-pl20 antibody (which also recognizes plOO) from Transduction Laboratories, rabbit anti-mouse IgG and Protein A Sepharose. The immune complex was washed five times and then dissociated by the addition of Laemmli sample buffer followed by heating at 100°C for 5 minutes. Proteins were precipitated by addition of four volumes of ethanol and incubation at -20°C for 16 hours. The precipitate was resolved by SDS-PAGE (6 % acryiamide) and proteins were visualized by Coomassie Blue. Protein corresponding to plOO was excised from the gel and digested with LysC. Peptides were separated by HPLC and sequenced. Mouse pl20 sequence was described by Reynolds et al., 1992. Clearly, human plOO is closely related to mouse pl20. In this figure the standard single letter amino acid code is used. Additionally "X" is used to designate any amino acid. In the Examples given later the following materials and methods were used:- Antibodiβs The anti-canine E-cadherin antibody rrl developed by Gumbiner and Simons (1986) was provided by Barry Gumbiner (Memorial Sloan-Kettering Cancer Center, NY) or obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, and the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, under contract N01-HD-2-3144 from the NICHD. The anti-human E-cadherin antibody HECD-1 (Shimoyama et al . , 1989) was from Takara Biomedicals (Shiga, Japan) . Anti-pl20 and anti-focal adhesion kinase (FAK) antibodies were from Transduction Laboratories (Lexington, KY) . The anti-pl20 antibody 2B12 (Kanner et al . , 1990) was a gift from J.T. Parsons (University of Virginia, Charlottesville, VA) . The peptide-directed antibodies against α- and β- catenin (Staddon et al . , J. Cell Sci . in press) were kindly provided by Kurt Herrenknecht (Eisai London Research) Laboratories Ltd., University College London, London UK). All secondary antibodies used for immunoprecipitation and immunocytochemistry were from Jackson Laboratories Inc. (West Grove, PA). HRP-conjugated secondary antibodies used for immunoblotting were from Amersham (Buckinghamshire, UK). Cells The following cells were cultured at 37°C in medium containing 100 U/mi p enicillin and 100 ug/ml streptomycin: Caco-2 (epithelial ceils derived from a human colonic tumour: 5 % CO2, MEM, 10 % FCS, 1 % non-essential amino-acids, 1 μg/ml insulin); ECV304 (a ceil line derived from human umbilical vein endothelial ceils: 5 % C0 2 , M199, 10 % FCS); LLC-PK1 (epithelial cells derived from porcine kidney: 5 % CO:, M199, 10 % FCS); MDBK (epithelial ceils derived from bovine kidney: 5 "., C0 2 , MEM. 10 FCS); Strain I MDCK cells (epithelial cells derived from canine kidney: 5 % CO 2, MEM, 10 FCS); RBE4 ceils (immortalized rat brain endothelial ceils (see Duπeu-Trautmann et al., 1993): 5 % CO 2, α-MEM: Ham s F10 (1:1), 10 X FCS, 0.3 mg/ ml geneticin. 1 ng/mi bFGF); Swiss 3T3 fibrobiasts ( 10 % CO;, DMEM, 10 FCS). Caco-2. ECV304. LLC-PK1 and MDBK ceils were obtained from the European Collection of Arumai Ceil Cultures (Salisbury, UK). MDCK ceils were provided by Barry Gumbiner. RBE4 ceils were from Pierre Couraud lUruversite Pans \TI. Pans, France) and Swiss 3T3 fibrobiasts were from Enriαue Rozer.curr (Imperial Cancer Research Fund, London. UK). Human umoilicai vein endothelial cells were from Clonetics (Palo Alto, CA) and cultured accorαmg to tne manuracturer s instructions. Primary cultures of bovine or porcine brain endothelial ceils were grown as described by Rubin et al. (1991). For experimental purposes, confluent cultures of Caco-2, MDBK, MDCK and brain endothelial cells were established on tissue culture- treated, polycarDonate Transwell filters (polycarbonate, 0.4 μm; Costar, Cambridge, MA). Other cells were grown on tissue culture plastic. Immunoblotting and Immunoprecipitation Whole ceil lysates from cultures maintained for 16-20 hours in 0.5 % serum were prepared bv rapidly replacing the medium with hot Laemmli sample buffer ( Laemmii. 1970) suppiementeα with 5 mM EDTA, followed by heating at 100°C for 5 minutes. Proteins were resolved by slab-gel electrophoresis as described by Laemmii, (1970). Tne geis were equilibrated in buffer containing 48 mM Tris, 39 mM glycine. 0.03 SDS (w/v) and 20 % methanol (v/v), and then transferred to nitrocellulose filters (Hybond ECL, Amersham). After Ponceau S staining, the filters were blocked in 5 % (w/v) non-fat dried milk in PBS at 4°C for 16-18 hours. Filters were then incubated with pπmary anhbody in PBS containing 0.05 % Tween-20 and 1 % BSA, followed by detecnon with appropriate HRP- conjugated seconαary annbody and chemilummescence (ECL, Amersham). Immunoprecipitations were performed at 4°C. Cultures were rinsed with PBS and then lyseα in either TX burfer (1 "'., (v/v) Triton X-100, 25 mM Hepes, 2 mM EDTA, 0.1 M XaCl. 25 mM XaF, 1 mM vanadate, 25 uM phenylarsine oxide. pH 7.6 (adjusted with XaOH), 1 M PMSF, 10 ug/ ml soybean trypsin inhibitor, 0.1 U/mi α2-macrcgιobuiin. 10 u ml leupeptim or TDS burfer, which was idenncai to the TX burfer except that it was supplemented with 0.5 n (w/ v) sodium deoxycπoiate ana 0.2 "'.. (w / v) SDS. The ceils were incubated with lysis buffer for 10-15 minutes ana then scraped. The lysates were collected and centπfugeα at 14.000 x g ror 20 minutes. The supernatant was precleared with Protein A Sepharose (Pharmacia. UK) for 1-2 hours and then incubated with primary annbody for 1 hour roilowed by a further 1 hour with Protein A Sepharose alone, in the case of rabbit antibodies, or together with rabbit anti- mouse antibodies ror the mouse monoclonal antibodies. After five washes in lysis buffer, immune complexes were dissociated by addition of Laemmii sample buffer followed by heanng at 100°C for 5 minutes. Protein analysis was by SDS-PAGE and immunoblothng as described above. For [ 35 S]methionine iabelling, the cultures were washed twice in methionine-free MEM supplemented with 0.5 % FCS. The ceils were incubated for 16-18 hours in this medium containing 50 uCi/mi [ 35 S]methionine (>1000 Ci/ m oi, Amersham ). Protein analysis was by SDS-PAGE, followed by fixation in 25 % methanol/ 10 acetic acid. Labelled protein was detected either by direct autoraάiography at room temperature or by fluorography at -80°C following impregnation of the gel with Amplify (Amersham). Immunocytochemistry Cells were fixed at room temperature for 15 minutes in 3 % paraform- aldehytie made up m PBS containing 0.3 mM CaCh and 0.5 mM MgSO-t. Fixed ceils were washeα and then rεrmeabilized by incubation with 0.5 % Triton X-100 :n PBS for 10 mmutes. After washing, the cells were incubated for 30 mmutes in PBS containing 10 °O caif serum and 0.1 M lysine, pH 7.4. Incubation with primary antibody was in PBS containing 10 "'. caif serum for 1 hour. After washing, the ceils were then incubated for 30-60 minutes with a 1:100 dilution of fluorophore- romugateti anti-mouse or anti-rabbit IgG, as appropriate, in PBS containing 10 °O caif serum. After washing, the niters were mounted with Citifluor (Cir.fluor Products. Canterbury, UK) and examined using a Xikon Microphot-FXA fluorescence microscope fitteα with 40 x and 60 x cbiectives. Photographs were taken using Kodak T-MAX film (400 ASA). For the reparanon of crvosections, brain and skeletal muscie from COτ- asphyxiated rats were removed and rapidly frozen in liquid nitrogen. Tissue blocks were mounted in Tissue Tek (R. Lamb, London. UK) and sections of 5-10 μm thickness were cut on a Bright cryostat. air-dried and stored for up to 4 weeks at -20°C. After thawing, the sections were fixed and permeabilized as described above. The sections were then washed, blocked with PBS containing 10% caif serum for 15 minutes and incubated with pπmarv antibodv diluted in 7BS containing 1 % caif serum ror 2 hours. After washing, they were incubated with F5Ξ containing 10% caif serum wirn eitner 10% goat serum or 10% donkey serum, as appropriate for t e r.ost cf the secondary antibody, for 15 minutes. Thev were men lr.cu p ate with seccncarv antibody diluted in PBS and serum for 1 hour. Sections were wasneα. mounreα and examined as described above. EXAMPLE 1 Antibody characterization: pl20 and D OQ proteins SDS lysates oi a vaπerv of ceii types were analyzed by immunoblotting for expression of pl20 using an ar.π-piZO annbody (Fig. 1 A). This antibody was raised against a rusion protein containing ammo acids 790-911 at the C-terminus oi pl20. The anπboαy cieariy rsacteα with two distinct bands of apparent moiecuiar mass 122 kDa ana I TO Da m extracts of Caco-2, ECV 304. RBE4, Swiss 3T3. LLC-PK1. MDΞK and brain endotheiial ceils. We wiil hereafter refer to these two banαs as pi22 zr.a. piOO. Αrh tr.e MDCK ceils a broad connnuous oanti was -etecrec. The relative mtensttv cr the staining of pI20 ana plOO depenaed on the eιi type. For example. iOO w.-.s more intense than pl20 in Caco-2 ceiis and in the Swiss 3T3 ceiis the reverse was ocserved. These blots were aiso probed with the rr.onocienai anπ-p !22 anπbccv 1212. r.-.iseα against a phosphotvrosine immunoprecipitate rrepareα rrom " iA" 1 - ' transrormeα chicken embryo nbroDia≤ts (Kanner et ai.. 1990V This anπboriv recognised oniy the upper of the two banαs (Fig. 1 5). 2B12 reacteα strongly with bovine protein, moderately with rodent protein and weakiy with human protein. However, it failed to react with extracts of the LLC-7K1 ( " porcine i ana MDCK (canine) ceils, rerlectmg limited cross-species reacπvirv. The reiaπonsnip σetween tne Known ppt>ύ i7ι: substrate pl20 and plOO ' was rurther examined bv immunoolot analysis of immunoprecipitates from cells lvsed in TDS burfer, used to minimize protein-protem interaction (Fig. 2 A). MDBK cells were used since both the anπ-pl20 and 2B12 antibodies worked weil (Fig. 1). In the ant-pl20 immunoprecipitates, pl20 and plOO were detected by biottmg with the anπ- l20 annboαy. 2B12 only reacted with pl20 in these immunoprecipitates. In 2B12 immunoprecipitates, blotting with both 2B12 and the antι-pl2D antibody revealed pl20. Thus, 2B12 recognized pl20, whereas the antι-pl20 annbody recognized the same pl20 protein as 2B12 and, in addition, a plOO protein. Autoradiographic analysis of immunoprecipitates of [ 35 S]methionine- labeiled MDBK ceils, as resoived by SDS-PAGE (Fig. 2 B), revealed mat the pl20 protein migrated as a cluster of bands that were identical in the both 2B12 immunoprecipitates and the ann-p!20 immunoprecipitates. However, in the anti- pl20 immunoprecipitates a similar cluster of additional bands corresponding to plOO was detecteα. Technically, detection by lmmunoblotting was light based (chemiiummescence) which αoes not have the greater resolution of direct autoradiography, hence the multiple bands seen in Fig. 2 B appear as a broad banα in Fig. 2A. The oasis of the muitipiicitv of the bands corresponding to pl20 ana plCO in tne MDBK ceils is not ciear. However. [ ? -P]phospnate-iabeιiιng of these ceiis raised the possibility that these bands mav represent differennaily phosphorylated protein (results not shown). Association with the cadherin/catenin complex: epithelial cells We next examined the possibility oi association of pl20 or plOO with other proteins. When TDS lysates ot [ 35 S]methiorune-iabelled MDBK cells were immunoprecipitated with the antι-pi20 antibody, pl20 and plOO were isolated as the maior proteins corresponded to tnose detected by lmmunoblotting (Fig. 2). Similarly, the maior Dantis seen in immunoprecipitates from TDS lysates of [ 35 S]mermonιne-iabelled MDCK ceils corresponαs to the broad band detected by immunoblorang (cf. Fig. 1 A and Fig. 3 A). In contrast, the ann-pl20 immunoprecipitates rrom MDCK ceils iysed in TX buffer, used to preserve macromolecular protein complexes, revealed additional distinct bands at approximately 130 kDa. 105 kDa and 97 kDa (Fig. 3 A). Proteins of similar moiecuiar mass were seen m E-caαheπn immunoprecipitates from similarly Iysed ceils (Fig. 3 A). In this case, the 130 kDa band corresponds to E-cadheπn, the 97 kDa band to β-catenm, and the 105 kDa band to α-catenin, which is dissociated from the compiex by iysis in TDS buffer (see McCrea and Gumbiner, 1991). Thus, it appeared that the anπ-pl20 antibody may be capable of immunopreάpitating the cadherin/catenin compiex. Caco-2 ceils were also [ 35 S]methionine-labelled, Iysed in TX buffer and immunoprecipitanons were performed using the anti-E-cadherin antibody HECD-1, anti-β-catenin annbody or the anti-pl20 annbody. HECD-1 or anti-β- catenin ciearly immunoprecipitated four major bands corresponding in order of increasing mobility to E-cacheπn. a.-, β-, and γ-catenin. Similar bands were immunoprecipitated. but to a lesser extent, with the anti-pl20 antibody (Fig. 3 B). These results (not shown ) were ai≤o obtained in MDBK cells when β-catenin immunoprecipitates were co r.rareα witn those obtained with the anπ-pl20 annbody. Thus, it appears tnat the anπ-pl20 antibody can immunoprecipitate proteins that comigrate with those of the cadherm/catenin complex from a variety of epithelial cell lines. The comigranon or bancs in the antι-pl20 and anti-E-cadheπn immunoprecipitates suggests, cut does not prove, that antι-pl20 is capable of immunopreαpitanng the cadherin/ catenin complex. Therefore, the idennty of the proteins in the E-cadheπn and antι-pl20 immunoprecipitates was examined by immimoblotting (Fig. 4). As expected. E-cadheπn and β-catenin were both present in the rrl immunoprecipitates. An antι-pl20 reactive band displaying a mobility that was similar to that of β-catenin was also present in the rrl immuno¬ precipitates. In the antι-pl20 immunoprecipitates, anti-pl20 reactive material was also present, unsurprisingly, but to a greater extent and migrated as a broader band than that seen in the rrl immunoprecipitates. In the antι-pl20 immunoprecipitates, E-cadheπn and 3-catenin also were cieariy detected. Omission of primary anhbody did not resuit in E-cadherin, anπ-pl20 reachvitv or β-catenin in the immunoprecipitates. Immunoprecipitanon with anti-FAK antibody, of the same species and isotype as the anti-pl20 annbody, immunoprecipitated FAK but not E-cadherin, β-catenin or anti-pl20 reactivity ι ' Fig. 4). It should be noted that although 3-catenin and the antι-pl20 immunoreactive band seen in the anti-E-cadheπn immunoprecipitates have a similar mobility (Fig. 4), this does not represent cross-reactivity of the anti-pl20 annbody with β-catemn. Thus, in β-catenin immunoprecipitates from MDCK cells lvsed in TDS buffer, which resuiteα :n dissociaπon of anti-pl20 reacπve material from the complex of 3-catenin and E-cadheπn (see Fig. 3 A), the anπ-pi20 antibody failed to react with β -catenin. Conversely, anπ-pl20 immunoprecipitates from TDS iysed ceils faii to contain 3-catemn immunoreactivity (results not shown). We aiso ocserveti the antι-r i2Q immunoreactive band, as seen in the rrl immunoprecipitates ( Fig. 4 in α-catenin and β-catenin immunoprecipitates 'results not shown). Thererore. on the basis oi the comigranon of [ 35 S]methιomne-iabeiled bands seen in anti-E-caαheπn ana :.r.tι-pi20 immunoprecipitates of ceiis Iysed in TX buffer, it appeared that the anπ-pl20 antibody couid immunoprecipitate the cadheπn/ catenin complex. By lmmunooiotting, the unequivocal identification of E-cadheπn and β -catenin in the antι- l20 immunoprecipitates verified this interpretation. Association with me cadherin/catenin compiex: enciotheiial cells Ξnαotne-ii. ceiis form :uncπonai complexes of similar composition to those found in e itr.aiiai ceils ■ ; for review, see Rubin. 1992). As shown (Fig. 1), endotheiiai ceiis a.so contain p:20 and piOO proteins. We therefore asked whether pl00/pi20 is a.so associated with the cadherin/catenin compiex in endothelial ceiis. Brain endotheiiai ceiis were iabeiled with [^Sjmethionine, Iysed with TX buffer and immunoprecipitated with either anri-3-catenιn antibody, anπ-pl20 annbody or, because of its strong reactivity against bovine protein (see Fig. 1), 2B12. The ann-3-ca rerun annbody cieariy immunoprecipitated three maior proteins (Fig. 5), :rrrεsponcing in orcer of increasing mobility to a 120-130 kDa band, probabiy reprεsenπng a cadherin, and α- (105 kDa) and β-catenin (97 kDa). The ann-pl20 anπrcdv immunoprecipitated proteins (but to a lesser extent than those ccserveά in tr.e i-caremn immunoprecipitates ) that comigrated with α- and 3-catenin and a brraα 120 kDa rand, representing pl20 (see later), that migrated between the presumptive cadherin and α-catenin (Fig. 5). In the 2B12 immunorrecipit.-te≤. proteins that comigrated with a- and 3-catemn were also observed but to a rr.ucn i-≤ser extent man observed in the 3-catenin immunoprecipitates. Again, a oroaα 120 kDa band was observed in these immunoprecipitates. From TDS ivsates. anti-3 -catenin antibody could still precipitate 3-catemn. rut an:ι- i22 antibody failed to do so. Lnstead. a diffuse band was opserveα that igrr.teα siightiv more slowly than 3-caten . and this was absent in the 2Ξ12 immunoprecipitates. therefore representing plOO. In the 2B12 immunoprecintates. oniv the 120 kDa band was observed. Thus, assoαation between the catemns ana i20 or piOO appeared to be observed in TX Ivsates, but not TDS Ivsates, or brain enαotheiiai ceiis. To expiore tr.e generality or these observations, similar expeπments were performed using human umciiicai vein endotheiiai ceils (Fig. ό A) and ECN304 ceiis ( Fig. 6 B) but using immunoolot analysis. Thus, from ceils Iysed in TX buffer. ann-3-c≥ rerun anπooαv lmmunoprecipitateα 3-catemn. as expected, but also nvo banαs or anr:-pi2-; immunoreacπvity, corresoonαmg to plOO and pi20. Simiiariv. the anr.-ri20 anπcoαy immunoprecipttateα piOO ana pi20. as expected, and 3- arerun. However, as for the cram enαctneiiai ceiis ana the epithelial ceils. TDS buffer resuiteα in dissociaπcn of 3-catemn ana r 100 /p 120. These data corroborate those obtained from tne analysis of the immunoprecipitates rrom [ 35 S]methionιne- labeileά brain encotneiiai ceiis (Fig. 5). EXAMPLE 3 Localization to ceil-ceil junctions To invesπgate the cellular localization of pl00/pl20, immunocyrochemicai analysis was performed. Unforninateiv, 2B12 did not label ceils under the fixation conditions that were trieα i raraformaidchvαe and methanol). However, the anπ- pl20 antibody iw cn reccgmzes pi20 ana plOO) stained ceiis weil. Double- labelling or MDCK ceils with antι-3-catemn ana the anπ-pi20 anπbocy reveaied a striking co-iocaiizar.on oi 2-caremn and anπ-pl20 immunoreacπvity ' Fig. 7 a,b). Similar results were round rcr the brain enαotheuai ceils i Fig. 7 c ). Irr.mur.ocvrccr.erm≤rrv was rurtr.er perrcrmeα on rrozen sections of cram ana ≤ke.erai muse. ■ Fig -- • Λhtι-p ι22 immunoreactivity co-iocanzeα with α- caterun at inrerceiiu.ar junctions or choroid piexu≤ epitheiium and ventricular epenαvmai ceils i Fig. i a.r :. In addition, the antι-oi20 anπbooy staineα ter- ndotneuai :uncπcns in corn .arcre : ~ :~. ά c.d ) ana ≤mail blooα vessels or the brain Fig. ≤ e.r : ana blooa vessels or muscie tissue ( Fig. 5 g,h). At the given aetecπon level, anπ-ol20 immunoreactivity was limited to endotheiiai and epitheϋal ceils in the two types of nssue investigated. ' --. the present study, i munobiot analysis of a variety of different ceils reveaieα that the antι- l20 monoclonal antibodv 2512 'Kanner et al.. 1990) recognized a broaα band or approximately 120 kDa. Another anti-pl20 monoclonal antiboαv that r.ad been raiseα against the C-cerminai porπon of pl20 recognized the same bancs as 2B12 and. in addition, another cluster of bands at 100 kDa. The reason for the multiplicity of bands at 120 kDa and 100 kDa is not ciear, out obviously they are immunoioeicaiiv related. The pattern of appearance of these muitipie bancs ai≤o cependeα on me ceil type. For example in MDCK ceils, the bands were very ci fuse. whereas m MDBK ceils they were cieariy resoiveα. especially when visualized by t 3 -S]methionιne iabeiling in the absence of fluorographic reagent, it is possible mat post-transiationai modificaπon. such as phospnoryiation ana mvrisroviaπor. mav account for the appearance of muitipie bands. The relationship, apart from tr.e immunoiogical one. between p!20 and plOO is not yet ciear. The Xortnern blots described by Reynolds et al. 11992) suggesteα the possiciiitv or r ;22-reιared ene procucts or alternatively spiiced transcripts. Furthermore. Southern ;.r.a;vsι≤ apparently indicated that one or more pl20-reιateα genes exist i Revπ.oic≤ ct ai.. If 2). It follows that the ciuster of bands corresponding to pi20 and the similar cluster to plOO could aiso represent splice variants or. respectively. pi O and r 120. It is aiso possible that plOO could simpiy represent a degradation product cr p 120. although samples were prepared in denaturing ourfer ana immunoprecipitations were performeα in the presence of inhibitors of a broaα spectrum or proteases. Our results provide evidence that pl20/pl00 associates with the •"adhenn catenin compiex. Thus, rrom TX-soiubiiized epitheiial cells. ann-pl20 antibodv immunoprecipitateα f 3? S :methionιne-labeiled proteins that comigrated with those immunoprecipitateα bv caαhenn anπbodies (.Fig. 3). Immunobiot anaivsis verified the identity or these proteins as cadheπns and catemns (Fig. 4). Similarly, anπ-pi20 reacnve protein was seen in tne anti-E-cadherin immunoprecipitates from MDCK ceils, aithougn on the basis of mobility this appears to represent primarily plOO, not pi20 (Fig. 4). However, it should be noted that pi20 immunoreacπve protein was not unequivocally identified in the MDCK ceils because of the poor reactivity of the 2B12 annbody with canine protein. This apparent association between pl20/pl00 and the cadherin/catenin compiex was not restricted to epithelial ceils as similar results were obtained using endotheiiai ceils, both by [ 5 S]methiorune-labeliing (Fig. 5) and immunobiot analysis (Fig. 6). However, although our results cleariy show an interaction between catenins and pl20/pl00 in endothelial cells, we did not identify a cadherin in the anπ-pl20 immunoprecipitates. The interpretation of the biochemical analyses of the protein complexes in the epithelial and endothelial ceils is further supported by the lmmunocytochemicai study. In cultured ceils ana in tissue sections. antι-pl20 immunoreactivity was strongly associateα with intercellular junctions, displaying a localization that was remarkably similar to that obtameα with antι-3 -catenin antibodv (Figs. 7 and S). Here, we were resmcteα to using the anπ-pl20 anπboov wmch might recognize either p!20 or piOO, or both. Of course, one explanation for the ability of antι-pl20 antibody to immunoprecipitate the cadherin/ catenin compiex is that it cross-reacts with one oi the known components or tne complex. However, this is uniikeiy ror the ollowing reasons. TDS lysis oi ceils dissociates α-catenin ana γ-catenm from the cadherin and 3-catemn complex (see McCrea and Gumbiner, 1991). In antt-E- cadheπn immunoprecipitates from MDCK cells iysed in TDS buffer. anπ-pl20 annbodv faiied to react with E-cadheπn or the co-precipitatmg β-catenin. In anπ-α- cate n immunoprecipitates rrom MDCK ceils iysed in TDS burfer. ann-pl20 annbodv failed to react with α-catemn (results not shown). As shown in Fig. ό, β- caterun immunoprecipitates rrom TDS-lysed endothelial ceils, although clearly 24 containing 3-catemn, and presumably a cadherin, did not contain any anti-pl20 reacπviry. Cross-reacπvity with γ-catemn, a protein of approximately 85 kDa, is unlikeiy as the immunoblots shown in Fig. 1 fail to show reactivity with protein below 100 kDa. One problem that has to be addressed concerns the lack of any obvious [ 35 S]metnιonιne-iabeiled bands that correspond to pl20/pl00 in the cadherin immunoprecipitates from TX Iysed ceils. Thus, as shown in Fig. 3, rrl and the anti- pl20 immunoprecipitates from MDCK ceils look remarkably similar. However, it is clear from the ann-pl20 immunoprecipitates from TDS Iysed cells that the anti- pl20 reactive material migrates as a broad band, the bulk of which migrates between α- and β-catenin, with the remainder migrating slightly slower than α- catenin. In conjunction with the immunobiot analysis shown in Fig. 4, which demonstrates that oniy a fraction of the anπ-pl20 immunoreactive material is immunoprecipitated by rrl. ana the fact that pl20 contains about half of the number of methiomnes in α- and 3-catemn, if we assume steady-state labelling, then it is obvious that it would be difficult to see any labelling corresponding to the ann- l20 lmmunoreacnve material in me rrl immunoprecipitates. Similarly, with respect to the experiments with the orain endotheiiai ceils (Fig. 5), under the extracπon conditions emplo\ eα it appears that oniy a fraction of the pooi of the cadherm,' catemn complex associates with plOO, and even less with pI20. Thus, it would be difficult to see iabeiiing oi banαs corresponding to pl20 and plOO in the 3-catemn immunoprecipitates as this region of the gel is already occupied by other major bands. It is aiso difficult to discern a band corresponding to the cadherm seen in the 3-catemn immunoprecipitates in either the antι-pl20 or 2B12 immunoprecipitates because or the dominance of the labeiiing of pl20 (Fig. 5). We emphasize that there are aiso ciear quantitaπve differences with respect to the ability of 2B12 and antι-pl20 antibody to immunoprecipitate the cadherm/ catemn compiex. Due to poor reactivity of 2B12 with ca ne protein, this could not be addressed with MDCK ceils. In bovine brain endotheiiai ceils, the anti-pl20 annbodv immunoprecipitated the cadherin/catenin complex, although not as weii as anti- 3-catenin antibody. 2B12 was even less effective than anti-pl20. As anti-pl20 recognizes pl20 and plOO, and 2B12 only pl20, the difference in the amount of the compiex immunoprecipitated must be attributable to greater association of plOO with the catenins. As far as we can tell, the efficiency of anti- pl20 and 2B12 in immunoprecipitation was simiiar in this experiment (see Fig. 5 for the simiiar intensity of a band corresponding to pl20 in the anti-pl20 and 2B12 immunoprecipitates from ceiis Iysed in TX buffer . These data also indicate that independent complexes of plOO and pl20 exist with catenins, rather that a catenin/ pl00/pl20 complex. If the latter were the case, 2B12 would be expected to immunoprecipitate as much of the catemns as antι-pl20 antibody. Our study provides a link between pl20/pl00 and adherens junction proteins. It is possible that such an interaction, perhaps via the influence of regulatory kinases, such as src, lyn and yes (see Tsukita et al., 1991), may play a roie in the moduiar.cn of cadherin function, and thereby other cellular functions influenced by the adherens runction. With respect to phosphorylation. the tyrosine phosphatase inhibitor phenviarsine oxide was found to cause an increase in tight juncπon permeability in MDCK ceils ( Staddon et ai., /. C ll Sci. in press ). This inhibitor increased the tyrosine phosphoryiation of the antι-pl20 immunoreacπve mateπai ( a maior piOO band, a minor pi20 band) in these ceils, as analyzed by anti-pl20 lmmunoblotting of antι-pl20 immunoprecipitates from SDS lysates (results not shown: see Staddon et al.. /. Cell Sci. in press). The pl20/pl00 proteins could also be involved in the interaction between cadherins and the actin-based ytoskeieton. p!20/pl00 mav aiso oe part of a signalling cascade, communicating information about the state of ceil-ceil adhesiveness to the interior of the ceil. 3-catemn is an arm protein iMcCrea et al., 1991) and can associate with cadherins and the APC gene product (Rubinfeld et al., 1993; Su et al.. 1993), also an arm protein (see Peifer et al., 1994). pl20 is an arm protein (Reynolds et al., 1992; Peifer et ai.. 1994), and, as we describe here, plOO is an immunologically related protein. These proteins can interact with β-catenin. The exact nature of the interacπon benveen pl20/pl00 and the catenins remains to be established. These proteins may interact directiy, or associate with different regions of the cytoplasmic domam of cadherins. Other linking or intermediary binding proteins could also be involved. 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Claims: CLAIMS 1. A protein: (a) having a molecular weight of about 100 kDa and being associated in vivo with the catenin/cadherin complex of epithelial or endothelial cells, which is referred to herein as plOO, or (b) having substantial amino acid homology with plOO, or (c) being a fragment of plOO or of a protein having substantial amino acid homology with plOO. 2. A protein according to claim 1 which comprises one or more of the amino acid sequences given below: (i) NISFGRDQDNK (ii) HAIPNLV (iii) XVLINK (iv) XPIEDPANDTVDFPX (v) XPSGALRNLAVDARX, wherein the letters used are based on the standard single letter amino acid code, but wherein "X" designates any amino acid. 3. A nucleic acid molecule coding for a protein according to claim 1 or claim 2, or being complementary to a nucleic acid molecule according to claim 1 or claim 2. 4. A vector comprising a nucleic acid molecule according to claim 3. 5. A host cell comprising a vector according to claim 4. 6. The use of a protein according to claim 1 or claim 2 or of pl20 for studying cell-cell adhesion. 7. The use of a protein according to claim 1 or claim 2 or of pl20 for studying tight junction permeability. 8. An agent which alters or blocks association between plOO or pl20 and the catenin/cadherin complex of epithelial or endothelial cells for use in the treatment or diagnosis of a disease. 9. An agent according to claim 8, wherein the disease is selected from: tumour cell invasion and metastasis, breast cancer, colorectal cancer, gastric carcinoma, gynaecological cancer, lung cancer, oesophageal cancer, prostate cancer, scirrhous cancer, tissue regeneration or developmental disorders. 10. An agent according to claim 8 or claim 9 which is a mimetic. 11. The use of an agent according to any of claims 8 to 10 the manufacture of a medicament for the treatment of a disease selected from: tumour cell invasion and metastasis, breast cancer, colorectal cancer, gastric carcinoma, gynaecological cancer, lung cancer, oesophageal cancer, prostate cancer, scirrhous cancer, tissue regeneration or developmental disorders. 12. The protein plOO, substantially as hereinbefore described, with reference to the accompanying examples.

Inventors: STADDON JAMES MICHAEL, STADDON, JAMES, MICHAEL
Applicants: EISAI CO LTD, STADDON JAMES MICHAEL, EISAI CO., LTD., STADDON, JAMES, MICHAEL
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61P35, A61P43, C07K14, C12N5, C12N15, G01N33