cis Interaction of the Cell Adhesion Molecule CEACAM1 with Integrin β3
2001; Elsevier BV; Volume: 159; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)61725-7
ISSN1525-2191
AutoresJens Brümmer, Alireza Ebrahimnejad, Raid Flayeh, Udo Schumacher, Thomas Löning, Ana‐Maria Bamberger, Christoph Wagener,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoCEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin β3 as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin β3 interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin β3 at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin β3 complexes in cell invasion. CEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin β3 as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin β3 interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin β3 at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin β3 complexes in cell invasion. Formerly known as biliary glycoprotein and recently designated as CEACAM1,1Beauchemin N Draber P Dveksler G Gold P Gray-Owen S Grunert F Hammarström S Holmes KV Karlsson A Kuroki M Lin S-H Lucka L Najjar SM Neumaier M Obrink B Shively JE Skubitz KM Stanners CP Thomas P Virji M Von Kleist S Wagener C Watt S Zimmermann W Redefined nomenclature for members of the carcinoembryonic antigen family.Exp Cell Res. 1999; 25: 243-249Google Scholar the human homologue of Cell-CAM is an established cell adhesion molecule in the rat.2Aurivillius M Hansen OC Lazrek MB Bock E Öbrink B The cell adhesion molecule Cell-CAM 105 is an ecto-ATPase and a member of the immunoglobulin superfamily.FEBS Lett. 1990; 264: 267-269Crossref PubMed Scopus (100) Google Scholar, 3Lin SH Giudotti G Cloning and expression of a cDNA coding for a rat liver plasma ecto-ATPase: the primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I.J Biol Chem. 1989; 264: 14408-14414Abstract Full Text PDF PubMed Google Scholar This cell-surface immunoglobulin-like glycoprotein belongs to the carcinoembryonic antigen (CEA) family and is expressed in a wide range of human tissues such as liver, endometrium, mammary ducts as well as epithelial cells of the gastrointestinal tract, endothelial cells, and hemopoietic cells.4Prall F Nollau P Neumaier M Haubeck HD Drzeniek Z Helmchen U Löning T Wagener C CEACAM1 (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.J Histochem Cytochem. 1996; 44: 35-41Crossref PubMed Scopus (173) Google Scholar Like several other cell adhesion molecules, CEACAM1 also functions as a microbial receptor. Thus, the mouse hepatitis virus utilizes mouse CEACAM1 (Ceacam1) as its receptor5Dveksler GS Dieffenbach CW Cardellichio CB McCuaig K Pensiero MN Jiang GS Beauchemin N Holmes KV Several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-A59.J Virol. 1993; 67: 1-8Crossref PubMed Google Scholar and in human granulocytes and epithelial cells CEACAM1 is a receptor for bacterial proteins from Escherichia coli, Salmonella typhimurium, Neisseria gonorrhoeae, and Haemophilus influenzae.6Virji M Watt SM Barker S Makepeace K Doyonnas R The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitides and Neisseria gonorrhoeae.Mol Microbiol. 1996; 22: 929-939Crossref PubMed Scopus (218) Google Scholar, 7Chen T Grunert F Medina-Marino A Gotschlich EC Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins.J Exp Med. 1997; 185: 1557-1564Crossref PubMed Scopus (173) Google Scholar, 8Gray-Owen SD Dehio C Haude A Grunert F Meyer TF CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.EMBO J. 1997; 16: 3435-3445Crossref PubMed Scopus (193) Google Scholar, 9Leusch HG Drzeniek Z Markos-Pusztai Z Wagener C Binding of Escherichia coli and Salmonella strains to members of the carcinoembryonic antigen family: differential binding inhibition by aromatic α-glycosides of mannose.Infect Immun. 1991; 59: 2051-2057Crossref PubMed Google Scholar, 10Virji M Evans D Hadfield A Grunert F Teixeira AM Watt SM Critical determinants of host receptor targeting by Neisseria meningitides and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules.Mol Microbiol. 1999; 34: 538-551Crossref PubMed Scopus (147) Google Scholar A number of physiological functions have been ascribed to CEACAM1 in different tissues. In endothelial cells, CEACAM1 exhibits properties of an angiogenic factor and acts as a major effector of vascular endothelial growth factor (VEGF).11Ergün S Kilic N Ziegeler G Hansen A Nollau P Götze J Wurmbach J-H Horst A Weil J Malkanthi F Wagener C CEA-related cell adhesion molecule 1 (CEACAM1): a potent angiogenic factor and a major effector of vascular endothelial growth factor (VEGF).Mol Cell. 2000; 5: 311-320Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar In epithelial cells it is generally believed, that CEACAM1 acts as a growth suppressor, since the expression of CEACAM1 was shown to be lost or significantly down- or dysregulated in carcinomas of the liver,12Hixson DC McEntire KD Detection of an altered form of cell-CAM 105 on rat transplantable and primary hepatocellular carcinomas.Cancer Res. 1989; 49: 6788-6794PubMed Google Scholar, 13Hixson DC McEntire KD Obrink B Alterations in the expression of a hepatocyte cell adhesion molecule by transplantable rat hepatocellular carcinomas.Cancer Res. 1985; 45: 3742-3749PubMed Google Scholar prostate,14Kleinerman DI Troncoso P Lin SH Pisters LL Sherwood ER Brooks T von Eschenbach AC Hsieh JT Consistent expression of an epithelial cell adhesion molecule (C-CAM) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor.Cancer Res. 1995; 55: 1215-1220PubMed Google Scholar endometrium,15Bamberger A Riethdorf L Nollau P Naumann M Erdmann I Götze J Brümmer J Schulte HM Wagener C Löning T Dysregulated expression of CEACAM1 (BGP, C-CAM), an adhesion molecule of the CEA family, in endometrial cancer.Am J Pathol. 1998; 152: 1401-1406PubMed Google Scholar breast,16Riethdorf L Lisboa BW Henkel U Naumann M Wagener C Löning T Differential expression of CEACAM1 (BGP), a cell adhesion molecule of the carcinoembryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland.J Histochem Cytochem. 1997; 45: 957-963Crossref PubMed Scopus (96) Google Scholar and colon.17Neumaier M Paululat S Chan A Matthaes P Wagener C Biliary glycoprotein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas.Proc Natl Acad Sci USA. 1993; 90: 10744-10748Crossref PubMed Scopus (232) Google Scholar, 18Nollau P Scheller H Kona-Horstmann M Rohde S Hagenmüller F Wagener C Neumaier M Expression of CD66a (human C-CAM) and other members of the carcinoembryonic antigen gene family of adhesion molecules in human colorectal adenomas.Cancer Res. 1997; 57: 2354-2357PubMed Google Scholar, 19Zhang L Zhou W Velculescu VE Kern SE Hruban RH Hamilton SR Vogelstein B Kinzler KW Gene expression profiles in normal and cancer cells.Science. 1999; 276: 1268-1272Crossref Scopus (1242) Google Scholar Consistent with this notion it has been shown that CEACAM1 acts as a negative regulator of tumor cell growth in human prostate20Hsieh J-T Luo W Song W Wang Y Kleinerman DI Van NT Lin S-H Tumor suppressive role of an androgen-regulated epithelial cell adhesion molecule (C-CAM) in prostate carcinoma cell revealed by sense and antisense approaches.Cancer Res. 1995; 55: 190-197PubMed Google Scholar and breast carcinoma models.21Luo W Wood CG Earley K Hung M-C Lin S-H Suppression of tumorigenicity of breast cancer cells by an epithelial cell adhesion molecule (C-CAM1): the adhesion and growth suppression are mediated by different domains.Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (86) Google Scholar Furthermore, CEACAM1 is strongly expressed by the extravillous trophoblast during the invasion of the maternal endometrium, and might, as a tumor suppressor, be involved in the molecular mechanisms controlling this process and for differentiating them from those implicated in tumor progression.22Bamberger A-M Sudahl S Löning T Wagener C Bamberger C Drakakis P Coutifaris C Makrigiannakis A The adhesion molecule CEACAM1 (CD66a, C-CAM, BGP) is specifically expressed by the extravillous intermediate trophoblast.Am J Pathol. 2000; 156: 1165-1170Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar The growth-suppressive effect of CEACAM1 depends on the presence of its cytoplasmic domain. Although the mechanism of action is largely unresolved, several reports suggest that CEACAM1 participates in signal transduction by interacting with other membrane or cytoplasmic proteins via its cytoplasmic domain. The cytoplasmic domain (CEACAM1cyt) is phosphorylated by multiple protein kinases23Brümmer J Ganzer S Ebrahimnejad A Flayeh R Streichert T Wagener C The CD66 complex: activation of tyrosine kinases of the Src family. Leucocyte Typing VI.in: Kishimoto T Kikutani H A von dem Borne Goyert SM Mason DY Miyasaka M Moretta L Okumara K Shaw S Springer TA Sugamura K Zola H Garland Publishing, New York1997: 1011-1012Google Scholar and the phosphorylation on one or both of its two tyrosine residues (Tyr-488 and Tyr-515) is triggered by several physiological events.24Skubitz KM Campbell KD Ahmed K Skubitz AP CD66 family members are associated with tyrosine kinase activity in human neutrophils.J Immunol. 1995; 155: 5382-5390Crossref PubMed Google Scholar One or both of these tyrosine-phosphorylated residues are involved in the association with protein kinases of the Src family,25Brümmer J Neumaier M Göpfert C Wagener C Association of pp60c-src with biliary glycoprotein (CEACAM1), an adhesion molecule of the carcinoembryonic antigen family down regulated in colorectal carcinomas.Oncogene. 1995; 11: 1649-1655PubMed Google Scholar paxillin,26Ebrahimnejad A Flayeh R Unteregger G Wagener C Brümmer J The cell adhesion molecule CEACAM1 associates with paxillin in granulocytes, epithelial and endothelial Cells.Exp Cell Res. 2000; 260: 365-373Crossref PubMed Scopus (28) Google Scholar the protein-tyrosine phosphatases SHP-127Beauchemin N Kunath T Robitaille J Chow B Turbide C Daniels E Veilette A Association of biliary glycoprotein with protein tyrosine phosphatases SHP-1 in malignant colon epithelial cells.Oncogene. 1997; 14: 783-790Crossref PubMed Scopus (125) Google Scholar and SHP-2.28Huber M Izzi L Grondin P Houde C Kunath T Veillette A Beauchemin N The carboxyl-terminal region of biliary glycoprotein controls its tyrosine phosphorylation and association with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells.J Biol Chem. 1999; 274: 335-344Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar In the present study, we used differentially phosphorylated recombinant expressed CEACAM cytoplasmic domains and mutants to isolate integrin β3 as an associated protein with the tyrosine phosphorylated cytoplasmic CEACAM1 domain. In vitro, this association depends on the phosphorylation of Tyr-488 in CEACAM1cyt. Using coprecipitation studies and laser scanning confocal microscopy analyses, we demonstrate the association of both molecules in granulocytes and the colonic carcinoma cell line HT29. Furthermore, our findings of CEACAM1/integrin β3 co-expression and colocalization at the maternal-fetal interface during placental development plus at the invading front of primary melanoma suggest a functional role of CEACAM1-integrin β3 association in cell migration. Cloning of the cDNA coding for the wild-type cytoplasmic domain of CEACAM1 and CEACAM3 and generation of the mutant domains CEACAM1cytY488F, CEACAM1cytY515Fwas performed as described.25Brümmer J Neumaier M Göpfert C Wagener C Association of pp60c-src with biliary glycoprotein (CEACAM1), an adhesion molecule of the carcinoembryonic antigen family down regulated in colorectal carcinomas.Oncogene. 1995; 11: 1649-1655PubMed Google Scholar, 26Ebrahimnejad A Flayeh R Unteregger G Wagener C Brümmer J The cell adhesion molecule CEACAM1 associates with paxillin in granulocytes, epithelial and endothelial Cells.Exp Cell Res. 2000; 260: 365-373Crossref PubMed Scopus (28) Google Scholar Protein kinases PKC, CK II (Biomol, Hamburg, Germany), and Src (Upstate Biotech, Lake Placid, NY) were diluted in kinase dilution buffer according to manufacturers’ instructions and added to 500 μg of purified cytoplasmic CEACAM1 and CEACAM3 domains in kinase assay buffer. For radioactive labeling 50 μCi [γ-32P]ATP (30 Ci/mmol; 1 Ci = 37 GBq) was added. After incubation at 30°C for 1 hour, phosphotyrosyl proteins were purified using the PY Immunoaffinity system (Oncogene Science, Manhasset, NY). The purified phosphorylated or unphosphorylated CEACAM1 and CEACAM3 domains were adjusted to 250 μg/ml with a 50% slurry of Ni-nitrilotriacetic acid (NTA) resin (Qiagen, Hilden, Germany). After incubation for 2 hours at 4°C 2 ml of this suspension were transferred into a column and washed three times in 1 ml of wash buffer (300 mmol/L NaCl, 50 mmol/L Na-phosphate, pH 7.5). Granulocytes were isolated from buffy coat of normal donors by Ficoll-Paque gradient centrifugation (d = 1.119). Freshly isolated granulocytes (5.5 × 1020) were extracted with 1% Nonidet P-40 diluted in PBS containing proteinase inhibitors. After centrifugation (10,000 × g, 30 minutes) 1 ml of the Nonidet P-40-soluble supernatant containing 1.25 g/L protein was passed over the Ni-NTA immobilized CEACAM1 and CEACAM3 domains. The column was washed with 0.3 mol/L NaCl containing 0.05 mol/L phosphate (pH 6.0) and eluted using 0.3 mol/L NaCl, 0.05 mol/L phosphate (pH 3,0). SDS-PAGE, silver staining, and Western blots were performed as described.29Stoffel A Neumaier M Gaida F-J Fenger U Drzeniek Z Haubeck H-D Wagener C Monoclonal, anti-domain and anti-peptide antibodies assign the molecular weight 160,000 granulocyte membrane antigen of the CD66 cluster to a mRNA species encoded by the biliary glycoprotein gene, a member of the carcinoembryonic antigen gene family.J Immunol. 1993; 150: 4978-4984Crossref PubMed Google Scholar Bound proteins from granulocyte extracts were separated by SDS-PAGE and transferred to PVDF membrane. Protein bands were visualized by Serva blue R staining and excised. N-terminal sequences were determined by automated Edman degradation (Richter AG, Hamburg, Germany). Sequence library searches were performed against SWISS-PROT and PIR databases. Extracts from cells containing 250 μg of protein were incubated with approximately 5 μg of monoclonal antibody for 1 hour at 4°C. Subsequently, Protein G PLUS/Protein A-agarose (50 μl) was added. After incubation on a rocker platform at 4°C for 24 hours, the precipitates were washed four times with antibody (Ab)-wash buffer (1% Triton X-100, 10 mmol/L Tris-HCl pH 7.6, 5 mmol/L EDTA, 50 mmol/L NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 100 μmol/L sodium orthovanadate, 0.1% azide). Precipitated proteins were boiled in sample buffer, separated by SDS-PAGE electrophoresis and visualized by immunoblotting. Extracts from cells containing 250 μg of protein precleared by rotating at 4°C with 30 μl of a 50% slurry of Protein G PLUS/Protein A-agarose (Dianova, Hamburg, Germany) for 30 minutes and beads were removed by centrifugation (5000 rpm). Supernatants were incubated with approximately 5 μg of human integrin β3 mAb (Chemicon International Inc., Temecula, CA) for 1 hour at 4°C. Subsequently, Protein G PLUS/Protein A-agarose (50 μl) and radioactively labeled CEACAM domains adjusted to 50 μg/ml were added. After incubation on a rocker platform at 4°C for 2 hours, the precipitates were washed four times with antibody (Ab)-wash buffer. Precipitated proteins were boiled in sample buffer, separated by SDS-PAGE electrophoresis and visualized by autoradiography. Cells of the colonic carcinoma cell line HT29-D4 were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum for 6 days. After washing (PBS) cells were lysed in Triton X-100 lysis buffer (50 mmol/L Hepes pH 7,5, 150 mmol/L NaCl, 1% Triton-X-100, 2% aprotinin, 2 mmol/L EDTA, 50 mmol/L NaF, 10 mmol/L Na-pyruvate, 10% glycerine, 1 mmol/L Na-orthovanadate, 1 mmol/L PMSF). Lysis was carried out on ice for 30 minutes followed by clarification in a microfuge (13,000 rpm for 30 minutes at 4°C). Endometrial epithelial cells were prepared from endometrium of cycling women undergoing hysterectomies for leiomyomas. Briefly, endometrial tissue was minced thoroughly and digested in DMEM with 0.5 mg/ml collagenase-dispase (Boehringer-Mannheim, Mannheim, Germany) and 2.5 mg/ml desoxyribonuclease (Sigma, Deisenhofen, Germany) for up to 2 hours, with gentle pipetting every 20–30 minutes. The suspension was filtered through a nylon stocking to remove nondispersed tissue fragments. Cells were harvested by centrifugation at 400 × g for 6 minutes, and resuspended in plating media, a 1:1 mixture of DMEM and HF-12 containing 10% FCSDCC (FCS depleted of steroids by treatment with dextran-coated charcoal), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml insulin (Sigma), and 10−9 mol/L 17β-estradiol (E2; Sigma). Stromal and epithelial cells were separated by sieving the cell suspension through a steal sieve (38 μm pore size). The retentate containing the epithelial glands was washed out by inverting the sieve and rinsing it with plating media. Endometrial stromal cells were collected in the filtrate. After centrifugation at 400 g for 6 minutes, epithelial glands were plated in six-well plates. Medium was changed every 48–72 hours. For confocal laser scanning microscopy tissue material, routinely fixed in 4% buffered formalin and embedded in paraffin, was selected after histological review from the files of the Department of Gynecopathology and the Department of Neuroanatomy, University Hospital Eppendorf, Hamburg. For the present study three CEACAM1 positive primary melanomas along with three first trimester placentas were investigated. Processing of cells for CLSM was performed as described.26Ebrahimnejad A Flayeh R Unteregger G Wagener C Brümmer J The cell adhesion molecule CEACAM1 associates with paxillin in granulocytes, epithelial and endothelial Cells.Exp Cell Res. 2000; 260: 365-373Crossref PubMed Scopus (28) Google Scholar From the tissues material used serial sections of 4 to 6 μm were cut from the paraffin blocks and mounted on 3-aminopropyl-triethoxysilan (APES)-coated slides, deparaffinized in Rotihistol, and rehydrated in graded alcohol to PBS-buffered saline. The slides were microwaved five times for 2 minutes in 10 mmol/L citrate pH 6.0. After cooling down for 20 minutes, the slides were washed three times in PBS for 5 minutes. Unspecific binding was blocked by incubating the slides in 20% FCS/3% BSA in PBS for 30 minutes and the first antibody −4D1/C2 against CEACAM1 was added in adjusted dilution (1:25). After incubation overnight slides were washed three times in PBS and incubated with goat anti-mouse IgG (H+L, GaM Dianova) TRITC (1:50). After washing three times for 5 minutes with PBS the samples were further processed by blocking any remaining epitopes on the mouse Fc fragment with a goat anti-mouse IgG (H+L, Dianova) F(ab′) fragment for 30 minutes. After three washing steps with PBS the second primary antibody (anti-integrin β3 of DAKO, Glostrup, Denmark) was added, incubated for 60 minutes at room temperature. Integrin signals were detected by a second FITC-conjugated antibody (goat anti-mouse IgG H+L, FITC labeled, Dianova). To rule out any cross-reactions between the two primary antibodies we performed immunostaining even with an anti-integrin β3-antibody (Chemicon International) raised in rabbits. In this case, the integrin β3-signals were detected using a sheep-anti-rabbit (Fc)-FITC secondary antibody (Dianova). Since we obtained the same results with respect to colocalization even using this protocol as a specificity control, these data are not presented separately in the results section or as figures. To avoid photobleaching of the fluorochrome during fluorescence microscopy, the slides were embedded in anti-fade solution (BiomedDia, Zweibrücken, Germany). The double-fluorescent stained specimens were analyzed with a confocal laser scanning microscope equipped with an external argon laser (Zeiss Invers 410, Zeiss, Oberkochen, Germany). The cells were x/y scanned in the reflecting mode with a simultaneous excitation wavelength of 488 and 543 nm. Using the confocal mode, the pinhole was closed to approximately 25. To visualize the distribution of the fluorescence signals the specimens were monitored using the extended depth of focus mode (EDF). Details on the production and characterization of the monoclonal CEACAM1 4D1/C2 have been published.30Wagener C Clark BR Rickard KJ Shively JE Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay.J. Immunol. 1983; 130: 2302-2307PubMed Google Scholar, 31Neumaier M Fenger U Wagener C Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography.J. Immunol. 1985; 135: 3604-3609Crossref PubMed Google Scholar The CEACAM mAb 12-140-4 was a kind gift from Ole P. Børmer (Norwegian Radium Hospital, Oslo, Norway). Monoclonal antibodies to human integrin β3 (Chemicon International and DAKO) were purchased and used according to the manufacturers’ specifications. As shown in Figure 1A, lane 1, a defined set of proteins was purified using the in vitro tyrosine phosphorylated cytoplasmic CEACAM1 domain. These bands were not detectable in the control lanes using unphosphorylated domain of CEACAM1 (Figure 1A, lane 2), the tyrosine phosphorylated (Figure 1A, lane 3) or unphosphorylated (Figure 1A, lane 4) cytoplasmic domain of CEACAM3. Here we report on the band of approximately 95 kd, which was identified unanimously as the human integrin β3subunit by N-terminal sequencing. In Western blots performed with the eluted antigens, a monoclonal anti-integrin β3antibody detected the 95 kd protein only in the tyrosine phosphorylated CEACAM1cyt eluate (not shown). In conclusion, the purification of integrin β3 was observed with the tyrosine phosphorylated cytoplasmic domain of CEACAM1 only. To further investigate an association of CEACAM1 with integrin β3, co-immunoprecipitation experiments were performed from membrane extracts of granulocytes. Extracts were prepared and incubated with monoclonal antibodies against integrin β3 or CEACAM1 or polyclonal mouse IgG (control). Immunoprecipitated complexes were analyzed by immunoblotting with the monoclonal antibodies against either integrin β3 or CEACAM1. Complexes containing CEACAM1 and integrin β3 could be immunoprecipitated with both anti-CEACAM1 (Figure 1B, lane 1) and anti-integrin β3 antibodies (Figure 1B, lane 3). Furthermore, co-immunoprecipitation of CEACAM1-integrin β3complexes was also observed in the epithelial colonic carcinoma cell line HT29 (Figure 1B, lanes 2 and 4). Differences of the apparent molecular weight of CEACAM1 in different cell types are established and probably due to glycosylation-differences.11Ergün S Kilic N Ziegeler G Hansen A Nollau P Götze J Wurmbach J-H Horst A Weil J Malkanthi F Wagener C CEA-related cell adhesion molecule 1 (CEACAM1): a potent angiogenic factor and a major effector of vascular endothelial growth factor (VEGF).Mol Cell. 2000; 5: 311-320Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Bamberger A Riethdorf L Nollau P Naumann M Erdmann I Götze J Brümmer J Schulte HM Wagener C Löning T Dysregulated expression of CEACAM1 (BGP, C-CAM), an adhesion molecule of the CEA family, in endometrial cancer.Am J Pathol. 1998; 152: 1401-1406PubMed Google Scholar, 26Ebrahimnejad A Flayeh R Unteregger G Wagener C Brümmer J The cell adhesion molecule CEACAM1 associates with paxillin in granulocytes, epithelial and endothelial Cells.Exp Cell Res. 2000; 260: 365-373Crossref PubMed Scopus (28) Google Scholar, 32Drzeniek Z Lamerz R Fenger U Wagener C Haubeck H-D Identification of membrane antigen in granulocytes and colonic carcinoma cells by a monoclonal antibody specific for biliary glycoprotein, a member of the carcinoembryonic antigen family.Cancer Lett. 1991; 56: 173-179Crossref PubMed Scopus (49) Google Scholar Complexes containing detectable CEACAM1 or integrin β3could be not immunoprecipitated using polyclonal mouse IgG and vinculin as controls (not shown). The in vitro tyrosine phosphorylated wild-type cytoplasmic CEACAM1 domain is, after incubation with granulocyte extracts, co-immunoprecipitated with anti-integrin β3 mAb (Figure 1C, lane 1). We used different CEACAM1cyt mutant domains to determine whether phosphorylated tyrosine residues within CEACAM1cyt are required for binding to integrin β3. The (Y515F) mutation (CEACAM1cytY515F-P) did not interfere with the observed interaction (Figure 1C, lane 2), but the (Y488F) mutation (CEACAM1cytY488F-P) did almost completely abolish coprecipitation (Figure 1C, lane 3). The tyrosine phosphorylated CEACAM3 domain as well as serine/threonine phosphorylated wild-type and mutant (Y488F and Y515F) CEACAM1cyt did not co-immunoprecipitate with integrin β3 mAb (not shown). To substantiate an association of CEACAM1 and integrin β3 in vivo, we conducted double-fluorescent confocal laser scanning microscopy on human granulocytes. First, the cellular distribution and the relative intensity of fluorescence on the cells were assessed qualitatively. Immunofluorescence of TRITC localizing CEACAM1 (Figure 2A, red) and of FITC localizing anti-integrin β3 mAb (Figure 2B, green) revealed a strong membrane-associated granular pattern for each of the molecules. Using double-fluorescent labeling colocalization of CEACAM1 and integrin β3 in the same cells was indicated by yellow fluorescence (Figure 2C). In addition to granulocytes, immunoreactivity of 4D1/C2 (Figure 2D) and integrin β3 mAb (Figure 2E) were localized on cells of the colonic carcinoma cell line HT29. Double-fluorescent labeling correspondingly displayed a close association of CEACAM1 with integrin β3 in HT29 cells (Figure 2F). Since integrin β3 has a well established role in the invasion and metastasis of malignant melanomas33Hsu MY Shih DT Meier FE Van Belle P Hsu JY Elder DE Buck CA Herlyn M Adenoviral gene transfer of β3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.Am J Pathol. 1998; 153: 1435-1442Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 34Natali PG Hamby CV Felding-Habermann B Liang B Nicotra MR Di Filippo F Giannarelli D Temponi M Ferrone S Clinical significance of α (v) β3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions.Cancer Res. 1997; 57: 1554-1560PubMed Google Scholar and since CEACAM1 expression is clinically positively correlated with metastasis of melanomas (P < 0.0001; U. Schumacher, submitted), we investigated primary melanomas for CEACAM1/integrin β3 expression. Representative results of CEACAM1 and integrin β3 expression in CEACAM1 positive primary melanomas are displayed Figure 3. Whereas CEACAM1/integrin β3 colocalization is heterogeneous in the center of primary melanoma (Figure 3A). In contrast, horizontal (Figure 3B) and longitudinal (Figure 3, C–E) sections revealed concentrated co-expression and colocalization at the tumor-stroma interface of invading tumor masses. Interestingly the strongest co-expression and colocalization is detected at the invading front (Figure 3E). Colocalization appears
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