Title: Regulation of Homotypic Cell-Cell Adhesion by Branched N-Glycosylation of N-cadherin Extracellular EC2 and EC3 Domains
Abstract: The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked β(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412–52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some β(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion- mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherin-catenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling. The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked β(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412–52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some β(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion- mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherin-catenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling. IntroductionCadherins are single-pass transmembrane receptors that mediate calcium-dependent cell-cell adhesion at adherens junctions and play an essential role in regulating major cellular behaviors, including cell growth, motility, and differentiation (1.Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1251) Google Scholar, 2.Yagi T. Takeichi M. Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar). Several cadherins, including E-cadherin and N-cadherin, have in common an extracellular domain with five segments of repeated sequences and regulate cell-cell adhesion in a homotypic manner through their association of amino-terminal extracellular domains, such as EC1 (3.Shan W.S. Koch A. Murray J. Colman D.R. Shapiro L. Biophys. Chem. 1999; 82: 157-163Crossref PubMed Scopus (33) Google Scholar, 4.Koch A.W. Bozic D. Pertz O. Engel J. Curr. Opin. Struct. Biol. 1999; 9: 275-281Crossref PubMed Scopus (126) Google Scholar). Calcium binding to the extracellular domain triggers a conformation that initiates the homotypic binding of cadherin between cells. The conserved cytoplasmic domain of cadherin interacts with various proteins, collectively termed catenins, that link cadherins to the actin-based cytoskeleton and promote strong cell-cell adhesion (5.Hirano S. Kimoto N. Shimoyama Y. Hirohashi S. Takeichi M. Cell. 1992; 70: 293-301Abstract Full Text PDF PubMed Scopus (475) Google Scholar). Evidence indicates that the formation and tyrosine phosphorylation of the cadherin-catenin complex are critical for the maintenance of the stabilization of cell-cell adhesion (6.Irby R.B. Yeatman T.J. Cancer Res. 2002; 62: 2669-2674PubMed Google Scholar, 7.Conacci-Sorrell M. Zhurinsky J. Ben-Ze'ev A. J. Clin. Invest. 2002; 109: 987-991Crossref PubMed Scopus (517) Google Scholar). It has been well documented that cadherins are implicated in the regulation of tumor invasiveness and metastatic potential. For example, loss of E-cadherin in epithelial tumors results in reduced tumor cell-cell adhesion, leading to a less adhesive, more motile, and invasive phenotype (8.Birchmeier W. Behrens J. Biochim. Biophys. Acta. 1994; 1198: 11-26Crossref PubMed Scopus (926) Google Scholar, 9.Takeichi M. Curr. Opin. Cell Biol. 1993; 5: 806-811Crossref PubMed Scopus (828) Google Scholar). By contrast, up-regulation of N-cadherin expression has been demonstrated in some invasive tumor cell lines and human tumor tissues, including mammary and esophageal squamous cell carcinoma (10.Hazan R.B. Kang L. Whooley B.P. Borgen P.I. Cell Adhes. Commun. 1997; 4: 399-411Crossref PubMed Scopus (183) Google Scholar, 11.Nagi C. Guttman M. Jaffer S. Qiao R. Keren R. Triana A. Li M. Godbold J. Bleiweiss I.J. Hazan R.B. Breast Cancer Res. Treat. 2005; 94: 225-235Crossref PubMed Scopus (73) Google Scholar, 12.Li K. Wang X. He W. Lin N. Fan Q.X. Chin. J. Cancer. 2009; 28: 8-13Google Scholar), and its expression is associated with tumor aggressiveness and increased metastatic potential in these tumors. Expression of N-cadherin in human mammary tumor cells leads to increased cell migration and invasion (13.Nieman M.T. Prudoff R.S. Johnson K.R. Wheelock M.J. J. Cell Biol. 1999; 147: 631-644Crossref PubMed Scopus (642) Google Scholar, 14.Hazan R.B. Phillips G.R. Qiao R.F. Norton L. Aaronson S.A. J. Cell Biol. 2000; 148: 779-790Crossref PubMed Scopus (746) Google Scholar) through a sustained activation of the mitogen-activated protein kinase signaling pathway (15.Suyama K. Shapiro I. Guttman M. Hazan R.B. Cancer Cell. 2002; 2: 301-314Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). These results show that N-cadherin expression is also associated with tumor invasiveness and may contribute to tumor progression.One of the most important posttranslational modifications of cadherins is N-glycosylation, which results from the action of many glycosyltransferases. There is an increasing body of evidence showing that aberrant N-glycosylation affects the location and stability of cadherin, cadherin-mediated cell-cell adhesion, and intracellular signal transduction, leading to tumor progression and metastasis (16.Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8754-8758Crossref PubMed Scopus (254) Google Scholar, 17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 18.Kitada T. Miyoshi E. Noda K. Higashiyama S. Ihara H. Matsuura N. Hayashi N. Kawata S. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 2001; 276: 475-480Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 21.Zhao H. Sun L. Wang L. Xu Z. Zhou F. Su J. Jin J. Yang Y. Hu Y. Zha X. Acta Biochim. Biophys. Sin. 2008; 40: 140-148Crossref Scopus (13) Google Scholar). For example, removal of several N-glycans from E-cadherin was reported to increase interaction of E-cadherin-catenin complexes and stabilized cell-cell contacts (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), although the mechanism was not explored in detail. Moreover, overexpression of GnT-III, 3The abbreviations used are: GnT-III and -VN-acetylglucosaminyltransferase III and V (Mgat5EC 2.4.1.155)respectivelysiRNAsmall interfering RNAL-PHAleucoagglutinating phytohemagglutininDMEMDulbecco's modified Eagle's mediumNHS-LS-biotinsulfosuccinimidyl-b-[biotin-amido]hexanoateBS3bis(sulfosuccinimidyl) suberateERKextracellular signal-regulated kinaseBSAbovine serum albuminPBSphosphate-buffered salineNcadN-cadherinPNGase Fpeptide N-glycosidase F. an enzyme catalyzing formation of “bisected” N-linked glycans, in B16 mouse melanoma cells resulted in an altered glycosylation of E-cadherin. This change in glycosylation was associated with a reduced E-cadherin turnover rate at the cell surface and reduced metastatic potential of the melanoma cells by increasing cell-cell adhesion (16.Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8754-8758Crossref PubMed Scopus (254) Google Scholar, 17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). A more recent study showed that dense cultures of human salivary epidermoid carcinoma A253 cells exhibited elevated expression of DPAGT1, the gene that initiates protein N-glycosylation (22.Nita-Lazar M. Noonan V. Rebustini I. Walker J. Menko A.S. Kukuruzinska M.A. Cancer Res. 2009; 69: 5673-5680Crossref PubMed Scopus (60) Google Scholar). Partial inhibition of DPAGT1 with small interfering RNA reduced the complex N-glycans of E-cadherin and increased the abundance of α-catenin and stabilizing proteins in adherens junctions (22.Nita-Lazar M. Noonan V. Rebustini I. Walker J. Menko A.S. Kukuruzinska M.A. Cancer Res. 2009; 69: 5673-5680Crossref PubMed Scopus (60) Google Scholar). Altered adhesion has also been observed for N-cadherin-expressing cells when N-glycosylation was altered. A study from an N-cadherin-expressing line subcloned from Madin-Darby canine kidney cells showed that incubation of these cells with tunicamycin, an inhibitor of N-glycosylation, or treatment with the O-glycosylation inhibitor BAG showed a lower molecular weight N-cadherin only after tunicamycin treatment, interpreted to mean that N-cadherin is N- but not O-glycosylated in these cells. Deletion of N-glycans by tunicamycin treatment had no impact on N-cadherin trafficking to the plasma membrane but altered its binding to catenins, consequently affecting the formation of cell-cell junctions (23.Youn Y.H. Hong J. Burke J.M. Invest. Ophthalmol. Vis. Sci. 2006; 47: 2675-2685Crossref PubMed Scopus (15) Google Scholar).Our laboratory reported that N-cadherin function was modulated by expression levels of GnT-V (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), a glycosyltransferase that functions in the synthesis of multiantennary N-glycans during glycoprotein biosynthesis. GnT-V catalyzes formation of the β(1,6)-branched N-acetylglucosamine on N-glycans (24.Brockhausen I. Carver J.P. Schachter H. Biochem. Cell Biol. 1988; 66: 1134-1151Crossref PubMed Scopus (93) Google Scholar), one of the most commonly up-regulated N-glycan structures during malignant transformation (25.Hakomori S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10231-10233Crossref PubMed Scopus (794) Google Scholar). In HT1080 human fibrosarcoma cells, overexpression of GnT-V caused increased β(1,6)-GlcNAc on N-cadherin and reduced cell-cell adhesion by promoting phosphorylation of catenins through EGFR and Src signaling pathways, resulting in a more motile phenotype. Moreover, GnT-V-deficient embryo fibroblasts isolated from GnT-V homozygous null mice (GnT-V(−/−)) showed significantly increased levels of N-cadherin-based cell-cell adhesion compared with those from GnT-V(+/+) mice (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). These results indicated that levels of particular N-linked glycans can modulate N-cadherin-associated, homotypic cell-cell adhesion and signaling, which play a crucial role in regulating cellular motility and invasiveness.N-cadherin is widely present in mesenchymal and neural cells, endothelia, and skeletal myocytes. Human N-cadherin contains eight putative N-linked glycosylation sites, although it is not known which of these sites are utilized. In the present study, we present a detailed investigation of the effects of N-cadherin N-glycosylation on cadherin-mediated cellular behaviors by using siRNA and site-directed mutagenesis strategies. Three of eight potential N-glycosylation sites on N-cadherin were identified to be N-glycosylated, present in domains EC2 and EC3, in contrast to the report on E-cadherin N-glycans (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Complex N-glycans with β(1,6) branching were identified at all of these three sites, as evidenced by binding by the lectin, L-PHA. In further contrast to the E-cadherin study, we provide evidence that inhibition or deletion of the N-glycosylation on N-cadherin had no significant effects on either N-cadherin expression on the cell surface or formation of cadherin-catenin complexes but instead led to increased cis-dimerization of N-cadherins, which led to stabilization of cell-cell contacts, increased cell-cell adhesion-mediated intracellular (outside-in) signaling, and reduced cell migration.DISCUSSIONIn the present study, a detailed investigation of the regulation of N-cadherin-mediated function by changes in N-glycosylation was performed using siRNA and site-directed mutagenesis of exogenously expressed N-cadherin. In addition, we dissected individual N-glycosylation site occupancy of N-cadherin and focused on the effects of deleting sites that expressed N-glycans, individually and in groups, on N-cadherin-related adhesive functions. Our results revealed consistently that inhibition of N-glycan expression on N-cadherin had no apparent effect on N-cadherin expression on the cell surface but stimulated N-cadherin-mediated cell-cell adhesion and suppressed migration. Moreover, we identified three potential N-glycosylation sites in extracellular domains EC2 and EC3 that expressed N-glycans that were bound by L-PHA, demonstrating that they contained β(1,6)-branched glycans.Oncogenesis is often associated with decreased cell-cell adhesion, alterations in adhesion-mediated signaling pathways, and changes in organization of the cytoskeleton that influence cell migration and invasiveness. Studies have shown that cadherin-induced cell-cell adhesion can be regulated by the level of N-glycosylation, including β(1,6) branching formed by GnT-V (16.Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8754-8758Crossref PubMed Scopus (254) Google Scholar, 17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 18.Kitada T. Miyoshi E. Noda K. Higashiyama S. Ihara H. Matsuura N. Hayashi N. Kawata S. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 2001; 276: 475-480Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 21.Zhao H. Sun L. Wang L. Xu Z. Zhou F. Su J. Jin J. Yang Y. Hu Y. Zha X. Acta Biochim. Biophys. Sin. 2008; 40: 140-148Crossref Scopus (13) Google Scholar). GnT-V, a rate-limiting and oncogene-regulated enzyme in the processing of multiantennary N-glycans during glycoprotein biosynthesis, catalyzes the formation of (GlcNAcβ(1,6)Man) branches on N-glycans (24.Brockhausen I. Carver J.P. Schachter H. Biochem. Cell Biol. 1988; 66: 1134-1151Crossref PubMed Scopus (93) Google Scholar, 25.Hakomori S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10231-10233Crossref PubMed Scopus (794) Google Scholar). Both in vitro and in vivo studies have implicated GnT-V in regulating tumor invasiveness and, in some cases, metastatic potential (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31.Guo H.B. Lee I. Kamar M. Akiyama S.K. Pierce M. Cancer Res. 2002; 62: 6837-6845PubMed Google Scholar, 44.Demetriou M. Nabi I.R. Coppolino M. Dedhar S. Dennis J.W. J. Cell Biol. 1995; 130: 383-392Crossref PubMed Scopus (250) Google Scholar, 45.Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (461) Google Scholar, 46.Seelentag W.K. Li W.P. Schmitz S.F. Metzger U. Aeberhard P. Heitz P.U. Roth J. Cancer Res. 1998; 58: 5559-5564PubMed Google Scholar, 47.Yamamoto H. Swoger J. Greene S. Saito T. Hurh J. Sweeley C. Leestma J. Mkrdichian E. Cerullo L. Nishikawa A. Ihara Y. Taniguchi N. Moskal J.R. Cancer Res. 2000; 60: 134-142PubMed Google Scholar). In an earlier study, we found that N-cadherin glycans were modified by GnT-V, and increased GnT-V expression in HT1080 cells inhibited clustering of cell surface N-cadherin. This effect on receptor clustering, in turn, enhanced the susceptibility of β-catenin and p120ctn phosphorylation by growth factors and the src oncogene, leading to decreased cell-cell adhesion, consistent with the increased migratory phenotype observed for GnT-V-overexpressing cells (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). These results suggested that the level of β(1,6) branching positively regulated tumor invasiveness-related phenotypes by altering cell-cell aggregation. Supporting this conclusion, further evidence was presented in the present study that reduced levels of GnT-V activity, resulting from GnT-V siRNA expression, enhanced homotypic cell-cell adhesion and adhesion-mediated ERK signaling and reduced cell migration/invasion of HT1080 cells. Knockdown of GnT-V in these cells had no effect on N-cadherin cell surface expression but did cause decreased expression levels of β(1,6) branching on N-cadherin. These results indicated that altered cell-cell adhesion was most likely due to changes in adhesive properties of N-cadherin regulated by GnT-V expression levels (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) rather than changes in the cell surface turnover of cadherin, which was noted for E-cadherin when GnT-III was overexpressed in melanoma cells (17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar).The regulation of N-cadherin-mediated cell-cell contact and cell migration by expression of N-glycans was further confirmed in CHO-K1 cells when exogenous N-cadherin was expressed. In CHO-K1 cells expressing exogenous N-cadherin, cell migration was inhibited due to increased cell-cell contact mediated by N-cadherin. Treatment of N-cadherin-expressing CHO-K1 cells with the N-glycan inhibitor, tunicamycin, resulted in promotion of cell-cell adhesion without affecting trafficking of N-cadherin to the cell surface, consistent with a previous report that treatment with tunicamycin induced an epithelioid phenotype in a Madin-Darby canine kidney subclonal line, clone-YH, by stabilizing homotypic N-cadherin-mediated cell junctions (23.Youn Y.H. Hong J. Burke J.M. Invest. Ophthalmol. Vis. Sci. 2006; 47: 2675-2685Crossref PubMed Scopus (15) Google Scholar). Although cell surface expression of N-cadherin was not affected by treatment of cells with N-glycosylation inhibitors, cell migration was significantly reduced, indicating that N-glycan expression can regulate N-cadherin-mediated migratory behavior. Inhibition of N-glycosylation or decreased expression of β(1,6) branching on N-cadherin both led to increased cell-cell contact and reduced cell migration. A recent study has shown that N-glycosylation of E-cadherin was cell density-dependent (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The complexity of N-glycans on E-cadherin, detected by N-glycosidase treatments and lectin binding, was decreased in dense cultures of Madin-Darby canine kidney cells with more stable adhesion junction formation, compared with sparse cultures. Another study, also using Madin-Darby canine kidney cells, showed that inhibition of N-glycan branching by swainsonine treatment tightened and stabilized cell-cell junctions (48.Vagin O. Tokhtaeva E. Yakubov I. Shevchenko E. Sachs G. J. Biol. Chem. 2008; 283: 2192-2202Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The normal development of cell-cell adhesion in these cells was associated with reduced complexity of E-cadherin N-glycans as well as those on the Na/K-ATPase β1 subunit, a membrane transport enzyme that has been found important for intercellular adhesion. Interestingly, these changes were found to be associated with increased expression of GnT-III, which forms bisecting GlcNAc on hybrid or complex N-glycan structures and consequently reduces branched glycan formation, but decreased expression of GnT-IV and GnT-V mRNA. Our results are in agreement with these observations and suggest that the regulation of cell-cell contact formation by changes in N-glycosylation or reduction of expression of GnT-V-modified N-glycans may be a general means to affect cadherin function that includes N- and E-cadherin and other adhesion-related membrane proteins, such as Na/K-ATPase.An increasing number of studies have explored the effects of altering the N-glycosylation of particular cell surface adhesion molecules using site-directed mutagenesis, including integrins (49.Sato Y. Isaji T. Tajiri M. Yoshida-Yamamoto S. Yoshinaka T. Somehara T. Fukuda T. Wada Y. Gu J. J. Biol. Chem. 2009; 284: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 50.Isaji T. Sato Y. Fukuda T. Gu J. J. Biol. Chem. 2009; 284: 12207-12216Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 51.Isaji T. Sato Y. Zhao Y. Miyoshi E. Wada Y. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 33258-33267Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), cadherins (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and Na,K-ATPase β1 (48.Vagin O. Tokhtaeva E. Yakubov I. Shevchenko E. Sachs G. J. Biol. Chem. 2008; 283: 2192-2202Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Structural studies have shown that cadherin-induced cell-cell contacts are mediated by the extracellular portion of cadherins, which is divided into five homologous repeats (∼110 amino acids) of extracellular cadherin domains (EC1–EC5) (52.Takeichi M. Annu. Rev. Biochem. 1990; 59: 237-252Crossref PubMed Scopus (1108) Google Scholar). EC1 and EC2 have been most implicated in being responsible for cadherin adhesive activity via different mechanisms (53.Chappuis-Flament S. Wong E. Hicks L.D. Kay C.M. Gumbiner B.M. J. Cell Biol. 2001; 154: 231-243Crossref PubMed Scopus (188) Google Scholar, 54.Pertz O. Bozic D. Koch A.W. Fauser C. Brancaccio A. Engel J. EMBO J. 1999; 18: 1738-1747Crossref PubMed Scopus (341) Google Scholar). EC1 was also found to be responsible for the binding specificity of cadherins (55.Shan W.S. Tanaka H. Phillips G.R. Arndt K. Yoshida M. Colman D.R. Shapiro L. J. Cell Biol. 2000; 148: 579-590Crossref PubMed Scopus (165) Google Scholar). EC4 is believed to play a major role in regulating N-cadherin-mediated epithelial to mesenchymal transition and increased motility (56.Kim J.B. Islam S. Kim Y.J. Prudoff R.S. Sass K.M. Wheelock M.J. Johnson K.R. J. Cell Biol. 2000; 151: 1193-1206Crossref PubMed Scopus (177) Google Scholar). Although the amino acid sequence of N-cadherin contains eight putative N-linked glycosylation sites through EC1–EC5, no information was available on the number of these sites that are glycosylated. To examine the site occupancy of N-glycosylation, we generated different N-glycosylation mutants in which Asn of the consensus site, Asn-Xaa-Ser/Thr, was mutated to Gln, where the least perturbation was expected (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 40.Ray K. Clapp P. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1998; 273: 34558-34567Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), using site mutagenesis. Based on the observation of consistent molecular shifts of mutant N-cadherins compared with the native form, measured from multiple SDS-PAGE experiments, we found that three of eight putative sites were N-glycosylated, Asn207 and Asn325 in EC2 and Asn402 in EC3. By contrast, mutation of the remaining sites did not reveal consistent band shifts upon SDS-PAGE, although these sites were located in the adhesion-regulating domain EC1 (Asn190) and migration-regulating domain EC4 (Asn572) or were conserved (Asn572 and Asn651) among all six of the common human cadherins. Interestingly, the mutant Nall, in which all eight potential sites were mutated, still showed a faster migration of N-cadherin on the gel (Fig. 3C) when compared with that of the N2/3/4 mutant, indicating that some of sites that were not glycosylated in the native protein may acquire N-glycosylation in a mutant in which all glycosylation sites that are normally utilized have been mutated. This compensatory N-glycosylation has been observed (40.Ray K. Clapp P. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1998; 273: 34558-34567Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and may suggest a cellular response to disruption of N-glycosylation of proteins or simply an alteration in protein structures that makes available normally non-utilized sites. Results from glycosidase digestions of N-cadherins showed that both native N-cadherins and N-cadherins in which at least one N-glycan was expressed were all PNGase F-sensitive, with little or no sensitivity to endoglycosidase H, indicating that N-cadherin expressed complex N-glycans. Interestingly, PNGase F digestion of N-cadherins seemed to be incomplete (Fig. 6). However, when Asn402 was mutated (mutants N4, N2/4, N3/4, and N2/3/4), deglycosylated N-cadherins showed increased mobility on SDS-polyacrylamide gel in two different cell lines (CHO-K1 and MCF-7), which was identical to the mobility of N-cadherins from tunicamycin-treated cells (Fig. 6, A and B), indicating that the resistance of N-glycosylation on Asn402 to PNGase F digestion was not cell type-dependent and either that this glycosylation site might contain some specific N-glycan structures that resist PNGase F digestion or that its removal may render some conformation changes that consequently make mutants more sensitive to PNGase F digestion. The finding that the three utilized sites in N-cadherin expressed complex N-glycans was further confirmed by L-PHA precipitation experiments in both CHO-K1 and MCF-7 cells, showing that all three active sites contained complex N-glycans with β(1,6) branching.We also found that mutation of single employed N-glycosylation sites did not significantly increase N-cadherin-mediated aggregation of CHO-K1 cells expressing N-cadherin. This result is probably due to the insensitivity of the assay employed (visual inspection). However, removal of the three utilized sites and all putative sites caused a remarkable enhancement of cell-cell adhesion, calcium-dependent cell-cell contact-mediated outside-in ERK signaling, and a consequent reduction in the rate of wound closure in the cell migration assay. These results are consistent with our observations on the effects on cell-cell adhesion of HT1080 cells with reduced branched N-glycan expression from expression of siRNA of GnT-V and the results of N-glycan inhibitor-treated CHO cells expressing N-cadherin, confirming the hypothesis that reduced N-glycosylation branching or deletion stabilizes cell-cell contact and inhibits cell migration.A previous study showed that overexpression of GnT-III in B16 mouse melanoma cells resulted in an altered glycosylation of E-cadherin (increased levels of the “bisected” N-linked glycan but decreased expression of the N-linked β(1,6) branch) that consequently delayed the E-cadherin turnover rate at the cell surface and reduced the lung metastatic potential of the melanoma cells (17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Another recent report, however, showed a reduction in total levels of E-cadherin expression after N-glycosylation sites were mutated (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In our study, by contrast, N-cadherin expression levels were not significantly affected by either GnT-V-directed siRNA expression or deletion of N-glycosylation by either N-glycan inhibitor treatment or site mutation, suggesting that altered N-cadherin adhesive function was not due to changes in N-cadherin expression but most likely due to changes in cell-cell cadherin binding.Moreover, in contrast to the work on E-cadherin, our results with N-cadherin demonstrate that the three sites with N-glycosylation are in domains EC2 and EC3, not domains EC4 and EC5 (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and that the mechanism of regulation does not involve catenin-cadherin interactions; rather, the regulation point appears to be in dimerization of the cadherin monomers prior to the trans-binding of dimers on apposing cell surfaces. It is generally believed that cis-dimerization of cadherins yields the functional unit of cadherin adhesion, formed by binding of two cadherin extracellular domains on the same cell surface. The interaction of a cadherin cis-dimer on one cell with a cis-dimer on an adjacent cell leads to formation of low affinity trans-dimers of cadherin, which initiate cell-cell adhesion (57.Niessen C.M. Gottardi C.J. Biochim. Biophys. Acta. 2008; 1778: 562-571Crossref PubMed Scopus (189) Google Scholar, 58.Gumbiner B.M. J. Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (687) Google Scholar). EC1 and EC2 have been reported to be involved in cadherin cis- and trans-dimer formation (59.Boggon T.J. Murray J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. Science. 2002; 296: 1308-1313Crossref PubMed Scopus (538) Google Scholar, 60.Häussinger D. Ahrens T. Aberle T. Engel J. Stetefeld J. Grzesiek S. EMBO J. 2004; 23: 1699-1708Crossref PubMed Scopus (114) Google Scholar, 61.Zhang Y. Sivasankar S. Nelson W.J. Chu S. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 109-114Crossref PubMed Scopus (137) Google Scholar). Furthermore, cadherins form the adherens junction via association of their cytoplasmic tails with several intracellular proteins known as the catenins, which link cadherins to the actin-based cytoskeleton and promote stronger cell-cell adhesion (62.Cavallaro U. Christofori G. Nat. Rev. Cancer. 2004; 4: 118-132Crossref PubMed Scopus (1037) Google Scholar, 63.Wheelock M.J. Johnson K.R. Curr. Opin. Cell Biol. 2003; 15: 509-514Crossref PubMed Scopus (231) Google Scholar). Although studies showed that deletion of N-glycans caused increased interaction of cadherin with catenins and vinculin (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 23.Youn Y.H. Hong J. Burke J.M. Invest. Ophthalmol. Vis. Sci. 2006; 47: 2675-2685Crossref PubMed Scopus (15) Google Scholar), in the present study, the interaction of cadherin and catenins appeared to be unaffected by inhibition of N-glycosylation.To test the hypothesis that the effects of N-glycan expression on N-cadherin function involved cis-dimer formation, chemical cross-linking experiments were performed. The results showed that increased cell-cell contact caused by inhibition of N-glycosylation resulted most likely from altered cis-dimer formation of N-cadherin after inhibition of N-glycosylation.A recent study from our laboratory using SH-SY5Y neuroblastoma cells showed that increased activity of GnT-Vb, a paralog of GnT-V whose expression is restricted mainly to neural cells, promoted the addition of the O-mannosyl-linked HNK-1 modification found on the developmentally regulated and neuron-specific receptor protein-tyrosine phosphatase β (64.Abbott K.L. Matthews R.T. Pierce M. J. Biol. Chem. 2008; 283: 33026-33035Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). These changes in glycosylation resulted in decreased cell-cell adhesion and increased rates of migration on laminin, accompanied by increased phosphorylated β-catenin. Expression of siRNA directed toward GnT-Vb transcripts in these cells had the opposite effects, as expected. Moreover, overexpression of GnT-V in HT1080 human fibrosarcoma cells caused increased β(1,6)-GlcNAc on N-cadherin and reduced cell-cell adhesion by enhancing phosphorylation of catenins (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Altered phosphorylation of catenins, therefore, appears to be a common mechanism by which changes in GnT-V and GnT-Vb expression result in altered cell-cell adhesion and enhanced migration.In conclusion, our results, along with those from E-cadherin N-glycan studies, strongly support the conclusion that N-glycosylation plays a prominent role in regulating cadherin-mediated adhesion and its intracellular signaling pathways, which can contribute to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling. IntroductionCadherins are single-pass transmembrane receptors that mediate calcium-dependent cell-cell adhesion at adherens junctions and play an essential role in regulating major cellular behaviors, including cell growth, motility, and differentiation (1.Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1251) Google Scholar, 2.Yagi T. Takeichi M. Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar). Several cadherins, including E-cadherin and N-cadherin, have in common an extracellular domain with five segments of repeated sequences and regulate cell-cell adhesion in a homotypic manner through their association of amino-terminal extracellular domains, such as EC1 (3.Shan W.S. Koch A. Murray J. Colman D.R. Shapiro L. Biophys. Chem. 1999; 82: 157-163Crossref PubMed Scopus (33) Google Scholar, 4.Koch A.W. Bozic D. Pertz O. Engel J. Curr. Opin. Struct. Biol. 1999; 9: 275-281Crossref PubMed Scopus (126) Google Scholar). Calcium binding to the extracellular domain triggers a conformation that initiates the homotypic binding of cadherin between cells. The conserved cytoplasmic domain of cadherin interacts with various proteins, collectively termed catenins, that link cadherins to the actin-based cytoskeleton and promote strong cell-cell adhesion (5.Hirano S. Kimoto N. Shimoyama Y. Hirohashi S. Takeichi M. Cell. 1992; 70: 293-301Abstract Full Text PDF PubMed Scopus (475) Google Scholar). Evidence indicates that the formation and tyrosine phosphorylation of the cadherin-catenin complex are critical for the maintenance of the stabilization of cell-cell adhesion (6.Irby R.B. Yeatman T.J. Cancer Res. 2002; 62: 2669-2674PubMed Google Scholar, 7.Conacci-Sorrell M. Zhurinsky J. Ben-Ze'ev A. J. Clin. Invest. 2002; 109: 987-991Crossref PubMed Scopus (517) Google Scholar). It has been well documented that cadherins are implicated in the regulation of tumor invasiveness and metastatic potential. For example, loss of E-cadherin in epithelial tumors results in reduced tumor cell-cell adhesion, leading to a less adhesive, more motile, and invasive phenotype (8.Birchmeier W. Behrens J. Biochim. Biophys. Acta. 1994; 1198: 11-26Crossref PubMed Scopus (926) Google Scholar, 9.Takeichi M. Curr. Opin. Cell Biol. 1993; 5: 806-811Crossref PubMed Scopus (828) Google Scholar). By contrast, up-regulation of N-cadherin expression has been demonstrated in some invasive tumor cell lines and human tumor tissues, including mammary and esophageal squamous cell carcinoma (10.Hazan R.B. Kang L. Whooley B.P. Borgen P.I. Cell Adhes. Commun. 1997; 4: 399-411Crossref PubMed Scopus (183) Google Scholar, 11.Nagi C. Guttman M. Jaffer S. Qiao R. Keren R. Triana A. Li M. Godbold J. Bleiweiss I.J. Hazan R.B. Breast Cancer Res. Treat. 2005; 94: 225-235Crossref PubMed Scopus (73) Google Scholar, 12.Li K. Wang X. He W. Lin N. Fan Q.X. Chin. J. Cancer. 2009; 28: 8-13Google Scholar), and its expression is associated with tumor aggressiveness and increased metastatic potential in these tumors. Expression of N-cadherin in human mammary tumor cells leads to increased cell migration and invasion (13.Nieman M.T. Prudoff R.S. Johnson K.R. Wheelock M.J. J. Cell Biol. 1999; 147: 631-644Crossref PubMed Scopus (642) Google Scholar, 14.Hazan R.B. Phillips G.R. Qiao R.F. Norton L. Aaronson S.A. J. Cell Biol. 2000; 148: 779-790Crossref PubMed Scopus (746) Google Scholar) through a sustained activation of the mitogen-activated protein kinase signaling pathway (15.Suyama K. Shapiro I. Guttman M. Hazan R.B. Cancer Cell. 2002; 2: 301-314Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). These results show that N-cadherin expression is also associated with tumor invasiveness and may contribute to tumor progression.One of the most important posttranslational modifications of cadherins is N-glycosylation, which results from the action of many glycosyltransferases. There is an increasing body of evidence showing that aberrant N-glycosylation affects the location and stability of cadherin, cadherin-mediated cell-cell adhesion, and intracellular signal transduction, leading to tumor progression and metastasis (16.Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8754-8758Crossref PubMed Scopus (254) Google Scholar, 17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 18.Kitada T. Miyoshi E. Noda K. Higashiyama S. Ihara H. Matsuura N. Hayashi N. Kawata S. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 2001; 276: 475-480Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 21.Zhao H. Sun L. Wang L. Xu Z. Zhou F. Su J. Jin J. Yang Y. Hu Y. Zha X. Acta Biochim. Biophys. Sin. 2008; 40: 140-148Crossref Scopus (13) Google Scholar). For example, removal of several N-glycans from E-cadherin was reported to increase interaction of E-cadherin-catenin complexes and stabilized cell-cell contacts (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), although the mechanism was not explored in detail. Moreover, overexpression of GnT-III, 3The abbreviations used are: GnT-III and -VN-acetylglucosaminyltransferase III and V (Mgat5EC 2.4.1.155)respectivelysiRNAsmall interfering RNAL-PHAleucoagglutinating phytohemagglutininDMEMDulbecco's modified Eagle's mediumNHS-LS-biotinsulfosuccinimidyl-b-[biotin-amido]hexanoateBS3bis(sulfosuccinimidyl) suberateERKextracellular signal-regulated kinaseBSAbovine serum albuminPBSphosphate-buffered salineNcadN-cadherinPNGase Fpeptide N-glycosidase F. an enzyme catalyzing formation of “bisected” N-linked glycans, in B16 mouse melanoma cells resulted in an altered glycosylation of E-cadherin. This change in glycosylation was associated with a reduced E-cadherin turnover rate at the cell surface and reduced metastatic potential of the melanoma cells by increasing cell-cell adhesion (16.Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8754-8758Crossref PubMed Scopus (254) Google Scholar, 17.Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). A more recent study showed that dense cultures of human salivary epidermoid carcinoma A253 cells exhibited elevated expression of DPAGT1, the gene that initiates protein N-glycosylation (22.Nita-Lazar M. Noonan V. Rebustini I. Walker J. Menko A.S. Kukuruzinska M.A. Cancer Res. 2009; 69: 5673-5680Crossref PubMed Scopus (60) Google Scholar). Partial inhibition of DPAGT1 with small interfering RNA reduced the complex N-glycans of E-cadherin and increased the abundance of α-catenin and stabilizing proteins in adherens junctions (22.Nita-Lazar M. Noonan V. Rebustini I. Walker J. Menko A.S. Kukuruzinska M.A. Cancer Res. 2009; 69: 5673-5680Crossref PubMed Scopus (60) Google Scholar). Altered adhesion has also been observed for N-cadherin-expressing cells when N-glycosylation was altered. A study from an N-cadherin-expressing line subcloned from Madin-Darby canine kidney cells showed that incubation of these cells with tunicamycin, an inhibitor of N-glycosylation, or treatment with the O-glycosylation inhibitor BAG showed a lower molecular weight N-cadherin only after tunicamycin treatment, interpreted to mean that N-cadherin is N- but not O-glycosylated in these cells. Deletion of N-glycans by tunicamycin treatment had no impact on N-cadherin trafficking to the plasma membrane but altered its binding to catenins, consequently affecting the formation of cell-cell junctions (23.Youn Y.H. Hong J. Burke J.M. Invest. Ophthalmol. Vis. Sci. 2006; 47: 2675-2685Crossref PubMed Scopus (15) Google Scholar).Our laboratory reported that N-cadherin function was modulated by expression levels of GnT-V (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), a glycosyltransferase that functions in the synthesis of multiantennary N-glycans during glycoprotein biosynthesis. GnT-V catalyzes formation of the β(1,6)-branched N-acetylglucosamine on N-glycans (24.Brockhausen I. Carver J.P. Schachter H. Biochem. Cell Biol. 1988; 66: 1134-1151Crossref PubMed Scopus (93) Google Scholar), one of the most commonly up-regulated N-glycan structures during malignant transformation (25.Hakomori S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10231-10233Crossref PubMed Scopus (794) Google Scholar). In HT1080 human fibrosarcoma cells, overexpression of GnT-V caused increased β(1,6)-GlcNAc on N-cadherin and reduced cell-cell adhesion by promoting phosphorylation of catenins through EGFR and Src signaling pathways, resulting in a more motile phenotype. Moreover, GnT-V-deficient embryo fibroblasts isolated from GnT-V homozygous null mice (GnT-V(−/−)) showed significantly increased levels of N-cadherin-based cell-cell adhesion compared with those from GnT-V(+/+) mice (19.Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). These results indicated that levels of particular N-linked glycans can modulate N-cadherin-associated, homotypic cell-cell adhesion and signaling, which play a crucial role in regulating cellular motility and invasiveness.N-cadherin is widely present in mesenchymal and neural cells, endothelia, and skeletal myocytes. Human N-cadherin contains eight putative N-linked glycosylation sites, although it is not known which of these sites are utilized. In the present study, we present a detailed investigation of the effects of N-cadherin N-glycosylation on cadherin-mediated cellular behaviors by using siRNA and site-directed mutagenesis strategies. Three of eight potential N-glycosylation sites on N-cadherin were identified to be N-glycosylated, present in domains EC2 and EC3, in contrast to the report on E-cadherin N-glycans (20.Liwosz A. Lei T. Kukuruzinska M.A. J. Biol. Chem. 2006; 281: 23138-23149Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Complex N-glycans with β(1,6) branching were identified at all of these three sites, as evidenced by binding by the lectin, L-PHA. In further contrast to the E-cadherin study, we provide evidence that inhibition or deletion of the N-glycosylation on N-cadherin had no significant effects on either N-cadherin expression on the cell surface or formation of cadherin-catenin complexes but instead led to increased cis-dimerization of N-cadherins, which led to stabilization of cell-cell contacts, increased cell-cell adhesion-mediated intracellular (outside-in) signaling, and reduced cell migration.