DDR1 Regulates the Stabilization of Cell Surface E-Cadherin and E-Cadherin-Mediated Cell Aggregation RAJALAKSHMANAN ESWARAMOORTHY, 1 CHIH-KUANG WANG, 1,2 WEN-CHENG CHEN, 3 MING-JER TANG, 4 MEI-LING HO, 1,5 CHI-CHING HWANG, 6 HUI-MIN WANG, 7 AND CHAU-ZEN WANG 1,5 * 1 Orthopedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 3 School of Dentistry, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Department of Physiology, National Cheng-Kung University Medical College, Tainan, Taiwan 5 Department of Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung, Taiwan The stabilization of cell surface E-cadherin is important for the maintenance of apical junction complexes and epithelial polarity. Previously, we reported that discoidin domain receptor 1 (DDR1) forms a complex with E-cadherin at adhesive contacts; however, the regulatory role of DDR1 in the stabilization of cell surface E-cadherin and E-cadherin-mediated cell behaviors remained undefined. To gain insight into these questions, we utilized two stable clones depleted for DDR1 via the small interfering RNA (siRNA) technique, and we over-expressed DDR1 in MDCK cells. We performed Western blotting, immunofluorescence analysis, flow cytometry, and cell aggregation studies to investigate the effect of DDR1 on cell surface E-cadherin. The results showed that both DDR1/2 and E-cadherin use their extracellular domains to form DDR/E-cadherin complexes. Neither the depletion nor the over-expression of DDR1 changed the expression level of E-cadherin in MDCK cells. Collagen disrupted the formation of E-cadherin complexes and caused E-cadherin to accumulate in the cytoplasm; however, over-expression of DDR1 stabilized E-cadherin on the cell surface and decreased its cytoplasmic accumulation. Furthermore, independently of collagen stimulation, the depletion of DDR1 resulted in a decrease in the level of cell surface E-cadherin, which consequently caused its cytoplasmic accumulation and decreased E-cadherin-mediated cell aggregation. These results indicate that DDR1 can increase the stability of cell surface E-cadherin and promote MDCK cell aggregation, which may be mediated through the formation of DDR1/E-cadherin complexes. Overall, these findings have implications for the physiological roles of DDR1 in association with the maintenance of both the adhesion junction and epithelial polarity. J. Cell. Physiol. 224: 387–397, 2010. ß 2010 Wiley-Liss, Inc. Cadherins play a central role in intercellular adhesion, maintenance of epithelial polarity and tissue architecture (Takeichi, 1991). E-cadherin is a 120-kDa adhesive transmembrane protein that consists of a large extracellular domain and an intracellular cytoplasmic domain. Furthermore, E-cadherin is an essential component of adherens and tight junctions in polarized epithelial cells (Hajra and Fearon, 2002; Wheelock and Johnson, 2003). E-cadherin plays crucial roles in cell spreading, migration, and especially tumor suppression (van Roy and Berx, 2008). The regulation of cadherin-based adhesion has been described in numerous reports. For example, the cytoplasmic domain of E-cadherin has been shown to associate with catenins, and this association causes the formation of E-cadherin/catenin complexes, which stabilize adhesion junctions and support cell–cell adhesion (Yamada et al., 2005; Cavey et al., 2008). Hakai-mediated endocytosis of the E-cadherin complex dynamically remodels surface expressed E-cadherin at adhesive contacts (Fujita et al., 2002; Paterson et al., 2003), and tyrosine phosphorylation of the cadherin/ catenin complex causes it to dissociate (Gumbiner, 2000). Discoidin domain receptors (DDRs) are a distinct class of receptor tyrosine kinases (RTKs) that bind to collagen only as dimeric (and not as monomeric) constructs (Shrivastava et al., 1997; Leitinger, 2003; Flynn et al., 2009). Two types of DDRs have been identified, DDR1 and DDR2, which are widely expressed in fetal and adult organs (Vogel et al., 2006). In the adult mouse, the expression of DDR1 is restricted to epithelial cells, particularly in the brain, lung, kidney, and gastrointestinal Abbreviations: DDR, discoidin domain receptor; MDCK, Madin– Darby canine kidney cells; RTK, receptor tyrosine kinase; EGFR, epidermal growth factor receptor; IGFR, insulin growth factor receptor; HA, hemagglutinin; PI, propidium iodide; SDS, sodium dodecyl sulfate; FITC, fluorescein isothiocyanate. E. Rajalakshmanan and Chi-Ching Hwang contributed equally to the study. Contract grant sponsor: National Science Council of Taiwan; Contract grant number: NSC-98-2320-B-037-021. Contract grant sponsor: Economic Affairs of Taiwan; Contract grant number: 98-EC-17-A-17-S1-041. *Correspondence to: Chau-Zen Wang, Department of Physiology, College of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. E-mail: czwang@kmu.edu.tw Received 9 December 2009; Accepted 9 February 2010 Published online in Wiley InterScience (www.interscience.wiley.com.), 12 April 2010. DOI: 10.1002/jcp.22134 ORIGINAL ARTICLE 387 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.