Lens Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens SungKyoung Lee, Shalini Shatadal, and Anne E. Griep Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States Correspondence: Anne E. Griep, Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, 1111 Highland Avenue, Madison, WI 53705; aegriep@wisc.edu. Submitted: July 17, 2015 Accepted: January 14, 2016 Citation: Lee S, Shatadal S, Griep AE. Dlg-1 interacts with and regulates the activities of fibroblast growth factor receptors and EphA2 in the mouse lens. Invest Ophthalmol Vis Sci. 2016;57:707–718. DOI:10.1167/ iovs.15-17727 PURPOSE. We previously showed that Discs large-1 (Dlg-1) regulates lens fiber cell structure and the fibroblast growth factor receptor (Fgfr) signaling pathway, a pathway required for fiber cell differentiation. Herein, we investigated the mechanism through which Dlg-1 regulates Fgfr signaling. METHODS. Immunofluorescence was used to measure levels of Fgfr1, Fgfr2, and activated Fgfr signaling intermediates, pErk and pAkt, in control and Dlg-1–deficient lenses that were haplodeficient for Fgfr1 or Fgfr2. Immunoblotting was used to measure levels of N-cadherin, EphA2, b-catenin, and tyrosine-phosphorylated EphA2, Fgfr1, Fgfr2, and Fgfr3 in cytoskeletal- associated and cytosolic fractions of control and Dlg-1–deficient lenses. Complex formation between Dlg-1, N-cadherin, b-catenin, Fgfr1, Fgfr2, Fgfr3, and EphA2 was assessed by coimmunoprecipitation. RESULTS. Lenses deficient for Dlg-1 and haplodeficient for Fgfr1 or Fgfr2 showed increased levels of Fgfr2 or Fgfr1, respectively. Levels of pErk and pAkt correlated with the level of Fgfr2. N-cadherin was reduced in the cytoskeletal-associated fraction and increased in the cytosolic fraction of Dlg-1–deficient lenses. Dlg-1 complexed with b-catenin, EphA2, Fgfr1, Fgfr2, and Fgfr3. EphA2 complexed with N-cadherin, b-catenin, Fgfr1, Fgfr2, and Fgfr3. Levels of these interactions were altered in Dlg-1–deficient lenses. Loss of Dlg-1 led to changes in Fgfr1, Fgfr2, Fgfr3, and EphA2 levels and to greater changes in the levels of their activation. CONCLUSIONS. Dlg-1 complexes with and regulates the activities of EphA2, Fgfr1, Fgfr2, and Fgfr3. As EphA2 contains a Psd95/Dlg/ZO-1 (PDZ) binding motif, whereas Fgfrs do not, we propose that the PDZ protein, Dlg-1, modulates Fgfr signaling through regulation of EphA2. Keywords: Dlg-1, fibroblast growth factor receptor signaling, Eph signaling, mouse, lens fiber cell differentiation T he formation and maintenance of the architecture of specialized organs are dependent on growth factor receptor signaling pathways that regulate cell proliferation, differentiation, cell–cell adhesion, cytoskeletal structure, api- cal–basal polarity, and planar cell polarity (PCP). 1–3 The ocular lens is an organ whose distinctive structure is known to be dependent on multiple growth factor receptor signaling pathways that regulate these diverse biologic processes. Identifying factors that coordinate these signaling pathways is crucial to our understanding of normal lens development and the lens pathology that forms when signaling pathways are disrupted. Dlg-1 (Discs large-1), the mouse homolog of Drosophila dlg, is required for developmental processes in multiple organs 4–6 through its regulation of cell proliferation, cell–cell adhesion, cell shape, and apical–basal and PCP. 7–9 Previously, we showed that Dlg-1 is required for lens fiber cell differentiation and maintenance of the architecture of the lens 8 and also is a modulator of the fibroblast growth factor receptor (Fgfr) signaling pathway in the mouse lens. 10 In this study, we address the possibility that Dlg-1 is a factor that regulates the interaction of the ephrin/Eph 11–16 and Fgf/Fgfr 17–19 signaling pathways, two signaling pathway that are crucial for fiber cell structure and differentiation. The lens is composed of a monolayer of epithelial cells that overlies a mass of derivative, differentiated, highly elongated cells, the fiber cells. After formation of the lens vesicle by day E10.5, cells in the anterior form the lens epithelial cells, whereas cells in the posterior undergo a terminal differentia- tion process to form the primary fiber cells. Subsequently, cells in the periphery of the epithelium undergo differentiation to form secondary fibers. Primary and secondary fiber cell differentiation is characterized by cell cycle withdrawal, cell elongation, and expression of differentiation-specific proteins. Notably, the secondary fibers take on a distinct hexagonal structure and are tightly packed into organized rows of cells that migrate along the capsule and epithelial cells until they meet their counterpart forming the lens sutures. 20,21 Among the various growth factors that are expressed in the lens, Fgfs are the only known growth factors capable of inducing lens fiber cell differentiation. 2,22,23 In mice, simulta- neous deletion of Fgfrs 1, 2, and 3 led to complete arrest of lens fiber differentiation, demonstrating that Fgfr signaling is required for lens development. 24 EphA2, a member of another family of receptor tyrosine kinases (RTKs), is expressed in the cortical lens fiber cells. 12–14 EphA2 and ephrin A-5, the ligand for EphA2 in the lens, have been shown to be required for the iovs.arvojournals.org j ISSN: 1552-5783 707 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.