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• W2044304997 abstract "Close homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules expressed by subpopulations of neurons and glia in the central and peripheral nervous system. It promotes neurite outgrowth and neuronal survival in vitro. This study describes a novel function for CHL1 in potentiating integrin-dependent cell migration toward extracellular matrix proteins. Expression of CHL1 in HEK293 cells stimulated their haptotactic migration toward collagen I, fibronectin, laminin, and vitronectin substrates in Transwell assays. CHL1-potentiated cell migration to collagen I was dependent on α1β1 and α2β1 integrins, as shown with function blocking antibodies. Potentiated migration relied on the early integrin signaling intermediates c-Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Enhancement of migration was disrupted by mutation of a potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA) in the sixth immunoglobulin domain of CHL1, suggesting that CHL1 functionally interacts with β1 integrins through this domain. CHL1 was shown to associate with β1 integrins on the cell surface by antibody-induced co-capping. Through a cytoplasmic domain sequence containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Tyr), CHL1 recruited the actin cytoskeletal adapter protein ankyrin to the plasma membrane, and this sequence was necessary for promoting integrin-dependent migration to extracellular matrix proteins. These results support a role for CHL1 in integrin-dependent cell migration that may be physiologically important in regulating cell migration in nerve regeneration and cortical development. Close homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules expressed by subpopulations of neurons and glia in the central and peripheral nervous system. It promotes neurite outgrowth and neuronal survival in vitro. This study describes a novel function for CHL1 in potentiating integrin-dependent cell migration toward extracellular matrix proteins. Expression of CHL1 in HEK293 cells stimulated their haptotactic migration toward collagen I, fibronectin, laminin, and vitronectin substrates in Transwell assays. CHL1-potentiated cell migration to collagen I was dependent on α1β1 and α2β1 integrins, as shown with function blocking antibodies. Potentiated migration relied on the early integrin signaling intermediates c-Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Enhancement of migration was disrupted by mutation of a potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA) in the sixth immunoglobulin domain of CHL1, suggesting that CHL1 functionally interacts with β1 integrins through this domain. CHL1 was shown to associate with β1 integrins on the cell surface by antibody-induced co-capping. Through a cytoplasmic domain sequence containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Tyr), CHL1 recruited the actin cytoskeletal adapter protein ankyrin to the plasma membrane, and this sequence was necessary for promoting integrin-dependent migration to extracellular matrix proteins. These results support a role for CHL1 in integrin-dependent cell migration that may be physiologically important in regulating cell migration in nerve regeneration and cortical development. In vertebrates, the L1 family of cell adhesion molecules (CAMs) 1The abbreviations used are: CAM, cell adhesion molecule; CHL1, close homolog of L1; GFP, green fluorescent protein; MAP, mitogen-activated protein; MEK, MAP kinase/extracellular signal-regulated kinase kinase; PI, phosphatidylinositol; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HBSS, Hanks' balanced saline solution; TRITC, tetramethylrhodamine isothiocyanate. is comprised of four members: L1/NgCAM, close homolog of L1 (CHL1), neurofascin, and NrCAM, all serving multiple functions in the development and function of the nervous system. L1, the prototype of this family of transmembrane glycoproteins, has been shown to participate in cell migration, axon fasciculation, and guidance, as well as synaptogenesis and adult synaptic plasticity (1Schmid R.S. Maness P.F. Kalverboer A.F. Gramsbergen A. Handbook on Brain and Behaviour in Human Development. Kluwer Academic Publishers, Groningen, The Netherlands2001: 199-218Google Scholar). The importance of L1 function in human brain development is revealed by the association of mutations in the L1 gene with a syndromic form of mental retardation, the L1 syndrome, formerly named CRASH (corpus callosum agenesis, mental retardation, adducted thumbs, spasticity, and hydrocephalus) (2Rosenthal A. Jouet M. Kenwrick S. Nat. Genet. 1992; 2: 107-112Google Scholar, 3Fransen E. VanCamp G. Vits L. Willems P.J. Hum. Mol. Genet. 1997; 6: 1625-1632Google Scholar, 4Kenwrick S. Watkins A. Angelis E.D. Hum. Mol. Genet. 2000; 9: 879-886Google Scholar). L1 knockout mice display nervous system anomalies similar to those seen in human patients, although there appear to be additional genetic modifiers of the disease (5Dahme M. Bartsch U. Martini R. Anliker B. Schachner M. Mantei N. Nat. Genet. 1997; 17: 346-349Google Scholar, 6Cohen N.R. Taylor J.S.H. Scott L.B. Guillery R.W. Soriano P. Furley A.J.W. Curr. Biol. 1997; 8: 26-33Google Scholar, 7Demyanenko G. Tsai A. Maness P.F. J. Neurosci. 1999; 19: 4907-4920Google Scholar, 8Demyanenko G.P. Shibata Y. Maness P.F. Brain Res. Dev. Brain Res. 2001; 126: 21-30Google Scholar). L1 family members share a structural plan consisting of an extracellular region comprised of six Ig-like domains, four or five fibronectin type III domains, a single transmembrane segment, and a short, conserved cytoplasmic region (9Brummendorf T. Lemmon V. Curr. Opin. Cell Biol. 2001; 13: 611-618Google Scholar). The extracellular portion of these proteins is highly glycosylated and allows them to participate in both homophilic and heterophilic interactions with a variety of ligands, including other members of the Ig superfamily. The cytoplasmic domain of L1 family members interacts with components of the actin cytoskeleton (10Dickson T.C. Mintz C.D. Benson D.L. Salton S.R. J. Cell Biol. 2002; 157: 1105-1112Google Scholar), protein kinases (11Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Biol. Chem. 1996; 271: 18217-18223Google Scholar, 12Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Google Scholar, 13Kamiguchi H. Lemmon V. J. Neurosci. Res. 1997; 49: 1-8Google Scholar), and complexes associated with endocytosis and protein trafficking in a lipid raft-associated manner (14Kamiguchi H. Long K.E. Pendergast M. Schaefer A.W. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Google Scholar, 15Nakai Y. Kamiguchi H. J. Cell Biol. 2002; 159: 1097-1108Google Scholar). An important binding partner is ankyrin, a protein that binds to the subcortical actin/spectrin cytoskeleton (16Davis J.Q. Bennett V. J. Biol. Chem. 1994; 269: 27163-27166Google Scholar, 17Bennett V. Chen L. Curr. Opin. Cell Biol. 2001; 13: 61-67Google Scholar). The interaction of L1 with ankyrin occurs through a conserved FIGQY sequence (Phe-Ile-Gly-Gln-Tyr) within the cytoplasmic domain and is proposed to stabilize axonal membranes and/or intercellular connections (18Bennett V. Baines A.J. Physiol. Rev. 2001; 81: 1353-1392Google Scholar). This idea is supported by the finding that in mice lacking ankyrin B, the axons eventually degenerate (19Scotland P. Zhou D. Benveniste H. Bennett V. J. Cell Biol. 1998; 143: 1305-1315Google Scholar), although initial axon outgrowth and L1 targeting are relatively normal. CHL1 is a newly identified member of the L1 family that is expressed in subpopulations of developing neurons in the central and peripheral nervous systems and that persists at low levels in the mature brain in areas of high plasticity (20Hillenbrand R. Molthagen M. Montag D. Schachner M. Eur. J. Neurosci. 1999; 11: 813-826Google Scholar, 21Liu Q. Dwyer N.D. O'Leary D.D. J. Neurosci. 2000; 20: 7682-7690Google Scholar). CHL1 is also expressed by Schwann cells, astrocytes, and oligodendrocyte precursors (22Holm J. Hillenbrand R. Steuber V. Bartsch U. Moos M. Lubbert H. Montag D. Schachner M. Eur. J. Neurosci. 1996; 8: 1613-1629Google Scholar) and is strikingly up-regulated in Schwann cells and sensory neurons upon nerve crush injury (23Zhang Y. Roslan R. Lang D. Schachner M. Lieberman A.R. Anderson P.N. Mol. Cell. Neurosci. 2000; 16: 71-86Google Scholar). The CALL gene, the human ortholog of the CHL1 gene (24Wei M.H. Karavanova I. Ivanov S.V. Popescu N.C. Keck C.L. Pack S. Eisen J.A. Lerman M.I. Hum. Genet. 1998; 103: 355-364Google Scholar), is closely linked to the 3p– syndrome characterized by mental retardation (25Angeloni D. Lindor N.M. Pack S. Latif F. Wei M.H. Lerman M.I. Am. J. Med. Genet. 1999; 86: 482-485Google Scholar). The human CHL1 gene is mutated in a patient with mental retardation (26Frints S.G.M. Marynen P. Fryns J.P. Schachner M. Rolf B. D'Hooge R. De Deyn P.P. Hartmann D. Froyen G. J. Hum. Genet. 2003; (in press)Google Scholar). An increased risk for schizophrenia associated with a missense polymorphism has also been reported (27Sakurai K. Migita O. Toru M. Arinami T. Mol. Psychiatry. 2002; 7: 412-415Google Scholar). A recent study (28Montag-Sallaz M. Schachner M. Montag D. Mol. Cell. Biol. 2002; 22: 7967-7981Google Scholar) showed that CHL1-deficient mice display misguided axons within the hippocampus and olfactory tract and anomalies in behavior. These findings emphasize the importance of CHL1 in the nervous system, although its specific functions are yet unknown. CHL1 shares the basic structural plan of L1 family members (22Holm J. Hillenbrand R. Steuber V. Bartsch U. Moos M. Lubbert H. Montag D. Schachner M. Eur. J. Neurosci. 1996; 8: 1613-1629Google Scholar) and has strong neurite outgrowth promoting capacity (20Hillenbrand R. Molthagen M. Montag D. Schachner M. Eur. J. Neurosci. 1999; 11: 813-826Google Scholar). The sequence of CHL1 reveals ∼60% amino acid identity with L1 in the extracellular region and ∼40% identity in the cytoplasmic domain. Yet in contrast to L1, CHL1 is not capable of self-associating, nor does it bind heterophilically to L1, and an extracellular domain-binding partner remains to be found (20Hillenbrand R. Molthagen M. Montag D. Schachner M. Eur. J. Neurosci. 1999; 11: 813-826Google Scholar). The extracellular segment of CHL1 contains a potential integrin-binding motif Arg-Gly-Asp (RGD) in the second Ig-like domain, where it may be masked by the predicted horseshoe conformation of the molecule (29Freigang J. Proba K. Leder L. Diederichs K. Sonderegger P. Welte W. Cell. 2000; 101: 425-433Google Scholar, 30Kunz B. Lierheimer R. Rader C. Spirig M. Ziegler U. Sonderegger P. J. Biol. Chem. 2002; 277: 4551-4557Google Scholar), rather than within the sixth Ig-like domain as in L1. However, the sixth Ig domain of CHL1 contains another potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA), which has been reported to mediate integrin-collagen interactions in platelets (31Staatz W.D. Fok K.F. Zutter M.M. Adams S.P. Rodriguez B.A. Santoro S.A. J. Biol. Chem. 1991; 266: 7363-7367Google Scholar). Finally, CHL1 is the only L1 family member with an altered sequence (FIGAY) in the presumed ankyrin-binding domain, and it lacks the RSLE motif, which is characteristic of other family members and is involved in endocytosis. The structural resemblance and the differences between CHL1 and the other members of the L1 family of CAMs suggest that this molecule might have both similar and distinctive functions within cells. Recently it was shown that L1 promotes integrin-mediated haptotactic migration of cultured cells toward extracellular matrix proteins (32Ruppert M. Aigner S. Hubbe M. Yagita H. Altevogt P. J. Cell Biol. 1995; 131: 1881-1891Google Scholar, 33Ebeling O. Duczmal A. Aigner S. Geiger C. Schollhammer S. Kemshead J.T. Moller P. Schwartz-Albiez R. Altevogt P. Eur. J. Immunol. 1996; 26: 2508-2516Google Scholar, 34Montgomery A.M. Becker J.C. Siu C.H. Lemmon V.P. Cheresh D.A. Pancook J.D. Zhao X. Reisfeld R.A. J. Cell Biol. 1996; 132: 475-485Google Scholar, 35Felding-Habermann B. Silletti S. Mei F. Siu C.H. Yip P.M. Brooks P.C. Cheresh D.A. O'Toole T.E. Ginsberg M.H. Montgomery A.M. J. Cell Biol. 1997; 139: 1567-1581Google Scholar, 36Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Google Scholar, 37Thelen K. Kedar V. Panicker A.K. Schmid R.S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Google Scholar). Because of potential integrin interaction motifs in CHL1, it was speculated that CHL1 may have a role in integrin-mediated cell migration. Here it is shown that CHL1 promotes haptotactic cell migration toward extracellular matrix proteins, but this function differs from L1 in regard to preference of extracellular matrix substrate and integrin partners. These differences are related to the structural differences between CHL1 and L1 and may be important for differentially regulating cell migration during nerve regeneration and neuronal migration during development. Plasmids and Reagents—The following cDNAs were subcloned into pcDNA3 (Stratagene, La Jolla, CA): wild type mouse CHL1, a CHL1 mutant in which the RGD sequence in the Ig2 domain was mutated to KGE, a CHL1 mutant in which the DGEA sequence in the Ig6 domain was mutated to AGEV, a CHL1 mutant in which the cytoplasmic sequence FIGAY was mutated to FIGAA, and human wild type L1 (+RSLE) (from J. Hemperly, BD Technologies, Research Triangle Park, NC). A plasmid encoding ankyrin G fused to green fluorescent protein (ankyrin-GFP) was provided by Vann Bennett (Duke University, Durham, NC). A rabbit polyclonal antibody was made against mouse CHL1-Fc (22Holm J. Hillenbrand R. Steuber V. Bartsch U. Moos M. Lubbert H. Montag D. Schachner M. Eur. J. Neurosci. 1996; 8: 1613-1629Google Scholar). Mouse monoclonal antibody Neuro4 against an extracellular epitope of human L1 was a gift of J. Hemperly. The following antibodies were obtained from Chemicon (Temecula, CA): anti-human β1 integrin monoclonal antibody 2253Z (clone 6s6), an activating monoclonal antibody MAB2000 (clone HB1.1) against human β1 integrin, anti-human α1 integrin monoclonal antibody 1973Z (clone FB12), and anti-human α2 integrin monoclonal antibody 1950Z (clone P1E6). Nonimmune mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). Human vitronectin, human fibronectin, and murine laminin were from Invitrogen and Peninsula Laboratories (San Carlos, CA). BD Biosciences (Palo Alto, CA) provided type I collagen from rat tail. MEK inhibitor U0126 (Promega, Madison, WI), Src inhibitor PP2 and inactive analog PP3, and PI 3-kinase inhibitor Ly294002 (CalBiochem, San Diego, CA) were dissolved in Me2SO. Cell Culture and Haptotactic Migration Assay—HEK293 cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) (Mediatech Cellgro), 4.5 mg/ml glucose, 10% heat-inactivated fetal bovine serum, 50 μg/ml gentamicin, and 250 μg/ml kanamycin. Cultures at 80–90% confluence in 60-mm dishes were transfected for transient expression of CHL1 or L1 pcDNA3 plasmids (5 μg) using Lipofect-AMINE 2000 (Invitrogen) in Opti-MEM I (Invitrogen). After 18–24hat 37 °C, the transfected cells were used for haptotactic migration or ankyrin recruitment assays. Haptotactic migration of HEK293 cells transiently expressing CHL1 or L1 was assayed essentially as previously reported (37Thelen K. Kedar V. Panicker A.K. Schmid R.S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Google Scholar), using modified Boyden chambers with 8-μm pore filters (Transwells 3422, Corning/Costar, Acton, MA) in serum-free medium (DMEM, 0.4 mm MnCl2, 50 μg/ml gentamicin, 250 μg/ml kanamycin). In the absence of Mn2+, CHL1 potentiated haptotactic migration of HEK293 cells toward collagen I (1.9-fold) to nearly the same extent as in the presence of Mn2+ (2.3-fold). The bottom sides of the filters were precoated with extracellular matrix proteins (500 μl of 20 μg/ml) or 2% bovine serum albumin in PBS at 4 °C overnight and blocked in 2% bovine serum albumin. The cells were detached with 5 mm Na-EDTA in Hanks' balanced saline solution (HBSS) and plated at 20,000 cells/Transwell. In some experiments, the cells were preincubated in serum-free medium with anti-integrin antibodies (1–4 μg/ml) for 15–30 min at 4 °C prior to plating. The cells were allowed to migrate for 3– 8hat37 °CinaCO2 incubator. After migration cultures were fixed in 4% paraformaldehyde, rinsed in PBS, and then treated with blocking solution (10% goat serum, 0.2% fish skin gelatin, PBS) for either 1 h at room temperature or overnight at 4 °C. To score migration, cells from the upper or lower sides of filters were removed, and cells on the opposite side were stained by indirect immunofluorescence with purified CHL1 polyclonal antibodies (20 μg/ml) or L1 monoclonal antibody Neuro4 (5 μg/ml) in blocking solution for 4 h at room temperature, followed by fluorescein isothiocyanate-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) diluted 1:75 to 1:100 in blocking solution. The cells were counterstained with 10 μm bis-benzimide (Hoechst 33258; Molecular Probes, Eugene, OR). The filters were mounted with Vectashield (Vector Laboratories, Burlingame, CA) on glass slides, and the cells were scored on both top and bottom surfaces of filters under epifluorescence illumination. For each filter, at least 150 cells were scored from six or more randomly selected fields using a 20× objective. To obtain the total number of cells on each side of a filter, the mean number of cells/field was determined and multiplied by a factor based on the number and size of fields and a filter diameter of 6.5 mm. The percentage of CHL1-immunoreactive cells that transmigrated was calculated as the ratio of CHL1-positive cells on the bottom of filters to total (top and bottom) CHL1-positive cells. The percentage of CHL1-negative bis-benzimide-positive cells that transmigrated was determined similarly. The percentage of cells transmigrated was converted to the total number of cells migrated per Transwell by multiplying the percentage by the number of cells plated. The experiments were performed in duplicate or triplicate, and the results of each condition were averaged. The means and standard errors were determined for each condition. Significant differences between experimental groups was evaluated by Student's t test (p < 0.05, one-tailed). Transfection efficiency of HEK293 cells was determined as the number of fluorescein isothiocyanate-labeled cells (transfected) divided by the number of bis-benzimide-labeled cells (transfected plus nontransfected) and was 50–70%. There was no deleterious effect of integrin antibodies or inhibitors on cell adhesion, which was evaluated by counting the total number of cells recovered after each assay. Similarly, inhibitors used at concentrations indicated in the signaling experiments (PP2, PP3, Ly294002, and U0126) did not affect the viability or cell recovery. Co-capping Experiments—HEK293 cells transfected with the pcDNA3-CHL1 plasmid were dissociated in 5 mm EDTA in HBSS, washed with 10% fetal bovine serum in DMEM, and resuspended in DMEM. The cells (30,000 cells/100 μl) were incubated with anti-integrin β1 mouse monoclonal antibody MAB2000 (20 μg/ml) at 4 °C for 20 min. The cells were washed with ice-cold HBSS, resuspended in DMEM containing 5 μg/ml goat anti-mouse IgG (Fcγ fragment-specific), and incubated for 20 min at 4 °C. After washing in ice-cold HBSS, the cells were resuspended in 10% fetal bovine serum in DMEM, plated onto collagen-coated MatTek plates (MatTek Corp., Ashland, MA), and incubated for1hat37 °C to allow integrins to cluster. The cells were then fixed in 4% paraformaldehyde in PBS for 15 min, washed, and blocked with 10% donkey serum in PBS for 1 h at room temperature. To label CHL1, the cells were incubated with CHL1 rabbit polyclonal antibody (20 μg/ml in blocking buffer) for 2 h at room temperature, washed with PBS, and then incubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and TRITC-conjugated donkey anti-goat IgG diluted 1:100 in blocking buffer. Finally, the cells were rinsed, mounted in Vectashield, and examined using an Olympus FV500 laser confocal microscope at the Microscopy Service Laboratory (Dr. Robert Bagnell, Department of Pathology, University of North Carolina-Chapel Hill) using appropriate filter sets. Assay for Ankyrin Recruitment—Ankyrin recruitment to CHL1 in the plasma membrane was assayed essentially as previously described for L1 (38Needham L.K. Thelen K. Maness P.F. J. Neurosci. 2001; 21: 1490-1500Google Scholar) with the following modifications. HEK293 cells on poly-d-lysine-coated MatTek dishes were transfected with plasmid pEGFP-N1 expressing a fusion protein between ankyrin G and green fluorescent protein (ankyrin-GFP) (0.05 μg) with or without co-transfection of pcDNA3-CHL1 (0.1 μg) using LipofectAMINE 2000. After 24 h, the cells were fixed with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, for 15 min, washed, and incubated with blocking buffer (10% normal goat serum in PBS) for 30 min. To label CHL1 on the cell surface, the cells were incubated for 4 h at room temperature with rabbit polyclonal antibody against CHL1 (20 μg/ml in blocking buffer), then washed, and incubated for 2 h with TRITC-conjugated goat anti-rabbit IgG diluted 1:200 in blocking buffer. The cells were washed and mounted in Vectashield. GFP/immunofluorescence images were recorded on an Olympus FV500 laser confocal microscope. The ankyrin-GFP fluorescence was recorded using the 488-nm excitation line of the laser, whereas the TRITC-labeled CHL1 was examined using the 543-nm excitation line of the laser and the appropriate band pass filters. Each experiment was repeated four times. CHL1 Potentiates Haptotactic Cell Migration to Extracellular Matrix Proteins through β1Integrins—Extracellular matrix proteins are important in mediating cell migration and neuronal process growth in the developing nervous system (39Sobeih M.M. Corfas G. Int. J. Dev. Neurosci. 2002; 20: 349-357Google Scholar) and are implicated in nerve response to injury (40Frostick S.P. Yin Q. Kemp G.J. Microsurgery. 1998; 18: 397-405Google Scholar, 41Previtali S.C. Feltri M.L. Archelos J.J. Quattrini A. Wrabetz L. Hartung H. Prog. Neurobiol. 2001; 64: 35-49Google Scholar). The ability of CHL1 to promote haptotactic migration toward extracellular matrix proteins was studied in Transwell assays in which cells were allowed to migrate from top to bottom chambers through filters coated on the underside with purified matrix molecules (37Thelen K. Kedar V. Panicker A.K. Schmid R.S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Google Scholar). The human embryonic kidney cell line HEK293 was used for these studies, because it expresses defined integrin subunits including α1, α2, α3, α5, αv, and β1, which can serve as extracellular matrix protein receptors (42Bodary S.C. McLean J.W. J. Biol. Chem. 1990; 265: 5938-5941Google Scholar, 43Simon K.O. Nutt E.M. Abraham D.G. Rodan G.A. Duong L.T. J. Biol. Chem. 1997; 272: 29380-29389Google Scholar), and because it does not express detectable levels of cell adhesion molecules of the L1 family (38Needham L.K. Thelen K. Maness P.F. J. Neurosci. 2001; 21: 1490-1500Google Scholar). HEK293 cells were transfected for transient expression with pcDNA3 plasmids encoding CHL1 or L1, and haptotactic migration was assayed toward purified matrix proteins. Nontransfected HEK293 cells displayed greater migration toward collagen type I, fibronectin, and laminin compared with random migration toward bovine serum albumin but did not migrate significantly toward vitronectin (Fig. 1). Expression of CHL1 significantly enhanced haptotactic migration toward each of these substrates as well as to vitronectin (Fig. 1). In contrast to CHL1, L1 was clearly not capable of enhancing HEK293 cell migration to collagen I, similar to previous findings in rat B35 neuroblastoma cells (37Thelen K. Kedar V. Panicker A.K. Schmid R.S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Google Scholar), although L1 was as effective as CHL1 at potentiating cell migration to fibronectin (Fig. 1). These results showed that CHL1 can promote haptotactic cell migration toward a range of ECM substrates and suggested that CHL1 might functionally interact with integrins. To determine whether integrins were involved in CHL1-mediated migration, haptotactic migration of HEK293 cells toward collagen I was further evaluated in the presence of function-blocking integrin antibodies. Collagen I was chosen as a substrate, because collagen I, in addition to collagens III and IV, increases at the site of nerve injury and promotes Schwann cell migration (41Previtali S.C. Feltri M.L. Archelos J.J. Quattrini A. Wrabetz L. Hartung H. Prog. Neurobiol. 2001; 64: 35-49Google Scholar), and CHL1 is up-regulated in Schwann cells during peripheral nerve regeneration (20Hillenbrand R. Molthagen M. Montag D. Schachner M. Eur. J. Neurosci. 1999; 11: 813-826Google Scholar, 23Zhang Y. Roslan R. Lang D. Schachner M. Lieberman A.R. Anderson P.N. Mol. Cell. Neurosci. 2000; 16: 71-86Google Scholar). Treatment of cells with function-blocking β1 integrin antibodies (44Ni H. Wilkins J.A. Cell Adhes. Commun. 1998; 5: 257-271Google Scholar) strongly inhibited CHL1-mediated HEK293 cell migration toward collagen I, as well as CHL1-independent migration of HEK293 cells (Fig. 2). Residual migration of CHL1-HEK293 cells in the presence of anti-β1 integrin antibodies was low but significantly elevated over that of HEK293 cells (p < 0.05), suggesting that CHL1 might function to a small extent through a non-β1 containing collagen receptor. Because the principal collagen I receptors are known to be α1β1 and α2β1 integrins (45van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Google Scholar), function-blocking antibodies against α1 and α2 integrins were evaluated next for effects on migration. Antibodies against either α1 or α2 integrin strongly inhibited CHL1-mediated migration toward collagen I, whereas α1 and α2 integrin antibodies added together were as effective as β1 integrin antibodies (Fig. 2). The α1 and α2 integrin antibodies were not as effective as β1 integrin antibodies in inhibiting the basal migration of HEK293 cells, thus it is likely that these cells use α1β1 and α2β1 integrins in addition to an unidentified β1 integrin heterodimer for migration to collagen. In summary, these results indicated that CHL1 potentiated the migration of HEK293 cells toward collagen I primarily through α1β1 and α2β1 integrins. The DGEA Motif in the CHL1 Ig6 Domain Is Required for Potentiating Migration to Collagen I—The mouse CHL1 and human CALL proteins contain a conserved Asp-Gly-Glu-Ala (DGEA) motif in the Ig6 domain of their extracellular regions (22Holm J. Hillenbrand R. Steuber V. Bartsch U. Moos M. Lubbert H. Montag D. Schachner M. Eur. J. Neurosci. 1996; 8: 1613-1629Google Scholar, 24Wei M.H. Karavanova I. Ivanov S.V. Popescu N.C. Keck C.L. Pack S. Eisen J.A. Lerman M.I. Hum. Genet. 1998; 103: 355-364Google Scholar). Interestingly, the DGEA motif is also present in collagen I where it serves as a recognition site for α2β1 integrin in platelets (31Staatz W.D. Fok K.F. Zutter M.M. Adams S.P. Rodriguez B.A. Santoro S.A. J. Biol. Chem. 1991; 266: 7363-7367Google Scholar). Mutation of the DGEA sequence in the CHL1 Ig6 domain to Ala-Gly-Glu-Val (AGEV) effectively inhibited CHL1-potentiated migration of HEK293 cells toward collagen I, reducing migration to nearly the level of CHL1-nonexpressing HEK293 cells (Fig. 3). In the Ig6 domain of L1, an Arg-Gly-Asp (RGD) sequence is required for potentiating migration to fibronectin (37Thelen K. Kedar V. Panicker A.K. Schmid R.S. Midkiff B.R. Maness P.F. J. Neurosci. 2002; 22: 4918-4931Google Scholar). The Ig2 domain of CHL1 contains an Arg-Gly-Asp (RGD) motif (22Holm J. Hillenbrand R. Steuber V. Bartsch U. Moos M. Lubbert H. Montag D. Schachner M. Eur. J. Neurosci. 1996; 8: 1613-1629Google Scholar), and a corresponding sequence (Lys-Gly-Asp) in the human CALL protein is closely conserved though nonidentical (24Wei M.H. Karavanova I. Ivanov S.V. Popescu N.C. Keck C.L. Pack S. Eisen J.A. Lerman M.I. Hum. Genet. 1998; 103: 355-364Google Scholar). This motif might interact with integrins, because RGD is a known adhesion motif that is present in collagen I, fibronectin, and vitronectin, and it is recognized by at least eight integrins (46Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Google Scholar). However, mutation of the RGD in the CHL1 Ig2 domain to Lys-Gly-Glu (KGE) did not perturb CHL1-potentiated migration to collagen (Fig. 3). These results illustrated the importance of the DGEA sequence in the CHL1 Ig6 domain in mediating haptotactic migration toward collagen I through α1/α2 β1 integrins. CHL1 Enhances Migration through c-Src, PI 3-Kinase, and MAP Kinase—The nonreceptor tyrosine kinase c-Src, PI 3-kinase, and MAP kinase are key signaling intermediates down-stream of cell adhesion molecules such as L1 (47Juliano R.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 283-323Google Scholar, 48Schmid R.S. Pruitt W.M. Maness P.F. J. Neurosci. 2000; 11: 4177-4188Google Scholar). There is evidence that PI 3-kinase also has a role in integrin receptor recycling (49Ng T. Shima D. Squire A. Bastiaens P.I. Gschmeissner S. Humphries M.J. Parker P.J. EMBO J. 1999; 18: 3909-3923Google Scholar), whereas MAP kinase has well characterized functions in regulating cell migration through regulating new protein synthesis that may be required for synthesis of receptors (50Kolch W. Biochem. J." @default.
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• W2044304997 title "Close Homolog of L1 Is an Enhancer of Integrin-mediated Cell Migration" @default.
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