Cis-activation of L1-mediated Ankyrin Recruitment by TAG-1 Homophilic Cell Adhesion
1998; Elsevier BV; Volume: 273; Issue: 50 Linguagem: Inglês
10.1074/jbc.273.50.33354
ISSN1083-351X
AutoresJyoti Malhotra, Panayoula C. Tsiotra, Domna Karagogeos, Michael Hortsch,
Tópico(s)Signaling Pathways in Disease
ResumoNeural cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily mediate not only cell aggregation but also growth cone guidance and neurite outgrowth. In this study we demonstrate that two neural CAMs, L1-CAM and TAG-1, induce the homophilic aggregation of Drosophila S2 cells but are unable to interact with each other when expressed on different cells (trans-interaction). However, immunoprecipitations from cells co-expressing L1-CAM and TAG-1 showed a strong cis-interaction between the two molecules in the plane of the plasma membrane. TAG-1 is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor and therefore is unable to directly interact with cytoplasmic proteins. In contrast, L1-CAM-mediated homophilic cell adhesion induces the selective recruitment of the membrane skeleton protein ankyrin to areas of cell contact. Immunolabeling experiments in which S2 cells expressing TAG-1 were mixed with cells co-expressing L1-CAM and TAG-1 demonstrated that the homophilic interaction between TAG-1 molecules results in the cis-activation of L1-CAM to bind ankyrin. This TAG-1-dependent recruitment of the membrane skeleton provides an example of how GPI-anchored CAMs are able to transduce signals to the cytoplasm. Furthermore, such interactions might ultimately result in the recruitment and the activation of other signaling molecules at sites of cell contacts. Neural cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily mediate not only cell aggregation but also growth cone guidance and neurite outgrowth. In this study we demonstrate that two neural CAMs, L1-CAM and TAG-1, induce the homophilic aggregation of Drosophila S2 cells but are unable to interact with each other when expressed on different cells (trans-interaction). However, immunoprecipitations from cells co-expressing L1-CAM and TAG-1 showed a strong cis-interaction between the two molecules in the plane of the plasma membrane. TAG-1 is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor and therefore is unable to directly interact with cytoplasmic proteins. In contrast, L1-CAM-mediated homophilic cell adhesion induces the selective recruitment of the membrane skeleton protein ankyrin to areas of cell contact. Immunolabeling experiments in which S2 cells expressing TAG-1 were mixed with cells co-expressing L1-CAM and TAG-1 demonstrated that the homophilic interaction between TAG-1 molecules results in the cis-activation of L1-CAM to bind ankyrin. This TAG-1-dependent recruitment of the membrane skeleton provides an example of how GPI-anchored CAMs are able to transduce signals to the cytoplasm. Furthermore, such interactions might ultimately result in the recruitment and the activation of other signaling molecules at sites of cell contacts. The navigation of growth cones to their targets in the developing embryonic nervous system is a critical step in the patterning of neuronal projections. Substantial evidence suggests that axonal guidance depends in part on cell surface and on extracellular matrix molecules, which are expressed along the path of the advancing growth cones (1Dodd J. Jessell T.M. Science. 1988; 242: 692-699Crossref PubMed Scopus (554) Google Scholar, 2Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar). Axonal extension and pathway choices are influenced by membrane receptors to these molecules, which are expressed on the growth cone.One group of such molecules are cell adhesion molecules (CAMs) 1The abbreviations used are: CAM, cell adhesion molecule; DiI, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate; FGF, fibroblast growth factor; GPI, glycosylphosphatidylinositol; Ig, immunoglobulin; NCAM, neural cell adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule; TAG-1, transiently expressed axonal surface glycoprotein-1; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: CAM, cell adhesion molecule; DiI, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate; FGF, fibroblast growth factor; GPI, glycosylphosphatidylinositol; Ig, immunoglobulin; NCAM, neural cell adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule; TAG-1, transiently expressed axonal surface glycoprotein-1; PAGE, polyacrylamide gel electrophoresis. belonging to the immunoglobulin (Ig) superfamily that are expressed by advancing growth cones and are able to recognize and transduce environmental signals (3Brümmendorf T. Rathjen F.G. J. Neurochem. 1993; 61: 1207-1219Crossref PubMed Scopus (122) Google Scholar). Ig domain CAMs have been implicated to act both as receptors as well as substrates for growing axons (4Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Crossref PubMed Scopus (395) Google Scholar, 5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). For example, members of the L1 family of neural CAMs, such as mammalian L1-CAMs and chicken Ng-CAM, not only exhibit a strong Ca2+-independent homophilic adhesive activity (6Lemmon V. Farr K.L. Lagenaur C. Neuron. 1989; 2: 1597-1603Abstract Full Text PDF PubMed Scopus (379) Google Scholar, 7Grumet M. Hoffman S. Chuong C.-M. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7989-7993Crossref PubMed Scopus (81) Google Scholar), they also promote neurite outgrowth in culture, probably by the activation of neuronal FGF receptors (8Doherty P. Walsh F.S. Mol. Cell. Neurosci. 1996; 8: 99-111Crossref Scopus (321) Google Scholar). Through the interaction with the cytoplasmic linker protein ankyrin, L1 family members are also connected to the membrane skeleton (9Davis J.Q. Bennett V. J. Biol. Chem. 1994; 269: 27163-27166Abstract Full Text PDF PubMed Google Scholar). Human L1-CAM and the Drosophila L1 homologue neuroglian both recruit ankyrin and other components of the membrane skeleton to cell contact sites in Drosophila S2 cell aggregates (10Dubreuil R.R. MacVicar G. Dissanayake S. Liu C. Homer D. Hortsch M. J. Cell Biol. 1996; 133: 647-655Crossref PubMed Scopus (112) Google Scholar, 11Hortsch M. O'Shea K.S. Zhao G. Kim F. Vallejo Y. Dubreuil R.R. Cell Adhes. Commun. 1998; 5: 61-73Crossref PubMed Google Scholar). This interaction strictly depends on the extracellular L1 adhesive activity, and ankyrin binding in turn stabilizes the L1 adhesive interaction (12Hortsch M. Homer D. Dhar Malhotra J. Chang S. Frankel J. Jefford G. Dubreuil R.R. J. Cell Biol. 1998; 142: 251-261Crossref PubMed Scopus (65) Google Scholar).Members of another subgroup of Ig domain neural CAMs are anchored in the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety. These include TAG-1 in mammalian species and its chicken homologue axonin-1 (13Furley A.J. Morton S.B. Manalo D. Karagogeos D. Dodd J. Jessell T.M. Cell. 1990; 61: 157-170Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 14Zuellig R.A. Rader C. Schroeder A. Kalousek M.B. von Bohlen und Halbach F. Osterwalder T. Inan C. Stoeckli E.T. Affolter H.U. Fritz A. Hafen E. Sonderegger P. Eur. J. Biochem. 1992; 204: 453-463Crossref PubMed Scopus (119) Google Scholar). TAG-1/axonin-1 has been shown to mediate homophilic cell adhesion and is able to promote neurite outgrowth in culture (13Furley A.J. Morton S.B. Manalo D. Karagogeos D. Dodd J. Jessell T.M. Cell. 1990; 61: 157-170Abstract Full Text PDF PubMed Scopus (475) Google Scholar,15Stoeckli E.T. Kuhn T.B. Duc C.O. Ruegg M.A. Sonderegger P. J. Cell Biol. 1991; 112: 449-455Crossref PubMed Scopus (129) Google Scholar, 16Rader C. Stoeckli E.T. Ziegler U. Osterwalder T. Kunz B. Sonderegger P. Eur. J. Biochem. 1993; 215: 133-141Crossref PubMed Scopus (76) Google Scholar). However, TAG-1-induced neurite growth is not only mediated by but also requires other neuronal membrane proteins, such as L1- or β1 integrin-type molecules (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar). TAG-1/axonin-1 interacts with a number of different heterophilic binding partners, including several members of the L1 family, NCAM, nervous tissue-specific chondroitin sulfate proteoglycans, and several extracellular matrix molecules (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 19Milev P. Maurel P. Haring M. Margolis R.K. Margolis R.U. J. Biol. Chem. 1996; 271: 15716-15723Crossref PubMed Scopus (188) Google Scholar). It has been suggested that some of these heterophilic interactions might enable TAG-1 to induce or influence intracellular signaling processes (5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). Axonin-1 and Ng-CAM expressed in the same plasma membrane engage in a strong cis-interaction, forming larger multimeric complexes that are also associated with several intracellular protein kinase systems (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). This cis-interaction between TAG-1/axonin-1 and L1-type molecules is essential for the stimulation of neurite outgrowth on TAG-1/axonin-1 substrates in culture, and homophilic TAG-1 cell adhesion appears to activate TAG-1-associated L1-CAM molecules (16Rader C. Stoeckli E.T. Ziegler U. Osterwalder T. Kunz B. Sonderegger P. Eur. J. Biochem. 1993; 215: 133-141Crossref PubMed Scopus (76) Google Scholar, 17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar). Although several lines of evidence suggest that the interaction of TAG-1 with L1-CAM is an important link in TAG-1-initiated signal transduction, a direct demonstration that TAG-1 is capable of altering the functional state of L1-CAM has been missing. In our present study we demonstrate that TAG-1-mediated homophilic cell adhesion induces an intracellular restructuring of the membrane skeleton by the cis-activation of human L1-CAM.DISCUSSIONComplex homo- and heterophilic interactions between different adhesion molecules within the plane of the plasma membrane and between adjacent cells have been proposed to play an important role in regulating the growth and guidance of axons during embryonic neurogenesis (5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). Our present in vitro study addresses how two different CAMs, human TAG-1 and human L1-CAM, interact and functionally regulate each other. Some groups of CAMs are transmembrane proteins and therefore potentially able to directly influence intracellular processes, e.g. by activating second messenger signaling cascades and/or by reorganizing components of the cytoskeleton. However, other CAM families, such as the TAG-1/axonin-1 and the F3/F11 groups, are anchored in the plasma membrane by a GPI moiety and therefore lack the means to interact with cytoplasmic proteins without additional linker proteins. Nevertheless, GPI-anchored CAMs are also able to operate as signal-transducing molecules during neuronal development. Although TAG-1/axonin-1 is fully functional as a homophilic CAM without engaging in any heterophilic cis-interactions, its ability to associate with other membrane proteins, especially L1-CAM, appears to be essential for its neurite outgrowth-promoting function (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar). Several models similar to the one displayed in Fig.6 have been proposed in which TAG-1/axonin-1 associates with other membrane proteins expressed in the same plasma membrane in a cis-interaction to form a signal-transducing, multimeric protein complex (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar). A similar model has also been suggested to explain the adhesion-dependent activation of neuronal FGF receptors by L1-CAM, NCAM, and N-cadherin (8Doherty P. Walsh F.S. Mol. Cell. Neurosci. 1996; 8: 99-111Crossref Scopus (321) Google Scholar).Although several molecular details of the TAG-1 interaction with L1-CAM have been elucidated, its regulatory and functional aspects are not well understood. L1-CAM/Ng-CAM not only can be co-immunoprecipitated with TAG-1/axonin-1, but cross-linking experiments suggest that both molecules directly bind to each other (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). The part of the axonin-1 molecule responsible for this cis-interaction has been mapped to its amino-terminal Ig protein domains (32Rader C. Kunz B. Lierheimer R. Giger R.J. Berger P. Tittmann P. Gross H. Sonderegger P. EMBO J. 1996; 15: 2056-2068Crossref PubMed Scopus (54) Google Scholar), whereas the homophilic adhesive activity of TAG-1/axonin-1 is mediated by its fibronectin type III domains (23Tsiotra P.C. Theodorakis K. Papamatheakis J. Karagogeos D. J. Biol. Chem. 1996; 271: 29216-29222Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The protein domains of L1-CAM that mediate TAG-1 recognition and binding have not been identified yet. Also whether the homophilic adhesive activities of L1-CAM and TAG-1 regulate their cis-interaction is currently unknown. However, co-capping experiments reported by Buchstaller et al. (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar) and the quantity of L1-CAM that co-immunoprecipitated with TAG-1 in our experiments suggest that a significant fraction of L1-CAM molecules forms heterodimeric complexes with TAG-1 before the two CAMs engage in cell adhesion and L1-CAM binds to the membrane skeleton. After L1-CAM interacts with ankyrin, it becomes resistant to Triton X-100 extraction as used in our immunoprecipitation experiments and is unavailable for precipitation with antibodies (10Dubreuil R.R. MacVicar G. Dissanayake S. Liu C. Homer D. Hortsch M. J. Cell Biol. 1996; 133: 647-655Crossref PubMed Scopus (112) Google Scholar).TAG-1/axonin-1 and L1-CAM/Ng-CAM are co-expressed in several locations during nervous system development, suggesting that their interaction is physiologically relevant (31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar, 33Honig M.G. Kueter J. Dev. Biol. 1995; 167: 563-583Crossref PubMed Scopus (30) Google Scholar, 34Yamagata M. Herman J.P. Sanes J.R. J. Neurosci. 1995; 15: 4556-4571Crossref PubMed Google Scholar). In cultures of chicken dorsal root ganglion neurons Ng-CAM and axonin-1 protein are found in overlapping areas on growth cone membranes, and axonin-1 expression by these cells is required for neurite outgrowth on both axonin-1 and Ng-CAM substrata (31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar). In contrast, in other developing neurons,e.g. in the rat embryonic spinal cord, TAG-1 and L1-CAM expression appear to be locally and temporally segregated (22Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (639) Google Scholar, 35Karagogeos D. Morton S.B. Casano F. Dodd J. Jessell T.M. Development. 1991; 112: 51-67PubMed Google Scholar). This finding indicates that TAG-1/axonin-1 and L1-CAM/Ng-CAM are not obligatory co-receptors but are able to function independently. It might mean either that the signaling capabilities of TAG-1/axonin-1 are limited in certain areas of the developing nervous system or at certain developmental time points or that TAG-1/axonin-1 associates with other membrane proteins than L1-CAM/Ng-CAM to form functionally active protein complexes.Although the cis-activation of neuronal FGF receptors by L1-CAM is thought to be the initial step in adhesion-induced neurite outgrowth, processes such as the recruitment of other signaling molecules to cell contact sites might also have an important, more indirect role. Our findings presented here would support the hypothesis that TAG-1 homophilic adhesion might also activate neuronal FGF receptor activity via L1-CAM. Other cellular changes that appear to be regulated by the TAG-1/axonin-1 interaction with L1-CAM/Ng-CAM are the recruitment and the release of several different protein kinase activities that are associated with the two CAMs in chicken dorsal root ganglion neurons (20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). In the case of integrin- and cadherin-mediated cell adhesion, interactions with cytoskeletal elements induce the assembly of signal-generating and -processing multiprotein complexes at the cytoplasmic aspect of the adhesion contact site. This makes the observed assembly of membrane skeleton components in response to TAG-1-mediated homophilic cell adhesion especially significant. However, the most tantalizing aspect of the results reported here is the observation that TAG-1 homophilic cell adhesion directly regulates the functional status of L1-CAM. These findings suggest a mechanism that explains how GPI-anchored CAMs might actively participate in regulating the growth, organization, and differentiation of neuronal cells during development. The navigation of growth cones to their targets in the developing embryonic nervous system is a critical step in the patterning of neuronal projections. Substantial evidence suggests that axonal guidance depends in part on cell surface and on extracellular matrix molecules, which are expressed along the path of the advancing growth cones (1Dodd J. Jessell T.M. Science. 1988; 242: 692-699Crossref PubMed Scopus (554) Google Scholar, 2Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar). Axonal extension and pathway choices are influenced by membrane receptors to these molecules, which are expressed on the growth cone. One group of such molecules are cell adhesion molecules (CAMs) 1The abbreviations used are: CAM, cell adhesion molecule; DiI, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate; FGF, fibroblast growth factor; GPI, glycosylphosphatidylinositol; Ig, immunoglobulin; NCAM, neural cell adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule; TAG-1, transiently expressed axonal surface glycoprotein-1; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: CAM, cell adhesion molecule; DiI, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate; FGF, fibroblast growth factor; GPI, glycosylphosphatidylinositol; Ig, immunoglobulin; NCAM, neural cell adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule; TAG-1, transiently expressed axonal surface glycoprotein-1; PAGE, polyacrylamide gel electrophoresis. belonging to the immunoglobulin (Ig) superfamily that are expressed by advancing growth cones and are able to recognize and transduce environmental signals (3Brümmendorf T. Rathjen F.G. J. Neurochem. 1993; 61: 1207-1219Crossref PubMed Scopus (122) Google Scholar). Ig domain CAMs have been implicated to act both as receptors as well as substrates for growing axons (4Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Crossref PubMed Scopus (395) Google Scholar, 5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). For example, members of the L1 family of neural CAMs, such as mammalian L1-CAMs and chicken Ng-CAM, not only exhibit a strong Ca2+-independent homophilic adhesive activity (6Lemmon V. Farr K.L. Lagenaur C. Neuron. 1989; 2: 1597-1603Abstract Full Text PDF PubMed Scopus (379) Google Scholar, 7Grumet M. Hoffman S. Chuong C.-M. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7989-7993Crossref PubMed Scopus (81) Google Scholar), they also promote neurite outgrowth in culture, probably by the activation of neuronal FGF receptors (8Doherty P. Walsh F.S. Mol. Cell. Neurosci. 1996; 8: 99-111Crossref Scopus (321) Google Scholar). Through the interaction with the cytoplasmic linker protein ankyrin, L1 family members are also connected to the membrane skeleton (9Davis J.Q. Bennett V. J. Biol. Chem. 1994; 269: 27163-27166Abstract Full Text PDF PubMed Google Scholar). Human L1-CAM and the Drosophila L1 homologue neuroglian both recruit ankyrin and other components of the membrane skeleton to cell contact sites in Drosophila S2 cell aggregates (10Dubreuil R.R. MacVicar G. Dissanayake S. Liu C. Homer D. Hortsch M. J. Cell Biol. 1996; 133: 647-655Crossref PubMed Scopus (112) Google Scholar, 11Hortsch M. O'Shea K.S. Zhao G. Kim F. Vallejo Y. Dubreuil R.R. Cell Adhes. Commun. 1998; 5: 61-73Crossref PubMed Google Scholar). This interaction strictly depends on the extracellular L1 adhesive activity, and ankyrin binding in turn stabilizes the L1 adhesive interaction (12Hortsch M. Homer D. Dhar Malhotra J. Chang S. Frankel J. Jefford G. Dubreuil R.R. J. Cell Biol. 1998; 142: 251-261Crossref PubMed Scopus (65) Google Scholar). Members of another subgroup of Ig domain neural CAMs are anchored in the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety. These include TAG-1 in mammalian species and its chicken homologue axonin-1 (13Furley A.J. Morton S.B. Manalo D. Karagogeos D. Dodd J. Jessell T.M. Cell. 1990; 61: 157-170Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 14Zuellig R.A. Rader C. Schroeder A. Kalousek M.B. von Bohlen und Halbach F. Osterwalder T. Inan C. Stoeckli E.T. Affolter H.U. Fritz A. Hafen E. Sonderegger P. Eur. J. Biochem. 1992; 204: 453-463Crossref PubMed Scopus (119) Google Scholar). TAG-1/axonin-1 has been shown to mediate homophilic cell adhesion and is able to promote neurite outgrowth in culture (13Furley A.J. Morton S.B. Manalo D. Karagogeos D. Dodd J. Jessell T.M. Cell. 1990; 61: 157-170Abstract Full Text PDF PubMed Scopus (475) Google Scholar,15Stoeckli E.T. Kuhn T.B. Duc C.O. Ruegg M.A. Sonderegger P. J. Cell Biol. 1991; 112: 449-455Crossref PubMed Scopus (129) Google Scholar, 16Rader C. Stoeckli E.T. Ziegler U. Osterwalder T. Kunz B. Sonderegger P. Eur. J. Biochem. 1993; 215: 133-141Crossref PubMed Scopus (76) Google Scholar). However, TAG-1-induced neurite growth is not only mediated by but also requires other neuronal membrane proteins, such as L1- or β1 integrin-type molecules (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar). TAG-1/axonin-1 interacts with a number of different heterophilic binding partners, including several members of the L1 family, NCAM, nervous tissue-specific chondroitin sulfate proteoglycans, and several extracellular matrix molecules (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 19Milev P. Maurel P. Haring M. Margolis R.K. Margolis R.U. J. Biol. Chem. 1996; 271: 15716-15723Crossref PubMed Scopus (188) Google Scholar). It has been suggested that some of these heterophilic interactions might enable TAG-1 to induce or influence intracellular signaling processes (5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). Axonin-1 and Ng-CAM expressed in the same plasma membrane engage in a strong cis-interaction, forming larger multimeric complexes that are also associated with several intracellular protein kinase systems (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). This cis-interaction between TAG-1/axonin-1 and L1-type molecules is essential for the stimulation of neurite outgrowth on TAG-1/axonin-1 substrates in culture, and homophilic TAG-1 cell adhesion appears to activate TAG-1-associated L1-CAM molecules (16Rader C. Stoeckli E.T. Ziegler U. Osterwalder T. Kunz B. Sonderegger P. Eur. J. Biochem. 1993; 215: 133-141Crossref PubMed Scopus (76) Google Scholar, 17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar). Although several lines of evidence suggest that the interaction of TAG-1 with L1-CAM is an important link in TAG-1-initiated signal transduction, a direct demonstration that TAG-1 is capable of altering the functional state of L1-CAM has been missing. In our present study we demonstrate that TAG-1-mediated homophilic cell adhesion induces an intracellular restructuring of the membrane skeleton by the cis-activation of human L1-CAM. DISCUSSIONComplex homo- and heterophilic interactions between different adhesion molecules within the plane of the plasma membrane and between adjacent cells have been proposed to play an important role in regulating the growth and guidance of axons during embryonic neurogenesis (5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). Our present in vitro study addresses how two different CAMs, human TAG-1 and human L1-CAM, interact and functionally regulate each other. Some groups of CAMs are transmembrane proteins and therefore potentially able to directly influence intracellular processes, e.g. by activating second messenger signaling cascades and/or by reorganizing components of the cytoskeleton. However, other CAM families, such as the TAG-1/axonin-1 and the F3/F11 groups, are anchored in the plasma membrane by a GPI moiety and therefore lack the means to interact with cytoplasmic proteins without additional linker proteins. Nevertheless, GPI-anchored CAMs are also able to operate as signal-transducing molecules during neuronal development. Although TAG-1/axonin-1 is fully functional as a homophilic CAM without engaging in any heterophilic cis-interactions, its ability to associate with other membrane proteins, especially L1-CAM, appears to be essential for its neurite outgrowth-promoting function (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar). Several models similar to the one displayed in Fig.6 have been proposed in which TAG-1/axonin-1 associates with other membrane proteins expressed in the same plasma membrane in a cis-interaction to form a signal-transducing, multimeric protein complex (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar). A similar model has also been suggested to explain the adhesion-dependent activation of neuronal FGF receptors by L1-CAM, NCAM, and N-cadherin (8Doherty P. Walsh F.S. Mol. Cell. Neurosci. 1996; 8: 99-111Crossref Scopus (321) Google Scholar).Although several molecular details of the TAG-1 interaction with L1-CAM have been elucidated, its regulatory and functional aspects are not well understood. L1-CAM/Ng-CAM not only can be co-immunoprecipitated with TAG-1/axonin-1, but cross-linking experiments suggest that both molecules directly bind to each other (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). The part of the axonin-1 molecule responsible for this cis-interaction has been mapped to its amino-terminal Ig protein domains (32Rader C. Kunz B. Lierheimer R. Giger R.J. Berger P. Tittmann P. Gross H. Sonderegger P. EMBO J. 1996; 15: 2056-2068Crossref PubMed Scopus (54) Google Scholar), whereas the homophilic adhesive activity of TAG-1/axonin-1 is mediated by its fibronectin type III domains (23Tsiotra P.C. Theodorakis K. Papamatheakis J. Karagogeos D. J. Biol. Chem. 1996; 271: 29216-29222Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The protein domains of L1-CAM that mediate TAG-1 recognition and binding have not been identified yet. Also whether the homophilic adhesive activities of L1-CAM and TAG-1 regulate their cis-interaction is currently unknown. However, co-capping experiments reported by Buchstaller et al. (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar) and the quantity of L1-CAM that co-immunoprecipitated with TAG-1 in our experiments suggest that a significant fraction of L1-CAM molecules forms heterodimeric complexes with TAG-1 before the two CAMs engage in cell adhesion and L1-CAM binds to the membrane skeleton. After L1-CAM interacts with ankyrin, it becomes resistant to Triton X-100 extraction as used in our immunoprecipitation experiments and is unavailable for precipitation with antibodies (10Dubreuil R.R. MacVicar G. Dissanayake S. Liu C. Homer D. Hortsch M. J. Cell Biol. 1996; 133: 647-655Crossref PubMed Scopus (112) Google Scholar).TAG-1/axonin-1 and L1-CAM/Ng-CAM are co-expressed in several locations during nervous system development, suggesting that their interaction is physiologically relevant (31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar, 33Honig M.G. Kueter J. Dev. Biol. 1995; 167: 563-583Crossref PubMed Scopus (30) Google Scholar, 34Yamagata M. Herman J.P. Sanes J.R. J. Neurosci. 1995; 15: 4556-4571Crossref PubMed Google Scholar). In cultures of chicken dorsal root ganglion neurons Ng-CAM and axonin-1 protein are found in overlapping areas on growth cone membranes, and axonin-1 expression by these cells is required for neurite outgrowth on both axonin-1 and Ng-CAM substrata (31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar). In contrast, in other developing neurons,e.g. in the rat embryonic spinal cord, TAG-1 and L1-CAM expression appear to be locally and temporally segregated (22Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (639) Google Scholar, 35Karagogeos D. Morton S.B. Casano F. Dodd J. Jessell T.M. Development. 1991; 112: 51-67PubMed Google Scholar). This finding indicates that TAG-1/axonin-1 and L1-CAM/Ng-CAM are not obligatory co-receptors but are able to function independently. It might mean either that the signaling capabilities of TAG-1/axonin-1 are limited in certain areas of the developing nervous system or at certain developmental time points or that TAG-1/axonin-1 associates with other membrane proteins than L1-CAM/Ng-CAM to form functionally active protein complexes.Although the cis-activation of neuronal FGF receptors by L1-CAM is thought to be the initial step in adhesion-induced neurite outgrowth, processes such as the recruitment of other signaling molecules to cell contact sites might also have an important, more indirect role. Our findings presented here would support the hypothesis that TAG-1 homophilic adhesion might also activate neuronal FGF receptor activity via L1-CAM. Other cellular changes that appear to be regulated by the TAG-1/axonin-1 interaction with L1-CAM/Ng-CAM are the recruitment and the release of several different protein kinase activities that are associated with the two CAMs in chicken dorsal root ganglion neurons (20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). In the case of integrin- and cadherin-mediated cell adhesion, interactions with cytoskeletal elements induce the assembly of signal-generating and -processing multiprotein complexes at the cytoplasmic aspect of the adhesion contact site. This makes the observed assembly of membrane skeleton components in response to TAG-1-mediated homophilic cell adhesion especially significant. However, the most tantalizing aspect of the results reported here is the observation that TAG-1 homophilic cell adhesion directly regulates the functional status of L1-CAM. These findings suggest a mechanism that explains how GPI-anchored CAMs might actively participate in regulating the growth, organization, and differentiation of neuronal cells during development. Complex homo- and heterophilic interactions between different adhesion molecules within the plane of the plasma membrane and between adjacent cells have been proposed to play an important role in regulating the growth and guidance of axons during embryonic neurogenesis (5Sonderegger P. Rathjen F.G. J. Cell Biol. 1992; 119: 1387-1394Crossref PubMed Scopus (151) Google Scholar). Our present in vitro study addresses how two different CAMs, human TAG-1 and human L1-CAM, interact and functionally regulate each other. Some groups of CAMs are transmembrane proteins and therefore potentially able to directly influence intracellular processes, e.g. by activating second messenger signaling cascades and/or by reorganizing components of the cytoskeleton. However, other CAM families, such as the TAG-1/axonin-1 and the F3/F11 groups, are anchored in the plasma membrane by a GPI moiety and therefore lack the means to interact with cytoplasmic proteins without additional linker proteins. Nevertheless, GPI-anchored CAMs are also able to operate as signal-transducing molecules during neuronal development. Although TAG-1/axonin-1 is fully functional as a homophilic CAM without engaging in any heterophilic cis-interactions, its ability to associate with other membrane proteins, especially L1-CAM, appears to be essential for its neurite outgrowth-promoting function (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar). Several models similar to the one displayed in Fig.6 have been proposed in which TAG-1/axonin-1 associates with other membrane proteins expressed in the same plasma membrane in a cis-interaction to form a signal-transducing, multimeric protein complex (17Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar). A similar model has also been suggested to explain the adhesion-dependent activation of neuronal FGF receptors by L1-CAM, NCAM, and N-cadherin (8Doherty P. Walsh F.S. Mol. Cell. Neurosci. 1996; 8: 99-111Crossref Scopus (321) Google Scholar). Although several molecular details of the TAG-1 interaction with L1-CAM have been elucidated, its regulatory and functional aspects are not well understood. L1-CAM/Ng-CAM not only can be co-immunoprecipitated with TAG-1/axonin-1, but cross-linking experiments suggest that both molecules directly bind to each other (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar, 20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). The part of the axonin-1 molecule responsible for this cis-interaction has been mapped to its amino-terminal Ig protein domains (32Rader C. Kunz B. Lierheimer R. Giger R.J. Berger P. Tittmann P. Gross H. Sonderegger P. EMBO J. 1996; 15: 2056-2068Crossref PubMed Scopus (54) Google Scholar), whereas the homophilic adhesive activity of TAG-1/axonin-1 is mediated by its fibronectin type III domains (23Tsiotra P.C. Theodorakis K. Papamatheakis J. Karagogeos D. J. Biol. Chem. 1996; 271: 29216-29222Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The protein domains of L1-CAM that mediate TAG-1 recognition and binding have not been identified yet. Also whether the homophilic adhesive activities of L1-CAM and TAG-1 regulate their cis-interaction is currently unknown. However, co-capping experiments reported by Buchstaller et al. (18Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar) and the quantity of L1-CAM that co-immunoprecipitated with TAG-1 in our experiments suggest that a significant fraction of L1-CAM molecules forms heterodimeric complexes with TAG-1 before the two CAMs engage in cell adhesion and L1-CAM binds to the membrane skeleton. After L1-CAM interacts with ankyrin, it becomes resistant to Triton X-100 extraction as used in our immunoprecipitation experiments and is unavailable for precipitation with antibodies (10Dubreuil R.R. MacVicar G. Dissanayake S. Liu C. Homer D. Hortsch M. J. Cell Biol. 1996; 133: 647-655Crossref PubMed Scopus (112) Google Scholar). TAG-1/axonin-1 and L1-CAM/Ng-CAM are co-expressed in several locations during nervous system development, suggesting that their interaction is physiologically relevant (31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar, 33Honig M.G. Kueter J. Dev. Biol. 1995; 167: 563-583Crossref PubMed Scopus (30) Google Scholar, 34Yamagata M. Herman J.P. Sanes J.R. J. Neurosci. 1995; 15: 4556-4571Crossref PubMed Google Scholar). In cultures of chicken dorsal root ganglion neurons Ng-CAM and axonin-1 protein are found in overlapping areas on growth cone membranes, and axonin-1 expression by these cells is required for neurite outgrowth on both axonin-1 and Ng-CAM substrata (31Stoeckli E.T. Ziegler U. Bleiker A.J. Groscurth P. Sonderegger P. Dev. Biol. 1996; 177: 15-29Crossref PubMed Scopus (51) Google Scholar). In contrast, in other developing neurons,e.g. in the rat embryonic spinal cord, TAG-1 and L1-CAM expression appear to be locally and temporally segregated (22Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (639) Google Scholar, 35Karagogeos D. Morton S.B. Casano F. Dodd J. Jessell T.M. Development. 1991; 112: 51-67PubMed Google Scholar). This finding indicates that TAG-1/axonin-1 and L1-CAM/Ng-CAM are not obligatory co-receptors but are able to function independently. It might mean either that the signaling capabilities of TAG-1/axonin-1 are limited in certain areas of the developing nervous system or at certain developmental time points or that TAG-1/axonin-1 associates with other membrane proteins than L1-CAM/Ng-CAM to form functionally active protein complexes. Although the cis-activation of neuronal FGF receptors by L1-CAM is thought to be the initial step in adhesion-induced neurite outgrowth, processes such as the recruitment of other signaling molecules to cell contact sites might also have an important, more indirect role. Our findings presented here would support the hypothesis that TAG-1 homophilic adhesion might also activate neuronal FGF receptor activity via L1-CAM. Other cellular changes that appear to be regulated by the TAG-1/axonin-1 interaction with L1-CAM/Ng-CAM are the recruitment and the release of several different protein kinase activities that are associated with the two CAMs in chicken dorsal root ganglion neurons (20Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). In the case of integrin- and cadherin-mediated cell adhesion, interactions with cytoskeletal elements induce the assembly of signal-generating and -processing multiprotein complexes at the cytoplasmic aspect of the adhesion contact site. This makes the observed assembly of membrane skeleton components in response to TAG-1-mediated homophilic cell adhesion especially significant. However, the most tantalizing aspect of the results reported here is the observation that TAG-1 homophilic cell adhesion directly regulates the functional status of L1-CAM. These findings suggest a mechanism that explains how GPI-anchored CAMs might actively participate in regulating the growth, organization, and differentiation of neuronal cells during development. We thank Dr. Vance Lemmon for generously providing the rabbit anti-L1 antiserum and the human L1 cDNA and Drs. Stephen Ernst and Robert Chandler for a critical reading of the manuscript. D. K. would also like to thank Dr. Joseph Papamatheakis for his support.
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