The Proprotein Convertase PC5A and a Metalloprotease Are Involved in the Proteolytic Processing of the Neural Adhesion Molecule L1
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m208351200
ISSN1083-351X
AutoresIna Kalus, Birthe Schnegelsberg, Nabil G. Seidah, Ralf Kleene, Melitta Schachner,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe transmembrane and multidomain neural adhesion molecule L1 plays important functional roles in the developing and adult nervous system. L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different fragments. In this report, we present evidence that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. Analysis of mutations revealed Arg845 to be the site of cleavage generating the N-terminal 140-kDa fragment. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140-kDa L1 fragment was found to be tightly associated with the full-length 200-kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth. The transmembrane and multidomain neural adhesion molecule L1 plays important functional roles in the developing and adult nervous system. L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different fragments. In this report, we present evidence that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. Analysis of mutations revealed Arg845 to be the site of cleavage generating the N-terminal 140-kDa fragment. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140-kDa L1 fragment was found to be tightly associated with the full-length 200-kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth. fibronectin type III Proteolytic processing of cell-surface proteins is of prime importance for regulating the functional properties of these proteins (for reviews, see Refs. 1Hooper J.D. Clements J.A. Quigley J.P. Antalis T.M. J. Biol. Chem. 2001; 276: 857-860Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 2Turner A.J. Hooper N.M. Biochem. Soc. Trans. 1999; 27: 255-259Crossref PubMed Scopus (54) Google Scholar, 3Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar, 4Shiosaka S. Yoshida S. Neurosci. Res. 2000; 37: 85-89Crossref PubMed Scopus (47) Google Scholar, 5Yoshida S. Shiosaka S. Int. J. Mol. Med. 1999; 3: 405-409PubMed Google Scholar). Cleavage of recognition molecules at the cell surface has been implicated in neuronal migration, neurite outgrowth, and synaptic plasticity (6Galko M.J. Tessier-Lavigne M. Science. 2000; 289: 1365-1367Crossref PubMed Scopus (147) Google Scholar, 7Hattori M. Osterfield M. Flanagan J.G. Science. 2000; 289: 1360-1365Crossref PubMed Scopus (460) Google Scholar, 8Madani R. Hulo S. Toni N. Madani H. Steimer T. Muller D. Vassalli J.D. EMBO J. 1999; 18: 3007-3012Crossref PubMed Scopus (234) Google Scholar, 9Spira M.E. Oren R. Dormann A. Ilouz N. Lev S. Cell. Mol. Neurobiol. 2001; 21: 591-604Crossref PubMed Scopus (69) Google Scholar, 10Oka T. Akisada M. Okabe A. Sakurai K. Shiosaka S. Kato K. Neurosci. Lett. 2002; 321: 141-144Crossref PubMed Scopus (27) Google Scholar, 11Szklarczyk A. Lapinska J. Rylski M. McKay R.D. Kaczmarek L. J. Neurosci. 2002; 22: 920-930Crossref PubMed Google Scholar, 12Lu X. Wyszynski M. Sheng M. Baudry M. J. Neurochem. 2001; 77: 1553-1560Crossref PubMed Scopus (54) Google Scholar, 13Nakagami Y. Abe K. Nishiyama N. Matsuki N. J. Neurosci. 2000; 20: 2003-2010Crossref PubMed Google Scholar). Among the neural adhesion molecules, L1 has been shown to undergo proteolytic cleavage, which has been suggested to be involved in several functions of this molecule.L1 is a member of the immunoglobulin superfamily consisting of immunoglobulin-like domains and fibronectin type III repeats (for reviews, see Refs. 14Brümmendorf T. Kenwrick S. Rathjen F.G. Curr. Opin. Neurobiol. 1998; 8: 87-97Crossref PubMed Scopus (212) Google Scholar and 15Hortsch M. Mol. Cell. Neurosci. 2000; 15: 1-10Crossref PubMed Scopus (182) Google Scholar). In the central nervous system, L1 is expressed only by post-mitotic neurons and mainly on non-myelinated axons, whereas in the peripheral nervous system, it is expressed by neurons as well as by non-myelinating Schwann cells. L1 is also expressed by non-neural cells, including normal and transformed cells of hematopoietic and epithelial origin. L1 is involved in neuronal migration, neurite outgrowth, and myelination (for review, see Ref. 14Brümmendorf T. Kenwrick S. Rathjen F.G. Curr. Opin. Neurobiol. 1998; 8: 87-97Crossref PubMed Scopus (212) Google Scholar) as well as axon guidance, fasciculation, and regeneration (16Castellani V. Chedotal A. Schachner M. Faivre-Sarrailh C. Rougon G. Neuron. 2000; 27: 237-249Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 17Zhang Y. Roslan R. Lang D. Schachner M. Lieberman A.R. Anderson P.N. Mol. Cell. Neurosci. 2000; 16: 71-86Crossref PubMed Scopus (82) Google Scholar). Furthermore, it enhances cell survival (18Chen S. Mantei N. Dong L. Schachner M. J. Neurobiol. 1999; 38: 428-439Crossref PubMed Scopus (146) Google Scholar) and synaptic plasticity (19Lüthi A. Mohajeri H. Schachner M. Laurent J.P. J. Neurosci. Res. 1996; 46: 1-6Crossref PubMed Scopus (0) Google Scholar). The importance of L1 in nervous system development is underscored by the abnormal phenotypes of L1 mutations in humans and mice (for review, see Ref. 20Kamiguchi H. Hlavin M.L. Lemmon V. Mol. Cell. Neurosci. 1998; 12: 48-55Crossref PubMed Scopus (116) Google Scholar). L1 engages in homophilic and heterophilic cell interactions (for reviews, see Refs. 14Brümmendorf T. Kenwrick S. Rathjen F.G. Curr. Opin. Neurobiol. 1998; 8: 87-97Crossref PubMed Scopus (212) Google Scholar and 15Hortsch M. Mol. Cell. Neurosci. 2000; 15: 1-10Crossref PubMed Scopus (182) Google Scholar) Heterophilic binding partners are the RGD-binding integrins and TAG-1/axonin-1, F3/F11/contactin, NCAM, CD9, CD24, and phosphacan (Ref. 21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar and references therein). These interactions are likely to depend on the presentation of the L1 molecule either as a membrane-bound form or as a proteolytic fragment, which has been described in various forms (Ref.22Nayeem N. Silletti S. Yang X. Lemmon V.P. Reisfeld R.A. Stallcup W.B. Montgomery A.M. J. Cell Sci. 1999; 112: 4739-4749PubMed Google Scholar and references therein). The 140- and 80-kDa fragments resulting from cleavage within the third fibronectin type III (FNIII)1 domain (23Moos M. Tacke R. Scherer H. Teplow D. Früh K. Schachner M. Nature. 1988; 334: 701-703Crossref PubMed Scopus (508) Google Scholar) have been generated in vitro by trypsin (24Sadoul K. Sadoul R. Faissner A. Schachner M. J. Neurochem. 1988; 50: 510-521Crossref PubMed Scopus (76) Google Scholar) or plasmin (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). The third FNIII domain containing two RGD-independent integrin-binding sites (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar) is involved in homophilic binding (25Holm J. Appel F. Schachner M. J. Neurosci. Res. 1995; 42: 9-20Crossref PubMed Scopus (26) Google Scholar), multimerization (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar), and L1-dependent neurite outgrowth (26Stallcup W.B. J. Neurosci. Res. 2000; 61: 33-43Crossref PubMed Scopus (19) Google Scholar). Cleavage within this domain by plasmin reduces multimerization and RGD-independent integrin binding (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). The 180- and 50-kDa fragments result from membrane-proximal cleavage of the membrane-spanning 200- and 80-kDa L1 forms, respectively, by a metalloprotease, most likely of the ADAM (adisintegrin andmetalloprotease) family (27Beer S. Oleszewski M. Gutwein P. Geiger C. Altevogt P. J. Cell Sci. 1999; 112: 2667-2675Crossref PubMed Google Scholar, 28Gutwein P. Oleszewski M. Mechtersheimer S. Agmon-Levin N. Krauss K. Altevogt P. J. Biol. Chem. 2000; 275: 15490-15497Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). This cleavage step has been proposed to be required for cell migration (28Gutwein P. Oleszewski M. Mechtersheimer S. Agmon-Levin N. Krauss K. Altevogt P. J. Biol. Chem. 2000; 275: 15490-15497Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Because specific proteolytic processing of L1 is important for regulation of neuronal migration and neurite outgrowth, we have searched for the proteases responsible for cleaving L1 at the two sites and investigated some of the structural and functional consequences of this proteolytic cleavage.DISCUSSIONFull-length L1 is converted to N-terminal 140-kDa and C-terminal membrane-spanning 80-kDa complementary proteolytic fragments (24Sadoul K. Sadoul R. Faissner A. Schachner M. J. Neurochem. 1988; 50: 510-521Crossref PubMed Scopus (76) Google Scholar) by proteolytic cleavage in the third FNIII domain (23Moos M. Tacke R. Scherer H. Teplow D. Früh K. Schachner M. Nature. 1988; 334: 701-703Crossref PubMed Scopus (508) Google Scholar). This domain is functionally important because it contributes to homophilic binding (25Holm J. Appel F. Schachner M. J. Neurosci. Res. 1995; 42: 9-20Crossref PubMed Scopus (26) Google Scholar) and multimerization of L1, neurite outgrowth, and RGD-independent integrin binding to the β1-integrin subunit (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). Two motifs (821QVKGHLR827 and837GSQRKHSKR845; see Fig. 2 A) within the third FNIII domain are involved in RGD-independent integrin binding to L1. Substitution of the dibasic RK and KR in the837GSQRKHSKR845 motif results in reduced multimerization of L1 molecules and diminished integrin binding (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). The functional activity of this particular RGD-independent integrin-binding motif appears to be regulated by proteolytic cleavage within this sequence motif: cleavage by plasmin within this motif disrupts multimerization and RGD-independent integrin binding. Plasmin, the end product of the plasminogen/plasminogen activator cascade, cleaves L1 within the 837GSQRKHSKR845 motif at Lys841 and Lys844, leading to the generation and release of a 140-kDa fragment (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). These cleavage sites are both different from the cleavage site obtained by N-terminal sequencing of the 80-kDa form (23Moos M. Tacke R. Scherer H. Teplow D. Früh K. Schachner M. Nature. 1988; 334: 701-703Crossref PubMed Scopus (508) Google Scholar), indicating that proteolytic cleavage takes place mainly at Arg845. The sequence840RKHSKR845 preceding this cleavage site resembles the proprotein convertase recognition motif, (R/K)X 0,2,4,6(K/R) (for review, see Ref. 37Seidah N.G. Chretien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (682) Google Scholar). Here, we have provided evidence that the proprotein convertase PC5A is responsible for the proteolytic processing at Arg845, leading to the generation of the 140-kDa L1 fragment. Mutation of this putative recognition motif abolishes cleavage of L1 by PC5A, clearly demonstrating that this sequence is the recognition motif for PC5A. Furin is relatively inefficient in processing L1 to yield a 140-kDa fragment, and other proprotein proteases do not cleave L1. A similar result was obtained for the processing of integrin pro-α-subunits: PC5A is more active than furin, whereas PC1, PC2, PACE4, PC7, and PC5B are inactive (38Lissitzky J.C. Luis J. Munzer J.S. Benjannet S. Parat F. Chretien M. Marvaldi J. Seidah N.G. Biochem. J. 2000; 346: 133-138Crossref PubMed Scopus (98) Google Scholar). In addition, although furin and other proprotein convertases do not efficiently process members of the transforming growth factor-β superfamily, PC5A appears to be responsible for the in vivoprocessing of these proteins (49Ulloa L. Creemers J.W. Roy S. Liu S. Mason J. Tabibzadeh S. J. Biol. Chem. 2001; 276: 21387-21396Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The low processing efficiency of furin could be due to the fact that furin preferentially cleaves substrates with the recognition motif RX(R/K)R (50Molloy S.S. Bresnahan P.A. Leppla S.H. Klimpel K.R. Thomas G. J. Biol. Chem. 1992; 267: 16396-16402Abstract Full Text PDF PubMed Google Scholar), and the sequence HXKR, which is present in L1, is a poor substrate (51Chen Y. Molloy S.S. Thomas L. Gambee J. Bachinger H.P. Ferguson B. Zonana J. Thomas G. Morris N.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7218-7223Crossref PubMed Scopus (94) Google Scholar).The particular spatial and temporal expression patterns of PC5A in the brain (45Dong W. Marcinkiewicz M. Vieau D. Chretien M. Seidah N.G. Day R. J. Neurosci. 1995; 15: 1778-1796Crossref PubMed Google Scholar, 52Zheng M. Seidah N.G. Pintar J.E. Dev. Biol. 1997; 181: 268-283Crossref PubMed Scopus (41) Google Scholar, 53Villeneuve P. Seidah N.G. Beaudet A. Neuroscience. 1999; 92: 641-654Crossref PubMed Scopus (23) Google Scholar) overlap with those of L1 (54Faissner A. Kruse J. Nieke J. Schachner M. Brain Res. 1984; 317: 69-82Crossref PubMed Scopus (80) Google Scholar), whereas furin is ubiquitously expressed in all brain areas and cell types (55Schäfer M.K. Day R. Cullinan W.E. Chretien M. Seidah N.G. Watson S.J. J. Neurosci. 1993; 13: 1258-1279Crossref PubMed Google Scholar). In contrast to furin, PC5A expression is restricted to neurons (53Villeneuve P. Seidah N.G. Beaudet A. Neuroscience. 1999; 92: 641-654Crossref PubMed Scopus (23) Google Scholar), as has been shown for L1 (for review, see Ref. 20Kamiguchi H. Hlavin M.L. Lemmon V. Mol. Cell. Neurosci. 1998; 12: 48-55Crossref PubMed Scopus (116) Google Scholar). Proteolysis of L1 by PC5A in the hippocampus might be involved in synaptic plasticity underlying hippocampus-dependent spatial learning. Indeed, generation of the 140-kDa fragment is observed in the hippocampus, which expresses PC5A, whereas in the cerebellum, which does not show detectable PC5A expression, this fragment is not generated. Interestingly, both PC5A (56Marcinkiewicz M. Savaria D. Marcinkiewicz J. Brain Res. Mol. Brain Res. 1998; 59: 229-246Crossref PubMed Scopus (32) Google Scholar) and L1 are up-regulated after a lesion in the peripheral nervous system; and thus, the processing of L1 by PC5A could be relevant in regeneration, which is highly L1-dependent (see, for instance, Ref. 17Zhang Y. Roslan R. Lang D. Schachner M. Lieberman A.R. Anderson P.N. Mol. Cell. Neurosci. 2000; 16: 71-86Crossref PubMed Scopus (82) Google Scholar). However, our results do not exclude that other proteases, such as plasmin, cleave L1 in the third FNIII domain, resulting in the generation of different 140-kDa fragments.A second proteolytic site has been shown to exist in L1, leading to the formation of a 180-kDa fragment. This site within an unknown cleavage sequence is localized close to the plasma membrane and is susceptible to cleavage by a metalloprotease. Recently, it has been suggested that the metalloprotease ADAM10 cleaves L1 at this site and generates a 180-kDa fragment (57Mechtersheimer S. Gutwein P. Agmon-Levin N. Stoeck A. Oleszewski M. Riedle S. Fogel M. Lemmon V. Altevogt P. J. Cell Biol. 2001; 155: 661-673Crossref PubMed Scopus (28) Google Scholar). In the present study, we have shown that the metalloprotease inhibitor GM 6001 inhibits formation of this 180-kDa fragment and interferes with neurite outgrowth. Interestingly, calmodulin inhibitors enhance proteolytic cleavage at this site, as has been shown for other functionally important cell-surface receptors regulating cytokine, neurotrophin, and cell recognition (46Diaz-Rodriguez E. Esparis-Ogando A. Montero J.C. Yuste L. Pandiella A. Biochem. J. 2000; 346: 359-367Crossref PubMed Scopus (56) Google Scholar, 47Kahn J. Walcheck B. Migaki G.I. Jutila M.A. Kishimoto T.K. Cell. 1998; 92: 809-818Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Our observation thus provides further evidence that intracellular signaling via calcium influences release of recognition molecule fragments from the cell surface. These fragments may then diffuse into the extracellular matrix to interact with partner molecules and thereby modulate the cellular environment. The release of the 180-kDa fragment entails the release of the 140-kDa fragment, which remains tightly associated with the membrane by interacting with the full-length L1 molecule. A prerequisite for this concerted release of the complex of the 180- and 140-kDa fragments appears to be the dimerization of full-length L1 at the cell surface, as shown in this study.cis-Dimerization has been reported for other recognition molecules belonging to the families of cadherins (58Tamura K. Shan W.S. Hendrickson W.A. Colman D.R. Shapiro L. Neuron. 1998; 20: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar); integrins (for review, see Ref. 59Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar); selectins (60Ramachandran V. Yago T. Epperson T.K. Kobzdej M.M. Nollert M.U. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10166-10171Crossref PubMed Scopus (114) Google Scholar); and immunoglobulins, such as ICAM-1 (intercellular adhesionmolecule-1) (61Jun C.D. Carman C.V. Redick S.D. Shimaoka M. Erickson H.P. Springer T.A. J. Biol. Chem. 2001; 276: 29019-29027Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), P0 (62Thompson A.J. Cronin M.S. Kirschner D.A. J. Neurosci. Res. 2002; 67: 766-771Crossref PubMed Scopus (18) Google Scholar), PEACAM-1 (63Newton J.P. Hunter A.P. Simmons D.L. Buckley C.D. Harvey D.J. Biochem. Biophys. Res. Commun. 1999; 261: 283-291Crossref PubMed Scopus (48) Google Scholar), JAM-1 (64Kostrewa D. Brockhaus M. D'Arcy A. Dale G.E. Nelboeck P. Schmid G. Mueller F. Bazzoni G. Dejana E. Bartfai T. Winkler F.K. Hennig M. EMBO J. 2001; 20: 4391-4398Crossref PubMed Scopus (187) Google Scholar), nectin (65Mizoguchi A. Nakanishi H. Kimura K. Matsubara K. Ozaki-Kuroda K. Katata T. Honda T. Kiyohara Y. Heo K. Higashi M. Tsutsumi T. Sonoda S. Ide C. Takai Y. J. Cell Biol. 2002; 156: 555-565Crossref PubMed Scopus (238) Google Scholar), tractin (66Jie C. Xu Y. Wang D. Lukin D. Zipser B. Jellies J. Johansen K.M. Johansen J. Biochim. Biophys. Acta. 2000; 1479: 1-14Crossref PubMed Scopus (9) Google Scholar), CD4 (67Lynch G.W. Sloane A.J. Raso V. Lai A. Cunningham A.L. Eur. J. Immunol. 1999; 29: 2590-2602Crossref PubMed Scopus (46) Google Scholar), and CEACAM-1 and CEACAM-2 (68Hunter I. Sawa H. Edlund M. Obrink B. Biochem. J. 1996; 320: 847-853Crossref PubMed Scopus (49) Google Scholar). Dimerization thus appears to be important for signal transduction, as has been shown for E- and C-cadherins as well as for the neural N-cadherin (Ref. 58Tamura K. Shan W.S. Hendrickson W.A. Colman D.R. Shapiro L. Neuron. 1998; 20: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar and references therein). Furthermore, dimerization of selectins has been shown to enhance adhesive tethers (60Ramachandran V. Yago T. Epperson T.K. Kobzdej M.M. Nollert M.U. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10166-10171Crossref PubMed Scopus (114) Google Scholar). Interestingly, dimerization of CEACAM-1 and CEACAM-2 is regulated by calmodulin and calcium ions (68Hunter I. Sawa H. Edlund M. Obrink B. Biochem. J. 1996; 320: 847-853Crossref PubMed Scopus (49) Google Scholar), highlighting the importance of inside-out signaling mechanisms. ICAM-1 exists predominantly as a dimer at the cell surface and binds in this dimeric state with greatly enhanced affinity to the integrin LFA-1 compared with its monomeric form (43Reilly P.L. Woska J.R. Jeanfavre D.D. McNally E. Rothlein R. Bormann B.J. J. Immunol. 1995; 155: 529-532PubMed Google Scholar).The shedding of the L1 dimers from the cell surface could have several consequences. Because the 180/140-kDa dimer dissociates into its monomers after release from the cell surface, the soluble diffusible 140- and 180-kDa fragments could constitute important ingredients in the extracellular matrix, possibly playing different functional roles. It might thus be conceivable that L1-synthesizing cells build concentration gradients of either soluble or matrix-embedded adhesion molecules that modulate cell migration and axon guidance by "conditioning" the cellular environment for L1 homophilic and heterophilic interactions with the surface of adjacent cells. Another possibility is that the proteolytic processing uncovers binding sites of the residual transmembrane fragments for different ligands. We thus support the idea that proteolytically processed L1 may subserve at least two functions: modification of the extracellular milieu and of transmembrane signaling via the residual L1 receptor stumps. Proteolytic processing of cell-surface proteins is of prime importance for regulating the functional properties of these proteins (for reviews, see Refs. 1Hooper J.D. Clements J.A. Quigley J.P. Antalis T.M. J. Biol. Chem. 2001; 276: 857-860Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 2Turner A.J. Hooper N.M. Biochem. Soc. Trans. 1999; 27: 255-259Crossref PubMed Scopus (54) Google Scholar, 3Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar, 4Shiosaka S. Yoshida S. Neurosci. Res. 2000; 37: 85-89Crossref PubMed Scopus (47) Google Scholar, 5Yoshida S. Shiosaka S. Int. J. Mol. Med. 1999; 3: 405-409PubMed Google Scholar). Cleavage of recognition molecules at the cell surface has been implicated in neuronal migration, neurite outgrowth, and synaptic plasticity (6Galko M.J. Tessier-Lavigne M. Science. 2000; 289: 1365-1367Crossref PubMed Scopus (147) Google Scholar, 7Hattori M. Osterfield M. Flanagan J.G. Science. 2000; 289: 1360-1365Crossref PubMed Scopus (460) Google Scholar, 8Madani R. Hulo S. Toni N. Madani H. Steimer T. Muller D. Vassalli J.D. EMBO J. 1999; 18: 3007-3012Crossref PubMed Scopus (234) Google Scholar, 9Spira M.E. Oren R. Dormann A. Ilouz N. Lev S. Cell. Mol. Neurobiol. 2001; 21: 591-604Crossref PubMed Scopus (69) Google Scholar, 10Oka T. Akisada M. Okabe A. Sakurai K. Shiosaka S. Kato K. Neurosci. Lett. 2002; 321: 141-144Crossref PubMed Scopus (27) Google Scholar, 11Szklarczyk A. Lapinska J. Rylski M. McKay R.D. Kaczmarek L. J. Neurosci. 2002; 22: 920-930Crossref PubMed Google Scholar, 12Lu X. Wyszynski M. Sheng M. Baudry M. J. Neurochem. 2001; 77: 1553-1560Crossref PubMed Scopus (54) Google Scholar, 13Nakagami Y. Abe K. Nishiyama N. Matsuki N. J. Neurosci. 2000; 20: 2003-2010Crossref PubMed Google Scholar). Among the neural adhesion molecules, L1 has been shown to undergo proteolytic cleavage, which has been suggested to be involved in several functions of this molecule. L1 is a member of the immunoglobulin superfamily consisting of immunoglobulin-like domains and fibronectin type III repeats (for reviews, see Refs. 14Brümmendorf T. Kenwrick S. Rathjen F.G. Curr. Opin. Neurobiol. 1998; 8: 87-97Crossref PubMed Scopus (212) Google Scholar and 15Hortsch M. Mol. Cell. Neurosci. 2000; 15: 1-10Crossref PubMed Scopus (182) Google Scholar). In the central nervous system, L1 is expressed only by post-mitotic neurons and mainly on non-myelinated axons, whereas in the peripheral nervous system, it is expressed by neurons as well as by non-myelinating Schwann cells. L1 is also expressed by non-neural cells, including normal and transformed cells of hematopoietic and epithelial origin. L1 is involved in neuronal migration, neurite outgrowth, and myelination (for review, see Ref. 14Brümmendorf T. Kenwrick S. Rathjen F.G. Curr. Opin. Neurobiol. 1998; 8: 87-97Crossref PubMed Scopus (212) Google Scholar) as well as axon guidance, fasciculation, and regeneration (16Castellani V. Chedotal A. Schachner M. Faivre-Sarrailh C. Rougon G. Neuron. 2000; 27: 237-249Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 17Zhang Y. Roslan R. Lang D. Schachner M. Lieberman A.R. Anderson P.N. Mol. Cell. Neurosci. 2000; 16: 71-86Crossref PubMed Scopus (82) Google Scholar). Furthermore, it enhances cell survival (18Chen S. Mantei N. Dong L. Schachner M. J. Neurobiol. 1999; 38: 428-439Crossref PubMed Scopus (146) Google Scholar) and synaptic plasticity (19Lüthi A. Mohajeri H. Schachner M. Laurent J.P. J. Neurosci. Res. 1996; 46: 1-6Crossref PubMed Scopus (0) Google Scholar). The importance of L1 in nervous system development is underscored by the abnormal phenotypes of L1 mutations in humans and mice (for review, see Ref. 20Kamiguchi H. Hlavin M.L. Lemmon V. Mol. Cell. Neurosci. 1998; 12: 48-55Crossref PubMed Scopus (116) Google Scholar). L1 engages in homophilic and heterophilic cell interactions (for reviews, see Refs. 14Brümmendorf T. Kenwrick S. Rathjen F.G. Curr. Opin. Neurobiol. 1998; 8: 87-97Crossref PubMed Scopus (212) Google Scholar and 15Hortsch M. Mol. Cell. Neurosci. 2000; 15: 1-10Crossref PubMed Scopus (182) Google Scholar) Heterophilic binding partners are the RGD-binding integrins and TAG-1/axonin-1, F3/F11/contactin, NCAM, CD9, CD24, and phosphacan (Ref. 21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar and references therein). These interactions are likely to depend on the presentation of the L1 molecule either as a membrane-bound form or as a proteolytic fragment, which has been described in various forms (Ref.22Nayeem N. Silletti S. Yang X. Lemmon V.P. Reisfeld R.A. Stallcup W.B. Montgomery A.M. J. Cell Sci. 1999; 112: 4739-4749PubMed Google Scholar and references therein). The 140- and 80-kDa fragments resulting from cleavage within the third fibronectin type III (FNIII)1 domain (23Moos M. Tacke R. Scherer H. Teplow D. Früh K. Schachner M. Nature. 1988; 334: 701-703Crossref PubMed Scopus (508) Google Scholar) have been generated in vitro by trypsin (24Sadoul K. Sadoul R. Faissner A. Schachner M. J. Neurochem. 1988; 50: 510-521Crossref PubMed Scopus (76) Google Scholar) or plasmin (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). The third FNIII domain containing two RGD-independent integrin-binding sites (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar) is involved in homophilic binding (25Holm J. Appel F. Schachner M. J. Neurosci. Res. 1995; 42: 9-20Crossref PubMed Scopus (26) Google Scholar), multimerization (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar), and L1-dependent neurite outgrowth (26Stallcup W.B. J. Neurosci. Res. 2000; 61: 33-43Crossref PubMed Scopus (19) Google Scholar). Cleavage within this domain by plasmin reduces multimerization and RGD-independent integrin binding (21Siletti S. Mei F. Sheppard D. Montgomery A.M. J. Cell Biol. 2000; 149: 1485-1502Crossref PubMed Scopus (131) Google Scholar). The 180- and 50-kDa fragments result from membrane-proximal cleavage of the membrane-spanning 200- and 80-kDa L1 forms, respectively, by a metalloprotease, most likely of the ADAM (adisintegrin andmetalloprotease) family (27Beer S. Oleszewski M. Gutwein P. Geiger C. Altevogt P. J. Cell Sci. 1999; 112: 2667-2675Crossref PubMed Google Scholar, 28Gutwein P. Oleszewski M. Mechtersheimer S. Agmon-Levin N. Krauss K. Altevogt P. J. Biol. Chem. 2000; 275: 15490-15497Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). This cleavage step has been proposed to be required for cell migration (28Gutwein P. Oleszewski M. Mechtersheimer S. Agmon-Levin N. Krauss K. Altevogt P. J. Biol. Chem. 2000; 275: 15490-15497Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Because specific proteolytic processing of L1 is important for regulation of neuronal migration and neurite outgrowth, we have searched for the proteases responsible for cleaving L1 at the two sites and investigated some of the structural and functional consequences of this proteolytic cleavage. DISCUSSIONFull-length L1 is converted to N-terminal 140-kDa and C-terminal membrane-spanning 80-kDa complementary proteolytic fragments (24Sadoul K. Sadoul R. Faissner A. Schachner M. J. Neurochem. 1988; 50: 510-521Crossref PubMed Scopus (76) Google Scholar) by proteo
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