Disruption of the GDNF Binding Site in NCAM Dissociates Ligand Binding and Homophilic Cell Adhesion

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor α1, shedding light on the molecular architecture of a multicomponent GDNF receptor. Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor α1, shedding light on the molecular architecture of a multicomponent GDNF receptor. Members of the glial cell line-derived neurotrophic factor (GDNF) 3The abbreviations used are: GDNF, glial cell line-derived neurotrophic factor; GFRα, GDNF family receptor α; NCAM, neural cell adhesion molecule; HA, hemagglutinin; Ig, immunoglobulin; PBS, phosphate-buffered saline. family regulate cell survival, differentiation, and migration in the peripheral and central nervous systems as well as in a few peripheral organs. The four members of this ligand family, i.e. GDNF, Neurturin, Artemin, and Persephin, share ∼40% of their amino acid sequence. GDNF and Artemin are being developed as therapeutic agents against Parkinson disease and peripheral neuropathies, respectively (1Barker R.A. Lancet Neurol. 2006; 5: 285-286Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 2Gill S.S. Patel N.K. Hotton G.R. O'Sullivan K. McCarter R. Bunnage M. Brooks D.J. Svendsen C.N. Heywood P. Nat. Med. 2003; 9: 589-595Crossref PubMed Scopus (1133) Google Scholar, 3Gardell L.R. Wang R. 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Development. 1999; 126: 2785-2797PubMed Google Scholar, 15Natarajan D. Marcos-Gutierrez C. Pachnis V. de Graaff E. Development. 2002; 129: 5151-5160Crossref PubMed Google Scholar) and ureter morphogenesis during kidney development (16Sainio K. Suvanto P. Davies J. Wartiovaara J. Wartiovaara K. Saarma M. Arumae U. Meng X.J. Lindahl M. Pachnis V. Sariola H. Development. 1997; 124: 4077-4087Crossref PubMed Google Scholar, 17de Graaff E. Srinivas S. Kilkenny C. D'Agati V. Mankoo B.S. Costantini F. Pachnis V. Genes Dev. 2001; 15: 2433-2444Crossref PubMed Scopus (202) Google Scholar). On the other hand, in the presence of NCAM, GDNF and GFRα1 stimulate neurite outgrowth in vitro (12Paratcha G. Ledda F. Ibáñez C.F. Cell. 2003; 113: 867-879Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar) and synaptogenesis in vitro and in vivo (18Ledda F. Paratcha G. Sandoval-Guzmán T. Ibáñez C.F. Nat. Neurosci. 2007; 10: 293-300Crossref PubMed Scopus (129) Google Scholar) in hippocampal neurons, stimulate migration of neuronal precursors in the rostral migratory stream (12Paratcha G. Ledda F. Ibáñez C.F. Cell. 2003; 113: 867-879Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 19Paratcha G. Ibáñez C.F. Ledda F. Mol. Cell Neurosci. 2005; 31: 505-514Crossref PubMed Scopus (125) Google Scholar), and regulate Schwann cell migration and function (12Paratcha G. Ledda F. Ibáñez C.F. Cell. 2003; 113: 867-879Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 20Iwase T. Jung C.G. Bae H. Zhang M. Soliven B. J. Neurochem. 2005; 94: 1488-1499Crossref PubMed Scopus (113) Google Scholar). Despite the importance of GDNF signaling for normal development and its possible therapeutic applications, the molecular architecture of these receptor complexes is not yet understood. Crystal structures have been described for GDNF (21Eigenbrot C. Gerber N. Nature Struct. Biol. 1997; 4: 435-438Crossref PubMed Scopus (104) Google Scholar), a fragment of the ligand binding domain of GFRα1 (22Leppanen V.M. Bespalov M.M. Runeberg-Roos P. Puurand U. Merits A. Saarma M. Goldman A. EMBO J. 2004; 23: 1452-1462Crossref PubMed Scopus (52) Google Scholar), and the complex between Artemin and the ligand binding domain of its cognate GFRα3 receptor (23Wang X. Baloh R.H. Milbrandt J. Garcia K.C. Structure. 2006; 14: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The latter validated previous mutagenesis studies performed on GDNF ligands and GFRα receptors (24Baloh R.H. Tansey M.G. Johnson E.M. Milbrandt J. J. Biol. Chem. 2000; 275: 3412-3420Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 25Eketjäll S. Fainzilber M. Murray-Rust J. Ibáñez C.F. EMBO J. 1999; 18: 5901-5910Crossref PubMed Scopus (106) Google Scholar, 26Scott R.P. Ibáñez C.F. J. Biol. Chem. 2001; 276: 1450-1458Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and demonstrated that the ligand binding domain of GFRα receptors is formed by a single compact module that interacts with the poles of the elongated dimer of GDNF family ligands. In addition, a model of the extracellular region of RET based on four consecutive cadherin-like domains has been reported (27Anders J. Kjær S. Ibáñez C.F. J. Biol. Chem. 2001; 276: 35808-35817Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), but crystal structure data are still missing. The extracellular region of NCAM is composed of five immunoglobulin-like (Ig) and two fibronectin-like domains, and the crystal structure of the first three Ig domains has been reported (28Soroka V. Kolkova K. Kastrup J.S. Diederichs K. Breed J. Kiselyov V.V. Poulsen F.M. Larsen I.K. Welte W. Berezin V. Bock E. Kasper C. Structure. 2003; 11: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Previous work has indicated that NCAM, unlike RET, can interact directly with GDNF but that high affinity binding and downstream signaling requires co-expression with the GDNF co-receptor GFRα1 (12Paratcha G. Ledda F. Ibáñez C.F. Cell. 2003; 113: 867-879Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). However, it has been unclear whether the ability of NCAM to bind GDNF is related to its adhesive properties or mediated by a distinct and specific protein-protein interaction interface. In this study, we set out to delineate GDNF binding determinants in NCAM and characterize their requirement for NCAM-mediated cell adhesion. Domain boundaries in the rat NCAM140 cDNA (Entrez ID X06564) (29Small S.J. Shull G.E. Santoni M.J. Akeson R. J. Cell Biol. 1987; 105: 2335-2345Crossref PubMed Scopus (92) Google Scholar) were defined based on the exon-intron structure of the chicken Ncam gene (Entrez ID AH005321) (30Owens G.C. Edelman G.M. Cunningham B.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 294-298Crossref PubMed Scopus (153) Google Scholar). The boundaries used for generation of Ncam deletion constructs were as follows (numbering refers to the mature protein, excluding the 19-residue signal peptide): domain 1, Leu1-Gln97; domain 2, Lys98-Val191; domain 3, Pro192-Ala288; domain 4, Lys289-Tyr396; domain 5, Ala397-Asp490; domain 6, Thr491-Arg591; domain 7, Glu592-Ala692. N-terminal deletion constructs were made by PCR, using Pfu DNA polymerase (Promega), with a sense primer corresponding to the beginning of the first domain after the deletion and an antisense primer corresponding to the end of the full-length rat NCAM140 cDNA. The sense and antisense primers contained SfiI and NotI restriction sites, respectively. The PCR fragments were digested with SfiI and NotI (New England Biolabs) and ligated into a SfiI/NotI-digested pSecTag 2A Hygro vector (Invitrogen) modified with a hemagglutinin (HA) tag insertion between the secretion tag and the SfiI site. C-terminal deletions (from the C-terminal end of the extracellular region) were made by fusing two PCR fragments. One fragment was made using the full-length sense primer and the antisense primer corresponding to the end of the most C-terminal domain to be present in the final construct (e.g. the end of domain 3 in the Δ4–7 construct). The other fragment was made using a sense primer corresponding to the first extracellular juxtamembrane residues following domain 7, together with the full-length antisense primer. The full-length sense and antisense primers contained SfiI and NotI restriction sites, respectively. The two fragments were digested with SfiI or NotI, respectively, and ligated into the SfiI/NotI-digested vector. The ligation site between the two fragments was kept blunt-ended to avoid the introduction of any additional amino acids. Single domain deletions were made similarly to the C-terminal deletions, the difference being that the second fragment was different for all constructs, beginning with the domain immediately 3′ of the one to be deleted (e.g. for the Δ3 construct, the second fragment begins with domain 4). Single domains were made in the same way as the N-terminal-truncated constructs but with the appropriate C-terminal deletion construct as a template (e.g. Δ5–7 for the “domain 4 only” construct). As pSecTag already contains an efficient signal sequence, the endogenous NCAM signal sequence was excluded from all constructs. The quadruple mutant constructs were made with the “QuikChange Multi” mutagenesis kit (Stratagene) using three primers simultaneously: one targeting both E236A and D242A, another for D250A, and a third for E272A. COS-7 cells grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in 100-mm plates were with of the appropriate DNA constructs with of after the plates were three with phosphate-buffered and with GDNF a of in binding bovine serum for 4 with for with plates were with and three with GDNF binding to the of of different constructs the cell we performed of cell in of cell were with in binding for The plates were with and with of and and cells were with a cell a 4 with were for was to the and the were for with The were four on and to were in a and the were with developed with and in a were made using cells were in plates with NCAM constructs together with either or using in of medium containing 10% fetal On the following of and cells were and with of medium in of and cell were and on a by Cell adhesion was as the of cells present in that contained used in molecular were from the with for domains of NCAM (28Soroka V. Kolkova K. Kastrup J.S. Diederichs K. Breed J. Kiselyov V.V. Poulsen F.M. Larsen I.K. Welte W. Berezin V. Bock E. Kasper C. Structure. 2003; 11: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and and for the GDNF dimer (21Eigenbrot C. Gerber N. Nature Struct. Biol. 1997; 4: 435-438Crossref PubMed Scopus (104) Google Scholar). Domain 3 of NCAM was to GDNF using with have a to for The complex was by to were from or were on the two proteins the same binding with the binding of NCAM domains and were to of domain binding with domain 3 and was as a On the other hand, of domain in a difference in binding with domain However, this interaction between GDNF and the of the NCAM as the C-terminal end of NCAM domain directly the of the GDNF the that the binding was to domain 3, in with We made a of the NCAM domain 3 with the four residues to in a of binding of In this the is the binding are The binding of domain 3 with the domain 3 or domain The of the of NCAM domain 3 and GDNF can be as a in the has been to bind to the poles of the GDNF dimer S. Fainzilber M. Murray-Rust J. Ibáñez C.F. EMBO J. 1999; 18: 5901-5910Crossref PubMed Scopus (106) Google Scholar). The crystal structure of a complex between GFRα3 and Artemin has been (23Wang X. Baloh R.H. Milbrandt J. Garcia K.C. Structure. 2006; 14: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) complex was used to the of GFRα1 the complex in to a first of the possible architecture of a The GFRα3 structure to was used as a template for a model of GFRα1 using The structures of GDNF and Artemin are for the sequence and However, the interface are different The of GFRα1 in to GDNF was by first the complex GDNF the GFRα1 was In the complex we have not the interface between GFRα1 and GDNF we it as a of the complex i.e. to whether model of the interaction is with a GDNF binding in NCAM were by the binding of a of NCAM deletion Four of NCAM constructs were made deletions from the deletions from the C-terminal end of the extracellular single domains or single domains on their all containing the and domains of the 1, As domain of the NCAM extracellular region is by two consecutive in the Ncam gene (30Owens G.C. Edelman G.M. Cunningham B.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 294-298Crossref PubMed Scopus (153) Google Scholar), we used the exon-intron structure to domain boundaries for binding of and NCAM present the cell were from their N-terminal by and to and of the N-terminal deletion a in GDNF binding after deletion of the third Ig domain The importance of this domain for GDNF binding was from the binding of the C-terminal deletion in which constructs containing this domain were capable of binding ligand constructs Ig domains 4 or Δ5–7 and GDNF binding with the full-length molecule The of are present in these domains C. E. T. M. M. B. Chem. 2004; PubMed Scopus Google Scholar, S. H. M. R. G. R. U. M. 2001; 11: PubMed Scopus Google Scholar), that NCAM GDNF The requirement of domains for ligand binding was by single domains from the NCAM deletion of the third domain the ability of NCAM to bind GDNF, indicating its requirement for ligand binding of either domains 4 or GDNF binding, indicating a of these domains on ligand the of NCAM domains for GDNF binding was using constructs single domains as the extracellular region of the molecule Domain 3 was to bind GDNF well on its other domains or not the from deletion that the third Ig domain of NCAM is both necessary and sufficient for GDNF binding and the ligand binding determinant in this receptor. the crystal structures of GDNF (21Eigenbrot C. Gerber N. Nature Struct. Biol. 1997; 4: 435-438Crossref PubMed Scopus (104) Google Scholar) and NCAM domain 3 (28Soroka V. Kolkova K. Kastrup J.S. Diederichs K. Breed J. Kiselyov V.V. Poulsen F.M. Larsen I.K. Welte W. Berezin V. Bock E. Kasper C. Structure. 2003; 11: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), we the interaction between the two with the molecular modeling was by from the GDNF all possible interaction of these the as by their binding all of which NCAM domain 3 on the between the two of the GDNF dimer and residues in the of the two The interface is by interactions, with four between and and from the first GDNF and from the with and respectively, from NCAM domain 3 of these four residues in the ligand binding interface of NCAM domain 3 to abolished the ability of this domain to bind GDNF in in the full-length NCAM the quadruple mutation GDNF binding to the effects of deletion of domain 3 In to to model of the these four specific residues the NCAM extracellular domain that are for its ability to interact with Most of NCAM interaction on the requirement of domain 3 for efficient NCAM-mediated cell adhesion (28Soroka V. Kolkova K. Kastrup J.S. Diederichs K. Breed J. Kiselyov V.V. Poulsen F.M. Larsen I.K. Welte W. Berezin V. Bock E. Kasper C. Structure. 2003; 11: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, G.C. Edelman G.M. Cunningham B.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, C. U. D. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We the effects of that GDNF binding on the ability of NCAM to mediate cell-cell grown in were with different NCAM constructs with for either or proteins and for their ability to containing and full-length NCAM the formation of cell in this deletion of domain 3 the ability of the molecule to cell adhesion by Domain 3 on its adhesive properties in this not In the quadruple mutant of NCAM of cell adhesion indicating that the four residues that are for GDNF binding are not in NCAM this that the two NCAM can be genetically and the to their in vivo for the generation of the of the of the GDNF we whether the interface we identified the formation of NCAM to the same GDNF NCAM domains be a GDNF binding to related on this was to the one for NCAM based on the crystal structure of domains (28Soroka V. Kolkova K. Kastrup J.S. Diederichs K. Breed J. Kiselyov V.V. Poulsen F.M. Larsen I.K. Welte W. Berezin V. Bock E. Kasper C. Structure. 2003; 11: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) with 2A from V. Kolkova K. Kastrup J.S. Diederichs K. Breed J. Kiselyov V.V. Poulsen F.M. Larsen I.K. Welte W. Berezin V. Bock E. Kasper C. Structure. 2003; 11: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), that it a for GDNF We whether model of the complex was with the of GFRα1 to Previous studies have the GFRα1 binding site to the two poles of the elongated GDNF dimer S. Fainzilber M. Murray-Rust J. Ibáñez C.F. EMBO J. 1999; 18: 5901-5910Crossref PubMed Scopus (106) Google Scholar), from the NCAM binding a that has been by the crystal structure of the GDNF Artemin in complex with the ligand binding domain of its cognate GFRα3 receptor (23Wang X. Baloh R.H. Milbrandt J. Garcia K.C. Structure. 2006; 14: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). the of that we the interaction between GDNF and the ligand binding fragment of on this two GFRα1 one end of the GDNF be the model to a complex containing the three proteins 4, and GFRα1 and NCAM are to interact with other (12Paratcha G. Ledda F. Ibáñez C.F. Cell. 2003; 113: 867-879Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar), interaction can be in the that it be mediated by other domains in the NCAM be that this model the of the GDNF binding domains of GFRα1 and NCAM in the possible of this complex to the plasma the of full-length GFRα1 and NCAM are to be to the plasma it has been that the of the ligand binding domain of GFRα3 the membrane (23Wang X. Baloh R.H. Milbrandt J. Garcia K.C. Structure. 2006; 14: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), both and in this domain are on the same of the an additional N-terminal domain still to be In the of NCAM, studies have indicated a of its extracellular domain S. Cunningham B.A. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A.K. U. J. Cell Biol. 1987; PubMed Scopus (59) Google Scholar). for the of a complex between GDNF, NCAM, and we performed and studies in cells with constructs NCAM and binding and complexes be after with containing that be by containing and that with containing a complex containing one molecule of GDNF, NCAM, and GFRα1 in be after NCAM and in cells that both NCAM and GFRα1 complexes corresponding to the of the complex in 4, and be 5, of multiple NCAM and interactions, this complex the same molecular as that of three NCAM together 5, In we have identified a and specific binding determinant in NCAM that is for its interaction with GDNF, a for the adhesive or properties of NCAM in GDNF We have demonstrated that ligand binding and cell adhesion can be genetically and the first into the molecular architecture of a multicomponent GDNF receptor. with