Three Distinct Molecular Surfaces in Ephrin-A5 Are Essential for a Functional Interaction with EphA3

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2·ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E α-helix, the E-F loop, the underlying H β-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes. Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2·ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E α-helix, the E-F loop, the underlying H β-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes. Signaling by Eph receptors (Ephs) 1The abbreviations used are: Ephs, Eph receptors; TEV, tobacco etch virus; HEK, human embryonic kidney; w/t, wild-type; IP, immunoprecipitation. and their cell surface-associated ephrin ligands forms an essential part of a highly conserved molecular mechanism coordinating cell migration and positioning during normal and oncogenic tissue development (1Poliakov A. Cotrina M. Wilkinson D.G. Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). In general, the path of Eph-expressing cells or axons is directed through contact-dependent cell-cell adhesion or repulsion (2Kullander K. Klein R. Nat. Rev. Mol. Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (970) Google Scholar), whereby competing interactions of neighboring Eph-expressing cells for ephrin targets govern the final cell position as the biological outcome (3Reber M. Burrola P. Lemke G. Nature. 2004; 431: 847-853Crossref PubMed Scopus (99) Google Scholar). Many biological effects attributed to Eph function require concurrent “forward” signaling in Eph-expressing cells and “reverse” signaling in ephrin-expressing cells (4Mellitzer G. Xu Q. Wilkinson D.G. Curr. Opin. Neuro. Biol. 2000; 10: 400-408Crossref PubMed Scopus (126) Google Scholar, 5.Boyd, A. W., and Lackmann, M. (2001) Science's STKE, http://stke.sciencemag.org/cgi/content/full/o_c_sigtrans;2001/112/re20,Google Scholar). In contrast to the prototypical activation mechanism of receptor tyrosine kinases, Eph signaling is activated by the assembly of ephrin/Eph oligomers into large clusters. Ephs are composed of conserved structural modules. They include a unique N-terminal ephrin binding domain (6Labrador J.P. Brambilla R. Klein R. EMBO J. 1997; 16: 3889-3897Crossref PubMed Scopus (64) Google Scholar, 7Lackmann M. Oates A.C. Dottori M. Smith F.M. Do C. Power M. Kravets L. Boyd A.W. J. Biol. Chem. 1998; 273: 20228-20237Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar) forming a globular β-barrel (9Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (95) Google Scholar), a cysteine-rich linker and epidermal growth factor-like region, and two fibronectin type III repeats. The cytoplasmic part contains an uninterrupted, tyrosine kinase domain (10Wybenga-Groot L.E. Baskin B. Ong S.H. Tong J. Pawson T. Sicheri F. Cell. 2001; 106: 745-757Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) and several protein-protein interaction modules, including Src homology 2-docking sites, a sterile-α-motif, and a C-terminal PDZ binding motif (11Himanen J.P. Nikolov D.B. Trends Neurosci. 2003; 26: 46-51Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Structural features broadly classify six glycophosphatidyl inositol membrane-anchored ephrins as A-type, which “promiscuously” can bind and activate nine type-A Ephs, as well as three transmembrane ephrins as B-type, which contain conserved cytoplasmic domains and activate six type-B Ephs (12Eph Nomenclature Committee letter Cell. 1997; 90: 403-404Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). It is now clear that this grouping is likely an oversimplification, and in particular, EphA4 and EphB2 bind and become activated by both A- and B-type ephrins (13Gale N.W. Holland S.J. Valenzuela D.M. Flenniken A. Pan L. Ryan T.E. Henkemeyer M. Strebhardt K. Hirai H. Wilkinson D.G. Pawson T. Davis S. Yancopoulos G.D. Neuron. 1996; 17: 9-19Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar, 14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar). This characteristic promiscuity of functionally relevant Eph/ephrin interactions is possibly because of the high structural conservation of Eph and ephrin binding domains. Crystal structures of the interacting domains of EphB2, ephrin-B2, and ephrin-A5 and of their complexes (8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar, 9Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (95) Google Scholar, 14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar, 15Toth J. Cutforth T. Gelinas A.D. Bethoney K.A. Bard J. Harrison C.J. Dev. Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) revealed that the initial 1:1 Eph/ephrin contacts (16Lackmann M. Mann R.J. Kravets L. Smith F.M. Bucci T.A. Maxwell K.F. Howlett G.J. Olsson J.E. Bos T.V. Cerretti D.P. Boyd A.W. J. Biol. Chem. 1997; 272: 16521-16530Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) are provided by a deep Eph surface channel formed by β-strands (D, E, G, J, M), which buries the extended, hydrophobic ephrin G-H loop (8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar). In the crystal structure of the EphB2·ephrin-B2 complex, a second lower affinity heterotetramerization interface facilitates formation of a 2:2 cyclic complex comprising two Eph/ephrin heterodimers (see Fig. 3). Although comparatively small, the tetramerization interface is critical for the assembly of stable, signaling-competent Eph clusters (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and in agreement with its postulated role of providing subclass binding specificities (8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar), is not present in the structure of the EphB2·ephrin-A5 complex (14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar). Although there is little doubt that the Eph/ephrin heterotetramers are the essential building block of Eph signaling complexes, downstream signaling requires the assembly of higher order oligomers (18Stein E. Lane A.A. Cerretti D.P. Schoecklmann H.O. Schroff A.D. Van Etten R.L. Daniel T.O. Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (368) Google Scholar). In vitro, this is routinely achieved through Eph activation by preclustered, tetravalent ephrin-Fc fusion proteins (19Davis S. Gale N.W. Aldrich T.H. Maisonpierre P.C. Lhotak V. Pawson T. Goldfarb M. Yancopoulos G.D. Science. 1994; 266: 816-819Crossref PubMed Scopus (634) Google Scholar). The available crystal structures leave unclear how Ephs and ephrins assemble into the oligomeric signaling complexes that are required for biological responses (11Himanen J.P. Nikolov D.B. Trends Neurosci. 2003; 26: 46-51Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 18Stein E. Lane A.A. Cerretti D.P. Schoecklmann H.O. Schroff A.D. Van Etten R.L. Daniel T.O. Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (368) Google Scholar) and suggest the involvement of Eph/ephrin contact regions outside the crystallized domains. Indeed, earlier studies indicated the presence of ephrin-independent Eph/Eph contacts located C-terminally of the globular domain that are important for EphA3 function (7Lackmann M. Oates A.C. Dottori M. Smith F.M. Do C. Power M. Kravets L. Boyd A.W. J. Biol. Chem. 1998; 273: 20228-20237Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Furthermore, a recent analysis of ephrin-A5 binding-compromised EphA3 mutants revealed, in addition to the two structurally defined ephrin binding sites, a third functional binding interface outside the crystallized domain (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This site, although contributing only modestly to ligand binding, is essential for receptor phosphorylation, recruitment of signaling molecules, and downstream responses, supporting the notion that the tetrameric Eph·ephrin complex observed in the crystal structure is necessary (but not sufficient) for signaling. The position of the newly identified binding site within the cysteine-rich linker that connects the ephrin binding and Eph-Eph dimerization domains suggests that ephrin binding may cause a reorientation of Ephs that facilitates their assembly into oligomeric clusters (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We have now applied the same random mutagenesis approach previously used to identify ephrin-A5-interacting residues in EphA3 to assign the molecular determinants of ephrin-A5 that mediate EphA3 binding. In this the recently crystal structure of ephrin-A5 in complex with EphB2 (14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar) the structural and functional analysis of EphA3 binding-compromised ephrin-A5 mutants from a library of random mutants the N-terminal receptor binding domain of analysis revealed a of critical residues that the two Eph binding sites in the crystal structure of the of the EphB2·ephrin-B2 complex (8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar). We a third the of which by the EphA3 mutagenesis (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This site, which likely Eph/ephrin the ephrin-A5 E and E-F loop, the underlying H β-strand, as well as the nearby B-C the dimerization and tetramerization sites, is with a corresponding interaction surface on EphA3 that is to the Eph globular and functional analysis of mutants the notion that of each of the three identified Eph binding sites of ephrin-A5 is required to responses in cells. The and the previously (16Lackmann M. Mann R.J. Kravets L. Smith F.M. Bucci T.A. Maxwell K.F. Howlett G.J. Olsson J.E. Bos T.V. Cerretti D.P. Boyd A.W. J. Biol. Chem. 1997; 272: 16521-16530Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, A.W. R.J. E. A. K. M. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and from and EphA3 and the EphA3 domain to the human and regions as previously (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). the of we a ephrin-A5 residues by the corresponding to the site of the tobacco etch by the and regions of human The into the unique site of the and into and the proteins from cell on and previously M. Bucci T. Mann R.J. Kravets E. Smith F. R.L. R.J. K. Boyd A.W. S. A. 1996; PubMed Scopus Google Scholar). large cells in as previously (14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar). ephrin-A5 by from the ephrin-A5 in corresponding to and and into a (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) This the ephrin and C-terminal and of the with the a from D. and mutants of the ephrin-A5 domain (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, R. EMBO J. PubMed Scopus Google Scholar). to or that with residues provided a of the library of random mutants in The from into the and ephrin-A5 proteins with the and and with as previously (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). from each of with and binding and for analysis of the ephrin-A5 relevant in critical ephrin-A5 into the by mutagenesis mutagenesis into cells and proteins from and as and by and by analysis with surfaces EphA3, and of interactions by surface on a as previously (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). and ephrin-A5 domain proteins on and The of high proteins determined from of ephrin-A5 domain in each from of each by the The cells that with EphA3 have previously S.H. P. M. Boyd A.W. Lackmann M. J. Cell 2002; PubMed Google Scholar, S.H. A. Lackmann M. J. Cell Biol. 2004; PubMed Scopus Google Scholar). of cells for 10 with preclustered, w/t, or cell and to with affinity or as previously S.H. P. M. Boyd A.W. Lackmann M. J. Cell 2002; PubMed Google Scholar). The and of the with and of each with for EphA3 define critical Eph binding and signal initiation residues in we of the domain to random only the N-terminal signal and C-terminal The experimental previously to assign critical EphA3 surfaces (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), in a of the ephrin-A5 This to a library of mutants, which are the that proteins both the N-terminal and the C-terminal with and for analysis of with an of two The ephrin-A5 proteins from for binding of that ephrins that EphA3 binding mutants this of the random mutagenesis of of of of in a The EphA3 an initial of their functional we the of human as well as of which bind with to EphA3 (16Lackmann M. Mann R.J. Kravets L. Smith F.M. Bucci T.A. Maxwell K.F. Howlett G.J. Olsson J.E. Bos T.V. Cerretti D.P. Boyd A.W. J. Biol. Chem. 1997; 272: 16521-16530Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, P. Rev. Neurosci. 1998; PubMed Scopus Google Scholar). of that the EphA3 binding to the crystallized of whereby of the corresponding residues are conserved in or of the six of are located within the or heterotetramerization molecular a cluster of that EphA3 binding within the highly conserved motif on the This the E and the E-F loop that additional Together with the nearby B-C loop, structure an ephrin surface the dimerization and tetramerization interfaces Fig. which is not in direct Eph contact in the (14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar). The of residues suggests that define a distinct third interaction site of the we the that not the binding to of the conserved residues of the and of or in the ephrin-A5 residues not EphA3 binding, that this region is not in Eph/ephrin of of the the ephrin-A5 structure in the of Eph/ephrin contacts to six mutants for functional including for each of the previously defined We into an ephrin-A5 to the part of human and by an site, of ephrin-A5 as well as from the same surface ephrins by and their binding to the EphA3 or as proteins on surfaces The analysis of the ephrin-A5 mutants revealed that a of the G-H loop with an the and the of the interaction in affinity binding This that the contribution to the binding affinity is provided by of the ephrin G-H loop into the ligand binding Eph channel the interface (8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar, F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). By on the ephrin and which the surface of the receptor the Eph/ephrin dimerization interaction only a in the binding affinity and of ephrin-A5 as into a a site, as and fusion from on and for as indicated in the to Fig. including and from for each as previously including and from for each as previously including and from for each as previously with and corresponding mutants to for the as into a a site, as and fusion from on and for as indicated in the to Fig. including and from for each as previously (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google with and corresponding mutants to for the in a a EphA3 and binding observed for the located the of the ephrin loop the tetramerization affinity from to the of a of the with ephrin-A5 two of the and located the previously dimerization and tetramerization surfaces (see Fig. EphA3 binding to an with that of the dimerization interface and the residues to the conserved mediate its the the ephrin-A5 molecular and are from the dimerization of to the of the G-H and tetramerization and is their the structure of ephrin binding only the molecular surface in particular, the positioning of the E-F loop and E to the underlying β-barrel We that this surface region, with the nearby B-C loop an additional Eph/ephrin interface that not in the crystal structures of the complex of their minimal interaction domains. EphA3 the binding of the ephrin-A5 mutants cause corresponding effects on EphA3 signaling and downstream responses by responses, including cell and as well as EphA3 and recruitment of or fusion proteins used for and their binding to EphA3 by analysis previously S.H. P. M. Boyd A.W. Lackmann M. J. Cell 2002; PubMed Google Scholar), of cells to in cell and By of of cells with the the cell cell as with cells. In recruitment not in cells. EphA3 only not in agreement with findings that EphA3 and recruitment are not S.H. P. M. Boyd A.W. Lackmann M. J. Cell 2002; PubMed Google Scholar). of cells to in the tetramerization interface or the H only cell and cell recruitment and EphA3 for both mutants to suggesting a role of the third ephrin interface for the of the Eph/ephrin signaling findings the functional of the and the heterotetramerization interfaces for EphA3 binding and activation by ephrin-A5 in suggest the essential involvement of a third previously Eph/ephrin contact unique of Eph signaling is the assembly of oligomeric signaling complexes (18Stein E. Lane A.A. Cerretti D.P. Schoecklmann H.O. Schroff A.D. Van Etten R.L. Daniel T.O. Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (368) Google Scholar, E. Lane A.A. H. Cerretti D.P. Daniel T.O. EMBO J. PubMed Google Scholar), which are required to cell surface of ephrins into responses of Eph-expressing cells (2Kullander K. Klein R. Nat. Rev. Mol. Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (970) Google Scholar, M. Burrola P. Lemke G. Nature. 2004; 431: 847-853Crossref PubMed Scopus (99) Google Scholar). analysis of the Eph and ephrin interaction domains a complex as an essential building block (8Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (276) Google Scholar) that additional molecular not in the crystal formed to assemble functional signaling clusters. we have identified a cluster of 10 ephrin-A5 residues that are part of the E and E-F loop, the underlying H β-strand, as well as the nearby B-C Together a surface the and the heterotetramerization providing a third EphA3 interaction site that essential for EphA3 signal In our we a library screening previously to assign the molecular determinants of EphA3 required for high affinity ephrin binding and formation of functional signaling complexes (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The is on the of a library of the providing an of the We that, in with and binding as functional for and proteins with binding for the interaction this approach of relevant interaction Indeed, of the same the that mutants the ephrin-A5 that, in addition to the high and affinity and tetramerization sites, an additional Eph/ephrin interface may required for receptor we that the ephrin-A5 surface located the previously identified and tetramerization sites, with the cysteine-rich linker of EphA3 (17Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) to assembly of tetrameric complexes into higher order signaling of the with are located to three EphA3 contact It is that the conserved motif of the which contains of the 10 the newly identified region, is and of the surface E and E-F loop the β-barrel (14Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (376) Google Scholar). is likely that EphA3 not interact with the ephrin-A5 with the E the E-F loop, and the B-C loop, which receptor likely for the that we identified a of in the is that there the of the E and E-F loop, providing large of the molecular Although we that the not the and heterotetramerization we this in the of structural However, we are that not the and of ephrin-A5 as well proteins that to a their receptor the of ephrin-A5 to corresponding to the heterotetramerization interface of that the high affinity complex the same as the structurally EphB2·ephrin-B2 In agreement with this the in EphA3 binding affinity and biological observed for the heterotetramerization surface EphA3 and the dimerization surface of EphA3 the effects that for of and high affinity binding sites, In this is to that, only effects on binding in the newly identified interaction site the of ephrin-A5 to EphA3 phosphorylation, and cell We that the structural role of the newly identified interaction surface in ephrins is to bind the cysteine-rich domain of an Eph receptor from an Eph/ephrin higher order signaling clusters. The of the on EphA3 binding suggests that of this to the tetramerization on high affinity Eph/ephrin We that the additional contact through the third interface an EphA3 that Eph/Eph It that is to an Eph·ephrin tetrameric complex three contact sites are within the interacting Eph and ephrin In this formation of higher order clusters on Eph-Eph a notion by the that Eph receptors the ephrin binding domain are into Eph signaling clusters S.H. A. Lackmann M. J. Cell Biol. 2004; PubMed Scopus Google Scholar). a of the molecular of the functional Eph/ephrin signaling clusters requires analysis of a complex their domains. with

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