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Silencing of EphA3 through a cis interaction with ephrinA5

Abstract

EphAs and ephrinAs are expressed in multiple areas of the developing brain in overlapping countergradients, notably in the retina and tectum. Here they are involved in targeting retinal axons to their correct topographic position in the tectum. We have used truncated versions of EphA3, single–amino acid point mutants of ephrinA5 and fluorescence resonance energy transfer technology to uncover a cis interaction between EphA3 and ephrinA5 that is independent of the established ligand-binding domain of EphA3. This cis interaction abolishes the induction of tyrosine phosphorylation of EphA3 and results in a loss of sensitivity of retinal axons to ephrinAs in trans. Our data suggest that formation of this complex transforms the uniform expression of EphAs in the nasal part of the retina into a gradient of functional EphAs and has a key role in controlling retinotectal mapping.

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Figure 1: Cis interaction between ephrinA5 and EphA3.
Figure 2: Characterization of single–amino acid changes in ephrinA5-GFP.
Figure 3: Control of EphA3 tyrosine phosphorylation by coexpressed ephrinAs occurs at the membrane.
Figure 4: Expression pattern and FRET analysis of ephrinA5-CFP and EphA3-YFP on retinal ganglion cells.
Figure 5: Expression of ephrinA5E129K abolishes sensitivity of temporal retinal axons for ephrinA5 applied in trans.

References

  1. McLaughlin, T. & O'Leary, D.D. Molecular gradients and development of retinotopic maps. Annu. Rev. Neurosci. 28, 327–355 (2005).

    Article  CAS  Google Scholar 

  2. Klein, R. Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr. Opin. Cell Biol. 16, 580–589 (2004).

    Article  CAS  Google Scholar 

  3. Poliakov, A., Cotrina, M. & Wilkinson, D.G. Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly. Dev. Cell 7, 465–480 (2004).

    Article  CAS  Google Scholar 

  4. Pasquale, E.B. Eph-ephrin promiscuity is now crystal clear. Nat. Neurosci. 7, 417–418 (2004).

    Article  CAS  Google Scholar 

  5. Kullander, K. & Klein, R. Mechanisms and functions of eph and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3, 475–486 (2002).

    Article  CAS  Google Scholar 

  6. Knöll, B. & Drescher, U. Ephrin-As as receptors in topographic projections. Trends Neurosci. 25, 145–149 (2002).

    Article  Google Scholar 

  7. McLaughlin, T., Hindges, R. & O'Leary, D.D. Regulation of axial patterning of the retina and its topographic mapping in the brain. Curr. Opin. Neurobiol. 13, 57–69 (2003).

    Article  CAS  Google Scholar 

  8. Rashid, T. et al. Opposing gradients of Ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system. Neuron 47, 57–69 (2005).

    Article  CAS  Google Scholar 

  9. Yates, P.A., Roskies, A.L., McLaughlin, T. & O'Leary, D.D. Topographic-specific axon branching controlled by ephrin-as is the critical event in retinotectal map development. J. Neurosci. 21, 8548–8563 (2001).

    Article  CAS  Google Scholar 

  10. Yates, P.A., Holub, A.D., McLaughlin, T., Sejnowski, T.J. & O'Leary, D.D. Computational modeling of retinotopic map development to define contributions of EphA-ephrinA gradients, axon-axon interactions, and patterned activity. J. Neurobiol. 59, 95–113 (2004).

    Article  CAS  Google Scholar 

  11. Hornberger, M.R. et al. Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons. Neuron 22, 731–742 (1999).

    Article  CAS  Google Scholar 

  12. Feldheim, D.A. et al. Topographic guidance labels in a sensory map to the forebrain. Neuron 21, 1303–1313 (1998).

    Article  CAS  Google Scholar 

  13. Erskine, L. et al. Retinal ganglion cell axon guidance in the mouse optic chiasm: expression and function of robos and slits. J. Neurosci. 20, 4975–4982 (2000).

    Article  CAS  Google Scholar 

  14. Niclou, S.P., Jia, L. & Raper, J.A. Slit2 is a repellent for retinal ganglion cell axons. J. Neurosci. 20, 4962–4974 (2000).

    Article  CAS  Google Scholar 

  15. Ringstedt, T. et al. Slit inhibition of retinal axon growth and its role in retinal axon pathfinding and innervation patterns in the diencephalon. J. Neurosci. 20, 4983–4991 (2000).

    Article  CAS  Google Scholar 

  16. Varela-Echavarria, A., Tucker, A., Puschel, A.W. & Guthrie, S. Motor axon subpopulations respond differentially to the chemorepellents netrin-1 and semaphorin D. Neuron 18, 193–207 (1997).

    Article  CAS  Google Scholar 

  17. Chen, H., Chedotal, A., He, Z., Goodman, C.S. & Tessier-Lavigne, M. Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 19, 547–559 (1997).

    Article  CAS  Google Scholar 

  18. Labrador, J.P., Brambilla, R. & Klein, R. The N-terminal globular domain of Eph receptors is sufficient for ligand binding and receptor signaling. EMBO J. 16, 3889–3897 (1997).

    Article  CAS  Google Scholar 

  19. Sobieszczuk, D.F. & Wilkinson, D.G. Masking of Eph receptors and ephrins. Curr. Biol. 9, R469–R470 (1999).

    Article  CAS  Google Scholar 

  20. Miao, H. et al. Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway. Nat. Cell Biol. 3, 527–530 (2001).

    Article  CAS  Google Scholar 

  21. Himanen, J.P. & Nikolov, D.B. Eph signaling: a structural view. Trends Neurosci. 26, 46–51 (2003).

    Article  CAS  Google Scholar 

  22. Himanen, J.P. et al. Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling. Nat. Neurosci. 7, 501–509 (2004).

    Article  CAS  Google Scholar 

  23. Ciossek, T., Lerch, M.M. & Ullrich, A. Cloning, characterization, and differential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of the eck/eph family. Oncogene 11, 2085–2095 (1995).

    CAS  Google Scholar 

  24. Scatchard, G. The attractions of protein for small molecules and ions Ann. NY. Acad. Sci. 51, 660–672 (1949).

    Article  CAS  Google Scholar 

  25. Marston, D.J., Dickinson, S. & Nobes, C.D. Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion. Nat. Cell Biol. 5, 879–888 (2003).

    Article  CAS  Google Scholar 

  26. Zimmer, M., Palmer, A., Kohler, J. & Klein, R. EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion. Nat. Cell Biol. 5, 869–878 (2003).

    Article  CAS  Google Scholar 

  27. Soond, S.M., Everson, B., Riches, D.W. & Murphy, G. ERK-mediated phosphorylation of Thr735 in TNFalpha-converting enzyme and its potential role in TACE protein trafficking. J. Cell Sci. 118, 2371–2380 (2005).

    Article  CAS  Google Scholar 

  28. Bastiaens, P.I. & Jovin, T.M. Fluorescence resonance energy transfer microscopy. in Cell Biology: A Laboratory Handbook (ed. Celis, J.E.) 136–146 (Academic Press, New York, 1998).

    Google Scholar 

  29. Ehehalt, R., Keller, P., Haass, C., Thiele, C. & Simons, K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J. Cell Biol. 160, 113–123 (2003).

    Article  CAS  Google Scholar 

  30. Wu, C., Butz, S., Ying, Y. & Anderson, R.G. Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane. J. Biol. Chem. 272, 3554–3559 (1997).

    Article  CAS  Google Scholar 

  31. Foster, L.J., De Hoog, C.L. & Mann, M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc. Natl. Acad. Sci. USA 100, 5813–5818 (2003).

    Article  CAS  Google Scholar 

  32. Yin, Y. et al. EphA receptor tyrosine kinases interact with co-expressed ephrin-A ligands in cis. Neurosci. Res. 48, 285–296 (2004).

    Article  CAS  Google Scholar 

  33. Gu, C. et al. The EphA8 receptor induces sustained MAP kinase activation to promote neurite outgrowth in neuronal cells. Oncogene 24, 4243–4256 (2005).

    Article  CAS  Google Scholar 

  34. Böhme, B. et al. Cell-cell adhesion mediated by binding of membrane-anchored ligand LERK-2 to the EPH-related receptor human embryonal kinase 2 promotes tyrosine kinase activity. J. Biol. Chem. 271, 24747–24752 (1996).

    Article  Google Scholar 

  35. Dravis, C. et al. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Dev. Biol. 271, 272–290 (2004).

    Article  CAS  Google Scholar 

  36. Holmberg, J., Clarke, D.L. & Frisen, J. Regulation of repulsion versus adhesion by different splice forms of an Eph receptor. Nature 408, 203–206 (2000).

    Article  CAS  Google Scholar 

  37. Marquardt, T. et al. Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains. Cell 121, 127–139 (2005).

    Article  CAS  Google Scholar 

  38. Davy, A. et al. Compartmentalized signaling by GPI-anchored ephrinA5 requires the fyn tyrosine kinase to regulate cellular adhesion. Gen. Dev. 13, 3125–3135 (1999).

    Article  CAS  Google Scholar 

  39. Paratcha, G. et al. Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts. Neuron 29, 171–184 (2001).

    Article  CAS  Google Scholar 

  40. Egea, J. et al. Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function. Neuron 47, 515–528 (2005).

    Article  CAS  Google Scholar 

  41. Reber, M., Burrola, P. & Lemke, G. A relative signalling model for the formation of a topographic neural map. Nature 431, 847–853 (2004).

    Article  CAS  Google Scholar 

  42. Goodhill, G.J., Gu, M. & Urbach, J.S. Predicting axonal response to molecular gradients with a computational model of filopodial dynamics. Neural Comput. 16, 2221–2243 (2004).

    Article  Google Scholar 

  43. Loschinger, J., Weth, F. & Bonhoeffer, F. Reading of concentration gradients by axonal growth cones. Phil. Trans. R. Soc. Lond. B 355, 971–982 (2000).

    Article  CAS  Google Scholar 

  44. Drescher, U. et al. In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359–370 (1995).

    Article  CAS  Google Scholar 

  45. Connor, R.J., Menzel, P. & Pasquale, E.B. Expression and tyrosine phosphorylation of Eph receptors suggest multiple mechanisms in patterning of the visual system. Dev. Biol. 193, 21–35 (1998).

    Article  CAS  Google Scholar 

  46. Burack, M.A., Silverman, M.A. & Banker, G. The role of selective transport in neuronal protein sorting. Neuron 26, 465–472 (2000).

    Article  CAS  Google Scholar 

  47. Monschau, B. et al. Shared and distinct functions of RAGS and ELF-1 in guiding retinal axons. EMBO J. 16, 1258–1267 (1997).

    Article  CAS  Google Scholar 

  48. Toth, J. et al. Crystal structure of an ephrin ectodomain. Dev. Cell 1, 83–92 (2001).

    Article  CAS  Google Scholar 

  49. Himanen, J.P. & Nikolov, D.B. Purification, crystallization and preliminary characterization of an Eph-B2/ephrin-B2 complex. Acta Crystallogr. D Biol. Crystallogr. 58, 533–535 (2002).

    Article  Google Scholar 

  50. Hansen, M.J., Dallal, G.E. & Flanagan, J.G. Retinal axon response to ephrinAs shows a graded, concentration-dependent transition from growth promotion to inhibition. Neuron 42, 717–730 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank V. Sundaresan (King's College, London) for providing Robo2-MYC and A. Ullrich (Max-Plank-Institut for Biochemistry, Munich) for the PDGF-R vector; S. Kümper and A. Snedden for cloning Eph and ephrin constructs; and C. Jarvis, P. Gordon-Weeks, and R. Drescher for critical reading of the manuscript. This work was supported by the Wellcome Trust. R. Carvalho is a student of the Gulbenkian PhD Program in Biomedicine, Portugal. M. Beutler was supported by Deutsche Forschungs Gemeinschaft (HE 3492/2-1), Schwerpunktprogram (SPP)1128 & Higher Education Funding Council England (HEFCE). T. Ng is supported by an endowment fund from the Richard Dimbleby Cancer Fund to King's College London.

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Correspondence to Uwe Drescher.

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Supplementary information

Supplementary Fig. 1

The PDGF receptor does not coimmunoprecipitate with ephrinA5. (PDF 419 kb)

Supplementary Fig. 2

Models for cis and trans interactions between EphAs and ephrinAs. (PDF 1502 kb)

Supplementary Fig. 3

Analysis of the availability of the LBD of EphA3 in cells coexpressing EphA3/ephrinA5wt (EphA3/ephrinA5E129K). (PDF 335 kb)

Supplementary Fig. 4

Characteristics of the binding of EphA3 to ephrinA5wt or ephrinA5E129K. (PDF 514 kb)

Supplementary Fig. 5

Donor (CFP) dequenching under acceptor (YFP) depletion. (PDF 876 kb)

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Carvalho, R., Beutler, M., Marler, K. et al. Silencing of EphA3 through a cis interaction with ephrinA5. Nat Neurosci 9, 322–330 (2006). https://doi.org/10.1038/nn1655

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