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Structurally encoded intraclass differences in EphA clusters drive distinct cell responses

Nature Structural & Molecular Biology volume 20, pages 958–964 (2013)Cite this article

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Abstract

Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties.

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Figure 1: Stimulation of EphA2 and EphA4 triggers different cell responses.
Figure 2: EphA4 ectodomain forms oligomeric units in crystal lattices.
Figure 3: Properties of the EphA4 HI-loop area and sushi dimerization region.
Figure 4: Eph ectodomains can control Eph clustering and function.
Figure 5: Non-ligand-bound EphA2 clustering depends on Eph-Eph ectodomain interactions.

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Acknowledgements

We thank Y. Zhao and W. Lu for protein expression and M. Jones and T.S. Walter for technical support. We are grateful to T. Gaitanos for help with confocal microscopy data acquisition, I. Davis and I.M. Dobbie for discussion and assistance at the single-molecule–localization facility in the Micron Advanced Bioimaging Unit, J. Erl and M. Ponserre for assistance with cell-based assays, K.J. Morris for providing access to MetaMorph software and R.M. Esnouf for aiding in protein structure analysis. We thank the staff of the Diamond Light Source for assistance with diffraction data collection and K. Diederichs for help with data integration. This research was funded by a Cancer Research United Kingdom grant to E.Y.J. (grant A10979). Localization microscopy facilities in the Micron Advanced Bioimaging Unit were funded by the Wellcome Trust (grant 091911). E.S. was funded by an Intra-European Fellowship (Marie Curie); D.d.T.R. was funded by an European Molecular Biology Organization long-term fellowship; N.M. is supported by a Wellcome Trust D.Phil. studentship; and A.R.A. was supported as a United Kingdom Medical Research Council Career Development Award Fellow. Partial funding was provided by the Deutsche Forschungsgemeinschaft (SFB870) to R. Klein and Wellcome Trust grant 090532/Z/09/Z supporting the Wellcome Trust Centre for Human Genetics.

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Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Elena Seiradake, Rainer Kaufmann, Nikolaos Mitakidis, Karl Harlos, A Radu Aricescu & E Yvonne Jones

  2. Max Planck Institute of Neurobiology, Martinsried, Germany

    Andreas Schaupp, Daniel del Toro Ruiz & Rüdiger Klein

  3. Department of Biochemistry, University of Oxford, Oxford, UK

    Rainer Kaufmann

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  1. Elena Seiradake
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Contributions

E.S. performed protein crystallization, structure analysis, cell rounding assays and Eph clustering experiments. A.S. contributed to time-lapse imaging experiments. D.d.T.R. performed stripe assays. R. Kaufman conducted localization microscopy data acquisition and analysis. N.M. contributed to protein crystallization. K.H. performed Eph crystal mounting for data collection. A.R.A., R. Klein and E.Y.J. contributed to discussion at all stages of the project. All authors contributed to writing of the manuscript.

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Correspondence to Rüdiger Klein or E Yvonne Jones.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Note (PDF 3739 kb)

Supplementary Movie 1

Phase-contrast images showing the response of an EphA2-mVenus transfected cell upon pre-clustered ephrinA5-Fc stimulation. Images were taken before and every 6 minutes after stimulation. (MOV 108 kb)

Supplementary Movie 2

Phase-contrast images showing the response of an EphA4-mVenus transfected cell upon pre-clustered ephrinA5-Fc stimulation. Images were taken before and every 6 minutes after stimulation. (MOV 157 kb)

Supplementary Movie 3

Confocal images of live COS7 cell time lapse experiments show mVenus-tagged EphA2 clustering upon ephrinA5-Fc stimulation. (MOV 275 kb)

Supplementary Movie 4

Confocal images of live COS7 cell time lapse experiments show mVenus-tagged EphA4 clustering upon ephrinA5-Fc stimulation. (MOV 908 kb)

Supplementary Movie 5

Confocal images of live COS7 cell time lapse experiments show mVenus-tagged chimeric Eph receptor A4A2 clustering upon ephrinA5-Fc stimulation. A4A2 contains an EphA4 ectodomain fused to EphA2 transmembrane and intracellular domains. (MOV 247 kb)

Supplementary Movie 6

Confocal images of live COS7 cell time lapse experiments show mVenus-tagged chimeric Eph receptor A2A4 clustering upon ephrinA5-Fc stimulation. A2A4 contains an EphA2 ectodomain fused to EphA4 transmembrane and intracellular domains. (MOV 434 kb)

Supplementary Movie 7

Confocal images of live COS7 cell time lapse experiments show the mVenus-tagged EphA2 sushi dimerization surface mutant (EphA2su) clustering upon ephrinA5-Fc stimulation. (MOV 284 kb)

Supplementary Movie 8

Confocal images of live COS7 cell time lapse experiments show the mVenus-tagged EphA2 sushi dimerization surface mutant (EphA2su) clustering upon ephrinA5-Fc stimulation. (MOV 401 kb)

Supplementary Movie 9

mCherry-tagged EphA2 was co-expressed in COS7 cells with the non-ephrin-binding mVenus-tagged mutant EphA2nb. Confocal images of time lapse experiments show mVenus-tagged EphA2nb co-clustering with mCherry-tagged EphA2 upon ephrinA5-Fc stimulation. (MOV 330 kb)

Supplementary Movie 10

mCherry-tagged EphA2 was co-expressed in COS7 cells with the mVenus-tagged non-ephrin-binding and sushi dimerization surface mutant EphA2nb–su. Confocal images of time lapse experiments show mVenus-tagged EphA2nb–su does not co-cluster with mCherry-tagged EphA2 upon ephrinA5-Fc stimulation. (MOV 352 kb)

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Seiradake, E., Schaupp, A., del Toro Ruiz, D. et al. Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nat Struct Mol Biol 20, 958–964 (2013). https://doi.org/10.1038/nsmb.2617

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