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Recruitment of Eph receptors into signaling clusters does not require ephrin contact

¹ Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
² Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
³ European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Address correspondence to Martin Lackmann, Dept. of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, Clayton, Victoria 3800, Australia. Tel.: 61 3 9905 3738. Fax: 61 3 9905 3726. email: Martin.Lackmann{at}med.monash.edu.au

Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions.

Key Words: fluorescence resonance energy transfer microscopy; EphA3 receptor; receptor protein tyrosine kinase; receptor aggregation; signal transduction

S.H. Wimmer-Kleikamp and M. Lackmann's present address is Dept. of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

Abbreviations used in this paper: 3YF EphA3, [Tyr₅₉₆-Phe, Tyr₆₀₂-Phe, Tyr₇₇₉-Phe] EphA3; Ephs, Eph receptor tyrosine kinases; FLIM, fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; HEK293, human epithelial kidney 293; nb-EphA3, [Phe₁₅₂-Leu, Val₁₃₃-Glu] EphA3; w/t, wild-type.

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